University of Nigeria · University of Nigeria Research Publications Author MONEKE, Nwabu Anene...
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University of Nigeria Research Publications MONEKE, Nwabu Anene Author PG/Ph.D/93/14521 Title Production, Purification, Immobilisation and Characterisation of Xvlose-Glucose Isomerases from Paenibacillus SP, and Alcaligenes Ruhlandi Isolated from Nigeria Soil Faculty Biological Sciences Department Microbiology Date August, 1997 Signature
Transcript of University of Nigeria · University of Nigeria Research Publications Author MONEKE, Nwabu Anene...
University of Nigeria Research Publications
MONEKE, Nwabu Anene
Aut
hor
PG/Ph.D/93/14521
Title
Production, Purification, Immobilisation and
Characterisation of Xvlose-Glucose Isomerases from Paenibacillus SP, and Alcaligenes Ruhlandi Isolated from
Nigeria Soil
Facu
lty
Biological Sciences
Dep
artm
ent
Microbiology
Dat
e August, 1997
Sign
atur
e
Production, Purification, Irnmobilisation and Characterisation of
xylose/glucose isomerases from Paenihacillus sp and Alcaligenes mhlandii
isolated from Nigerian soil.
Moneke, Nwabu Anene
PG / Ph.D.19311452 1
A thesis submitted to the Department of Microbiology, in the Faculty of
Biological Sciences, as a requirement for the award of the degree of
Doctor of Philosophy (Industrial and Food Microbiology) of the
University of Nigeria, Nsukka .
Supervisor : Professor S . K . C. Obi
August, 1997
CERTIFICATION
Mr Anene Nwabu Moneke, a post-graduate student in the Department of
Microbiology, has satisfactorily completed the requirements for the degree of
Doctor of Philosophy (Ph.D) in Industrial and Food Microbiology. The work
embodied in his thesis is original and has not been submitted in part or full for
any other diploma or degree,of this or any other University.
Supervisor :
Professor SI K . c . OBI
Department of Microbiology
University of Nigeria
Nsukka.
DEDICATION
To my dear mother, brothers and sisters, who encourag
my dear children and wife, who put up with me; and 11
father of blessed memory, who passed on while the strugg
me;
dear
vas on.
ACKNOWLEDGEMENTS
First, I must thank our Father Almighty for granting me stre
good health all through the course of this research work.
It is my pleasure to acknowledge the debt I owe to my colleagu
friends without whom this work would not have been possible.
supervisor, Professor S.K.C. Obi, advised, criticised and guided
through this work . He undertook the formidable task of reading
manuscript and assisted me in sundry other ways. My host in G
Professor Hans Bisswanger of the University of Tuebingen, Ger
most helpful in providing me with the working knowledge of bio
especially the practical aspect of the subject. He also gave me an
access to all the facilities at the Physiologisch-Chemie Institut, UI
Tuebingen. I have benefitted immensely from the expertise of tl
eminent Professors (Obi and Bisswanger) and working closely v
over the years has done much to shape my own scientific ideas.
IV
:th and
; and
Y
e all
le
many - any, was
!emistry,
nrestricted
irersity of
se two
h them
I also wish to thank my colleagues in Professor Bisswanger's laboratory,
Tuebingen, Germany - Qiang Liu, Stefan, Uli, Bernd, Christian, ~ a i n e r and
Victoria for their understanding, support and helpful suggestions during my
stay with them. I must thank Qiang Liu specially for helping me o&rcome !
my computer fright and coaching me on the use of computers. I
To my friend and colleague, Dr Bato Okolo, I cannot thank you enough for
not allowing me to abandon this programme. His frank and valuablk
comments on my work are appreciated. I am also very grateful to di- Lewis
Ezeogu for his assistance all through this work. To the entire st
Department of Microbiology, University of Nigeria, thank you al
me appreciate the essence of science and knowledge.
I received a lot of steadfast support and words of encouragen
numerous friends especially Mr C.T.Onyekwelu, Professor Obui
Ekwueme, Engr. Fred Okeke, Dr S.V.O. Shoyinka, Mr G. Ezekw
Uche Apakama (Paxs), IK. Ugwu, Tayo Adenaike and Emma Ez,
Finally, I must thank my dear mother, brothers and sisters for ti
patience, love and care. To my wonderful wife, Pamela and lo\
Kemy and Ninny, I cannot thank you enough for your love and
encouragement. I could not have wished for a more sympathetic I
while this work lasted
THANK YOU !
Anene N. Moneke
August, 1997
v
Bof the
for helping
nt from my
neme
:o,
lwanne.
:ir
~g kids,
:atment
TABLE OF CONTENTS
Title page ........ .... ......
Certification . . . .. . . . . . . . . . . . . .
Dedication . . . . . . . . . . . . . . . . . . . .
Acknowledgements . .. . . . . . . . . Table of Contents . . . . . . . . . .
List of Figures .... ...........
List of Tables ....... ...........
Abstract ,.,...,..,. ...... .....
CHAPTER 1
1.0 Introduction . . . . . , . . . .
1.1 Aims and Objectives . . . ..
CHAPTER 2
Literature Review .....
The Starch molecule .....
Enzyme conversion of Starch .. . . . . Chemical isomerisation of D-glucose ........
Types of xylose/glucose isomerases . . . . . .
Xylose/glucose isomerase production in bacteria . . . ... . . . Strain yield improvement .......... ......... ..,...... ....
2.6.1 Homologous hosts . . ... . . .... . . . ,............... ..,.......,.... ...
2.6.1.1 Homologous cloning in E. coli ................ ..................
2.6.1.2 Homologous cloning in Streptomyces spp. .. . ... . . .. . . ..
i . . 11
iii
iv
vi . . .
Xlll
xvi
xvii
1
4
6
6
9
11
12
16
19
2 1
23
24
vii
................................................ 2.6.2 Heterologous hosts
. ................... 2.6.2.1 Cloning from Bacillus subtilis to E coli ; I
....... 2.6.2.2 Heterologous cloning into other bacterial hosts I I
........................ 2.6.2.3 Heterologous cloning in yeasts I I
....................... 2.6.2.4 Heterologous cloning in plants I
2.7 Optimisation of fermentation medium .......... ............. i
2.7.1 Inducer ~ I ............................................................
2.7.2 Nitrogen source ..............................................
..................................... 2.7.3 pH and temperature optima
2.7.4 Metal ion requirement ..........................................
2.8 Immobilisation of xylose/glucose isomerase ...................
..................................... 2.8.1 Cell-free immobilisation
.................................... 2.8.2 Whole-cell immobilisation
2.9 Purification of xylose/g~ucose isomerase ..........................
2.10 Properties of xylose/glucose isomerase .........................
2.10.1 Substrate specificity .............................................
2.10.2 Metal ion requirement and inhibitors ........................
2.10.3 Subunit structure ..............................................
2.10.4 Optimum temperature and pH ................................
2.10.5 Active-site studies ................................................
2.11 Mechanism of action of xylose isomerase ..................
2.1 1.1 Chemical modification of xylose/glucose isomerase ....... i
2.1 1.2 X-ray crystallography ......................................... ;
2.1 1.3 Isotopic exchange .............................................. (
2.12 Genetic regulation of xylose/gl:lucose isomerase biosynthesis ...... I
I 2.12.1 Genetic organisation of xyl genes ............................ :
I
............................................ 2.12.2 Divergent promoters ; ~ 2.12.3 Catabolite repression ......................................... ! r 2.13 Genetic improvement of xylose/glucose isomerase by site di pcted
mutagenesis .....................................................
2.13.1 Thermal stabilisation . . . . . . . . . . . . ......................
2.13.2 Deciphering the role of metal ions ........................
......................... 2.13.3 Alteration of substrate specificity
2.13.4 Functional role of essential amino acid residues ..........
2.13.5 Alteration of pH optimum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.14 Identified problems and possible solutions .................
2.14.1 Enhancement of thermostability ..........................
2.14.2 Enrichment of fructose ......................................
2.14.3 Lowering of isomerisation pH ............................
2.14.4 Simultaneous isomerisation and fermentation of xylose . .
......................................................... 2.15 Future scope
CHAPTER 3
......................................... 3.0 Materials and Methods
........................................................... 3.1 Materials
..... 3.2 Collection of sample and isolation of microorganisms
............................ 3.2.1 Sample preparation ................
.................. 3.2.2 Isolation of microorganisms ................
.... 3.3 Screening test for xylose/glucose isomerase production
................. 3.3.1 Confirmation of xylose isomerase production
3.4 Enzyme assays ................................ ................
........................ 3.5 Determination of protein concenhatiol~
3.6 Identification of isolates ...........................................
3.6.1 Identification of actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. ............................... 3.6.2 Identification of true bacteria
3.7 Preliminary production of xylose isomerase in submerged
culture ....... .............. ...............................
3.8 Analysis of data ....................... ...............................
... 3.9 Extraction and purification of two xylose isomerase enzymes
...................................... 3.9.1 Preparation of crude extract
..... 3.9.2 Ion exchange chromatography on Whatrnan DE52 column
3.9.3 Ammonium sulphate fractionation ...................................
3.9.4 Gel filtration on Sephacryl S-200 HR .............................
3.9.5 Hydrophobic interaction chromatography on Phenyl superose
........ ....................................................... column
3.9.6 Gel filtration on Superose 6TM ......................................
3.10 Homogeneity of purified xylose/glucose isomerase ..............
3.11 Determination of molecular mass of the purified xylose/glucose
lsomerases ....................................... ...................
3.12 Enzyme characterisation .............................................
3.12.1 Effect of temperature on enzyme activity .........................
3.12.2 Effect of temperature on enzyme stability ........................
3.12.3 Enzyme decay ........................................................
....................................... 3.12.4 Effect of pH on enzyme activity
3.12.5 Effect of pH on enzyme stability ....................................
3.12.6 Effect of substrate concentration on D-xylose/glucose isomerase
activities ...................... ........................................
.................. 3.12.7 Effect of divalent metals on D-xylose isomerase
3.13 Inhibition of D-xylose isomerase activity .............................
.................................................. 3.13.1 Inhibition by EDTA
3.13.2 Inhibition by D-xylitol and D-lyxose ..............................
3.13.3 Inhibition of the xylose isomerase enzymes by copper ions in the
.......................................... presence of manganese ions
3.14 Immobilisation of the xylose/glucose isomerase enzymes ........
3.14.1 Polyacrylamide gel entrapment ........................................
3.14.2 Covalent bonding to controlled pore glass ..........................
3.14.3 Fixation on cyanogen bromide activated sepharose 4B ..........
3.15 Assay of the immobilised xylose/glucose isomerases ..........
3 . 16 Protein assay of immobilised enzymes ..............................
3.17 Characterisation of immobilised enzymes . . . . . . . . . . . . . . . . . . . . . . . .
3.17.1 Temperature stability of the immobilised enzymes ..............
3.17.2 pH stability of immobilised enzymes ..............................
3.17.3 Half-life study at 5S°C ......................... ..................
3.17.4 Activity yield of imtnobilised enzymes . . . . . . . . . . . . . . . . . . . . . . . .
CHAPTER 4
4.0 Results .................................................................... 85
4.1 Isolation and identification of microorganisms with xylosel
glucose isomerase activity ............................... ..............
....................... 4.2 Selection of strains ........................
4.3 Purification of the enzymes of Paenibacillus and Alcaligenes
ruhlandii ..................................................................
................................. 4.4 Molecular mass determination
....................... 4.5 Effect of temperature on enzyme activity
......................... 4.6 Thermal stability profiles of enzymes
............ 4.7 pH activity and stability profiles of the enzymes
4.8 Dependence of enzyme activities on substrate concentration ..
4.9 Effect of divalent metals ............................................
4.10 Effect of various concentrations of divalent metals on . .
..................................................... enzyme activlty
4.11 Inhibition studies ...............................................
4.11.1 EDTA .............................................................
4.1 1.2 D-xylitol ..........................................................
4.1 1.3 D-lyxose .......................................................
4.11.4 Competitive inhibition by copper ions in the presence of
manganese ions ......................... ............ .....
4.12 Immobilisation of the enzymes ..............................
4.12.1 pH stability studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CHAPTER 5
5.0 Discussion .......................................................
xii
CHAPTER 6
6.0 Conclusion .........................
...................................................... References
Appendices ..................................... ...................
.................... 1 . Statistical validation of treatment effects
............................ 2 . Stock solutions for SDS-PAGE
...................... 3 . Standard curve of D-xylulose ......
4 . Standard curve of D-fructose . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
.................. 5 . Standard curve of bovine serum albumin
LIST OF FIGURES
1 . HFCS production by D-xylose/glucose isomerase ......................
2 . A process flowsheet for starch liquefaction and saccharification ..
3 . Chemical isomerisation of glucose ..........................................
4 . Production of fructose syrup .............................................
5 . Reaction of glucose isomerase .........................................
6 . Mechanism of action of glucose isomerase ............................
7 . Elution profile of l'aenibacillus enzyme on Whatman DE52 .........
8 . Elution profile of A1caligene.v ruhlandii enzyme on Whatman DE52 ..
9 . Elution profile of Paenibacillus enzyme on Sephacryl S-200HR ....
10 . Elution profile of A . ruhlandii enzyme on Sephacryl S200HR ....
1 1 . Elution profile of PaenibuciNus enzyme on Phenyl superose ........
12 . Elution profile of A . ruhlandii enzyme on Phenyl superose .......
13 . Elution profile of Paenibaci1lu.s enzyme on Superose 6TM .............
14 . Elution profile of A . ruhlundii enzyme on Superose 6TM ............
15 . SDS-PAGE .................................................................
16 . Molecular weight determination on Superose 12TM ...................
..................... 17 . Molecular weight determination by SDS-PAGE
..................... 18 . Effect of temperature on activity of the enzymes
................... 19 . Arrhenius plot for I'aenibacillus xylose isomerase
. .................. 20 . Arrhenius plot for A ruhlandii xylose isomerase
..................... 2 1 . Effect of temperature on stability of the enzymes
22 . Enzyme decay at 55OC for Paenihacillus xylose isomerase ...........
.......... 23 . Enzyme decay at 55'C for A . ruhlandii xylose isomerase
...... 24 . Effect of pH on activity and stability of Paenibuillus enzyme
.... . 25 . Effect of pH on activity and stability of A ruhlundii enzyme
.... 26 . Lineweaver-Burke plot for the enzymes with xylose as substrate
.... 27 . Lineweaver-Burke plot for the enzymes with glucose as substrate
28 . Eadie-Hofstee diagram for dependence of Paenibacillus enzyme on
divalent metals .....................................................................
29 . Eadie-Hofstee diagram for dependence of A . rrrhlandii enzyme on
...................... divalent metals ......................... ..............
30 . Lineweaver-Burke plot for dependence of Paenibacillus enzyme on
divalent metals .............. ..................... ...........................
3 1 . Lineweaver-Burke plot for dependence of A . ruhlandii-enzyme on
.................................................................. divalent metals
32 . Effect of EDTA on Paenibacilltrs enzyme ...............................
33 . Effect of EDTA on A . ruhlandii enzyme ............................
34 . Inhibition of Paenibacilltrs enzyme by D-xylitol .....................
35 . Inhibition of A . ruhlandii enzyme by D-xylitol ..................
36 . Lineweaver-Burke plot of the inhibition of Paenibacillus enzyme by
D-xylitol at various xylose concentrations .................................
37 . Lineweaver-Burke plot of the inhibition of A . ruhlandii enzyme by
D-xylitol at various xylose concentrations ....................................
38 . Replot of slopes of D-xylitol inhibition of Paenibacillus enzyme ...
39 . Replot of slopes of D-xylitol inhibition of A . ruhlandii enzyme ....
40 . Inhibition of Paenibacillus enzyme by D-lyxose .........................
41 . Inhibition of A . ruhlandii enzyme by D-lyxose ..........................
42. Lineweaver-Burke plot of the inhibition of Paenibacillus enzyme by
D-lyxose at various xylose concentrations .............. ............. ... 134
43. Lineweaver-Burke plot of the inhibition of A. ruhlandii enzyme
by D-lyxose at various xylose concentrations ................. ........... 135
44. Replot of slopes of D-lyxose inhibition of F'aenibacillus enzyme .... 136
45. Replot of slopes of D-lyxose inhibition of A. ruhlandii enzyme .. 137
46. Eadie-Hofstee plot of the competitive inhibition of Mg2' by CU"
in the Paenrbacillus enzyme reaction ........ ............... .......... 138
47. Eadie-Hofstee plot of the competitive inhibition of MgZ' by CU"
in the A. ruhlandii enzyme reaction ............. ................ ... 139
48. Effect of pH on the stability of immobilised Paenibacillus xylose
isomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . , . . , . . . . . . .. . .. . . 142
49. Effect of pH on the stability of immobilised A. ruhlandii xylose
isomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................... ................... 143
LIST OF TABLES
................ 1 . Xylose/glucose isomerase producing organisms
2 . Commercial xylose/glucose isomerase producers ...............
3 . Production of xylose/glucose isomerase by various organisms ...
4 . Immobilised xylose/glucose isomerase of commercial importance ...
5 . Effect of isomerisation temperature on the concentration of fructose
6 . Isolated bacteria and their xylose isomerase activities ...............
7 . Isolated actinomycetes and their xylose isomerase activities ........
8 . Purification summary of I'aenihacillzrs xylose isomerase ............
9 . Purification summary of A . ruhlandli xylose isomerase ............
10 . Effect of divalent metals on metal-free Paenibaci1lu.s and
A . ruhlandii enzymes .....................................................
I I . Kinetic constants for the divalent metal ions ............................
12 . Summary of results of irnmobilisation experiments .................
ABSTRACT
D-xylose/glucose isomerases from two microbial isolates- a strain of
Alcaligenes ruhlandii, and a new strain of Paenibacillus sp isolated from soil
samples are described. The enzymes were purified to apparent homogeneity
by ion-exchange chromatography on DEAE-cellulose (Whatman DE52),
ammonium sulphate fractionation, gel filtration on Sephacryl-S200 HR,
phenyl-Superose hydrophobic interaction chromatography and a second gel
filtration on Superose 6TM. The Paenibacillus enzyme had a purification
factor of 6.74 and a yield of 18.69% while that of Alcaligenes ruhlandii had
a purification factor of 10.11 and a yield of 13.53%. Both D-xylose/glucose
isomerases were homotetramers with relative subunit molecular masses of
45,000 and 53,000 respectively as estimated by SDS-PAGE. The native
molecular masses as determined by Superose 1 2 ' ~ gel chromatography were
181,000 for the Paenibacillus enzyme and 199,000 for that ofAlcaligenes
ruhlanu'ji. Both enzymes showed requirements for divalent metal ions with
the D-xylose/glucose isomerase from Paenihacillus sp showing highest
activity with ~n~~ while that of Alcaligenes ruhlandii had preference for
~ g ~ ' . Both enzymes were also activated by co2' though to a lesser degree.
CU" was inhibitoly lo both enzymes. Enzyme binding to metal ions showed
biphasic characteristics as an indication for two non-identical binding sites
per subunit. D-glucose was converted to D-fructose at a rate 2 - 3 fold slower
than the rate for D-xylose isomerisation. D-xylitol and D-lyxose proved to be
competitive inhibitors of both enzymes. The Paenihacillus enzyme had a
pH optimum of 7 while that of Alcaligenes ruhlandii exhibited a pH
xviii
optimum of 6.5. The maximum activity temperature was 65°C for the
Paenibacillus enzyme and 65 - 70°C for that of Alcaligenes ruhlandii - with
activation energies of 39.61K~mol-' and 42.14 K~mol-' respectively. Half-
life studies at 55°C showed that 50% of the activity was retained after 4 days
for the Paenibacillus enzyme and after 6 days for the Alcaligenes ruhlandii
enzyme. Immobilisation of the Paenihacillus enzyme on contTolled pore
glass gave the highest yield of 76.45% and the half-life at 5 5 T was extended
to 7 days while immobilisation of the enzyme from Alcaligenes ruhlandii on
cyanogen bromide activated Sepharose 4B gave the highest yield of 82.6%
while extending the half-life at 55°C to 12 days. Both immobilised enzymes
were stable at 4OC and 25°C.
CHAPTER 1
1.0 INTRODUCTION
D-xylose/glucose isomerase enzyme (D-xylose ketol isomerase,
EC 5.3.1.5) catalyses the reversible isomerisation of glucose to fructose.
Most of the known glucose isomerases are intracellular enzymes whose
original function was apparently to catalyse the fo~mation of xylulose from
xylose (Chen, 1980).
By far the most successful application of enzymes on an industrial scale is
the production of high fructose corn syrup (H.F.C.S.) using D-xylosel
glucose isomerase (Figure 1). The annual world consumption of H.F.C.S. in
1995 was estimated to be 10 million tons (dry weight) (deRaadt et al., 1994).
Today, H.F.C.S. has almost completely replaced sucrose in the United States,
while the rest of the world recorded a moderate (3 - 4%) production growth
rate (Bhosale et al., 1996).
The aim of this process is to produce a material of equivalent or higher level
of sweetness to sucrose from a low cost raw material such as corn starch. This
conversion requires the sequential use of three enzymes : i) the starch
(a polymer of D-glucose) is liquefied using a bacterial alpha amylase, ii) the
liquefied material is saccharified by the action of glucoamylase to give a
solution where 94 -96% of the carbohydrate present is in the form of
D-glucose, and iii) isomerisation of the glucose solution by D-xylosel
glucose isomerase. Glucose has some applications in the food and
pharmaceutical industries but its use is restricted by its limited solubility at
high concentrations and its low sweetening power representing 70 -75% that
of sucrose (Gacesa & Hubble, 1987). However, D-fructose (levulose) the
other monosaccharide moiety of sucrose on the other hand, has twice the
sweetening power of sucrose and plays an important role in the diet of
diabetics as it is only slowly absorbed by the stomach and intestinal tract and
does not influence the blood glucose level (Crueger & Crueger, 1990).
The second major commercial interest in the D-xylose/glucose isomerase
enzyme is in the production of ethanol from xylose. The predominant sugar in
hemicellulosic agricultural residues is xylose, which occurs as a linear subunit
of xylan. Several yeasts particularly those belonging to the genera
I'achyLsole and Candida have been shown to convert xylose to ethanol.
However, the industrially important yeasts of the genus Saccharomyces are
unable to produce ethanol from D-xylose. To take advantage of the existing
yeast technology, work is currently directed towards the introduction of a
bacterial D-xylose isomerase gene into suitable yeast host (Henrick et a/.,
1989).
In view of their high industrial significance, D-xylose/glucose isomerases
from various microorganisms have been studied and their catalytic and
physicochemical properties reviewed (Chen, 1980). Cussently, most
commercially available xylose/glucose isomerases are derived from
mesophilic microorganisms such as Streptomyces, Actinoplanav, RaciNus, and
Flavobacterium species. The enzymes generally exhibit the properties of
themostability and their utilisation in the immobilised forms helps to enhance
their shelf-lives (Verhoff et al., 1985). The optimum temperature for
isomerisation varies from 40 to 90°C depending on the experimental
Corn
4 HFCS
T wet milling xylose z.somera,w
.1 T Starch -+ + an~ylase -+ Glucose
.+ Oil & -t Gluten ,fi.m~entation
4 Ethanol,
yeast
Figure I : HFCS production by D-xylose/glucose isomerase (Henrick et al., 1989)
conditions like pH, type of buffer, substrate concentrations, activators,
stabilizers and reaction time. The available commercial D-xylose/glucose
isomerases require metal ions for their activity and stability and their pH
activity optima are usually slightly basic depending on the source of the
enzyme. The reaction temperatures used in current industrial processes for
sweetener production do not exceed 60°C because of by-products and colour
formation during reaction at higher temperatures and alkaline pH (Vaheri &
Kauppinen, 1977).
Presently in Nigeria, a process for the enzymatic production of HFCS is
yet to be developed and commercialised. For this reason, crystalline sucrose
is still the prefered sweetener by most people. The country stands to gain
economically if we are to utilise our abundant cheap starch sources (corn,
cassava, sorghum, yam, et cetera) to produce HFCS using xylose/glucose
isomerase enzymes derived from our local microbial isolates.
1.1 AIMS AND OBJECTIVES
The present work was aimed at achieving the following objectives
1) to screen bacterial isolates ftom diverse ecological niches in the Nsukka
area of Nigeria for ability to produce xylose/glucose isomerase enzymes
active in the acid to neutral pH range.
2) to purify the enzymes to the point of homogeneity or near -homogeneity
and thereafter subject them to kinetic and physicochemical characterisation
3) to immobilise the purified enzymes on various solid supports and thereafter
assess the effect of immobilisation on certain catalytic and kinetic properties.
CHAPTER 2
2.0 LITERATURE REVIEW
2.1 THE STARCH MOLECULE
The basic raw material used for the production of carbohydrate-based
sweeteners is starch. Because starches are insoluble complex molecules they
must first undergo strnctural modification before they can be hydrolysed
enzymatically to release simple fermentable sugars. A flowsheet of a starch
liquefaction and saccharification process is given in Figure 2 (Antrim et al.,
1979).
Starch is a biopolymer composed of amylose and amylopectin polymers
that contain ant~ydroglucose units joined by only a- 1,4 linkages in the case
of amylose but a-1,4 and a-1,6 linkages in the case of amylopectin. The
amylose fraction contains nearly all a-1,4 linked glucose units. The
amylopectin fraction contains about 5% a-1,6 linkages; therefore a linear
chain is interupted on the average of every twenty glucose units by an a- 1,6
linkage (Hebeda, 1993). The relative amount of each fraction varies and
amylose/amylopectin ratio ranges from 01100 to 85/15 in different starches.
Regular corn starch contains about 27% amylose and 73% amylopectin
whereas high amylose corn starch, potato starch and waxy maize starch
contain 50 - 70%, 17 - 23% and < 2% amylose respectively (BeMiller, 1992).
Starch is present in plants as small granules that range in size from 0.5 to
Starch slurry
& t lime water
Feedtank t a-amylase (pH6 - 6.5)
& Liquefaction t steam (80-150°C upto 3h)
& t pH adjustment with acid
pH4 -5 ; 24-90h.+ Saccharification tglucoamylase;50-60°C
& t (90-96% dextrose,DB)
Filtration ; refining
& t salts & pH adjustment
40-50% dry substancc -+ lsomerisation t 65 - 65"C, pH7- 8.5
4 Refining, concentrating
4 Fructose syrup
Figure 2 : A process flowsheet for starch liquefaction and saccharification (Antrim et al., 1979)
175 pm depending on the source. The granules may be present in various
locations within the plant including the root, tuber, stem pith, leaf, seed, fruit,
pollen, etcetera.
Amylose is essentially a linear molecule in which the glucose units are
linked through a - 1,4 bonds and it has a double helical crystalline structure.
X-ray diffraction patterns suggest that the helix contains six D-glucose
molecules per turn with dimensions that enable an iodine molecule to be
accommodated within the helix. This gives rise to the characteristic blue
colour of the starch-iodine complex.
Amylopectin in contrast to amylose is a highly branched structure with
4 -6% a-1,6 bonds at branch points; the average length of the branch chains
is 20-25 glucose units. The individual amylopectin molecules are similar but
not identical in their branching configuration. A randomly branched " b u s h
form characterises the amylopectin molecules, having areas of both relatively
open and compact structures. Because there is a general lack of helical
structure, amylopectin unlike amylose does not form blue colour in
the presence of iodine. Amylopectin may have a molecular mass in excess of
10 ', making it the largest molecule in nature. In the raw state, starch granules
are round or irregular in shape and are between I and IOOpm long. The
granules are held together by internal hydrogen bonds so that they are able to
absorb very little water. The resultant crystalline structure is such that light is
refracted during its passage through the granule. The starch molecule exists
naturally as an entangled mass. In the granular form, the links act as if they
were magnetised being held together to form sphaerocrystals comprising
concentric layers of starchy materials deposited in a radial fashion, with a
central region known as helium (Hebeda, 1993). When heated in an aqueous
slurry, granules hydrate and swell resulting in a loss of crystallanity.
Depending on the type of starch, gelatinisation generally begins at between 50
and 68°C and is completed at between 64 and 7S°C. Regular corn starch, for
example, exhibits a gelatinisation temperature range of 62-70°C waxy maize
starch 63-72"C, potato starch 58-62T, tapioca starch 52-64°C and 70%
high amylose corn starch in excess of 100°C (BeMiller, 1992).
2.2 ENZYMIC CONVERSION OF STARCH
The primary enzymes used in the production of starch based sweeteners
are amylases and isomerases. The amylases such as a-amylase, 0-amylase,
glucoamylase and pullulanase have the ability to catalyse the hydrolysis of
a-1,4 and /or a-1,6 linkages in starch to produce lower molecular weight
saccharides.
During the production of starch based sweeteners, thermostable a-amylases
from bacterial sources are used at high temperatures to liquefy starch and
produce soluble dextrins. Glucoamylases from fungal sources catalyse the
saccharification process, converting the dextrins to dextrose. Other amylases
such as fungal a-amylase are often used in conjunction with glucoamylase to
increase the dextrose yield. Alpha and P-amylases from bacterial, fungal and
plant sources are used to saccharify dextrins to a wide range of syrups that
exhibit varied saccharide compositions. Xylose/glucose isomerase from a
number of different bacterial sources have been shown to isomerise dextrose
to fructose. There are a number of commercially available xylose/glucose
isomerase preparations. Some of these are based on heat fixed immobilised
cells while others are immobilised extracted enzymes.
In a typical process (Figure 2), the raw material (corn) is milled and the
resultant starch grains suspended to give a 30-35% (wlv) slurry. This material
is difficult to handle because of its high viscosity and suspended particulate
material. The next stage, liquefaction, is achieved using bacterial a-amylase.
The enzyme is mixed with the starch sluny and held at a temperature of 80-
150°C for a period of 2-3 hours at pH 6- 6.5. At this stage, calcium ions are
added to activate the a-amylase. The a-amylase catalyses the hydrolysis of
a-1,4 linked glucose units but is incapable of breaking branched chains and so
degradation is limited by the amount of branched chains. The resultant limit
dextrin material has to be broken down using a second enzyme, pullulanase.
Prior to the addition of glucoamylase, liquefied starch is cooled to 50-60°C
and the pH adjusted to 4 -5. The holding time for the material at this stage is
24 - 90 hours depending on the throughput and amount of enzyme added. At
the end of this, a product concentration of 90-96% dextrose is required for a
viable isomerisation stage.
Glucose is also produced from starch by means of acid hydrolysis.
2.3 CHEMICAL ISOMERISATION OF D-GLUCOSE
The chemical conversion of glucose to fructose at high temperatures and
alkaline conditions has been demonstrated. However, this approach has
turned out to be unattractive in view of the non specificity of the conversion
reaction which results in the formation of psicose, coloured substances and
formate (Figure 3). Moreover, it is difficult to attain a fructose concentration
of more than 40% by this method and the fructose so produced is fraught with
off flavours and reduced sweetness, which cannot be easily remedied (Barker
et al., 1973). As a result of these disadvantages, chemically obtained fructose
syrup has not been employed commercially.
Fructose was originally produced from invert sugar solutions using the
calcium fructonate method. This involved the mixing of calcium hydroxide
with an invert sugar, treatment with carbon dioxide, separation of CaC03,
:followed by vacuum evaporation and crystallisation (Crueger & Crueger,
1990). Since 1964, fructose has been produced on industrial scale using
cationic resins (Lauer, 1980).
Fructose has been produced biochemically from inulin, sucrose, or glucose
which comes from starch (Figure 4). Today, however, due to inulin shortages
and high prices, the inulin technique is no longer used (Kierstan, 1980).
Although the scientific discription of the enzymic isomerisation of glucose
to fructose is of a much earlier date, the first patent on "production of
fructose from glucose through the action of xylose/glucose isomerase" was
first published by Marshall in 1960 - U.S. Patent 2950288. Marshall & Kooi
(1957) discovered that in f'seudonlonas hydrophrla, isomerisation takes place
without phosphorylation.
In a cell with "normal" metabolism, carbohydrate isomerisation occurs
following a phospholylation step. A xylose/glucose isomerase technique for
the production of high fructose syrup (about 42% fructose) was first
developed in Japan and later in the U.S.A. Enzymatic isomerisation of
glucose to fructose was first established on industrial scale in 1967 by Clinton
Corn Processing Co. in the U.S.A., using in-house enzyme technology.
Around 1974, irnmobilised xylose/glucose isomerase became commercially
available. With the increasing acceptance of HFCS, especially in the soft
drink industry, the glucose isomerisation process was rapidly adopted by
practically all major starch processing companies in the western world
between 1975 and 1980. A substantial increase in HFCS consumption
occurred around 1978 with the introduction of fructose enrichment, a
chromatographic separation of fructose and glucose which makes possible the
production of HFCS with increased fructose content and sweetness. Today,
HFCS has almost completely replaced sucrose in the United States while the
enzyme, xyloselglucose Isomerase now commands the biggest market in the
food industry.
2.4 TYPES OF XYLOSEIGLUCOSE ISOMERASES
Four different types of enzymes are able to convert glucose to fructose
(Figure 5). The first type is a glucose-phosphate isomerase (D-glucose-6-
phosphate-ketol-isomerase, EC 5.3.1.9). Producers of this enzyme include
Escherichia intermedia, E. ,fretmdii, Aerohacter aerogenes and A. cloacae.
The enzymes need arsenate to form a glucose - arsenate complex which is
isomerised as follows :
Glucose + arsenate ++tt glucose-arsenate
Glucose -anenate + enzyme tt++ glucose-arsenate-enzyme
Glucose-arsenate-enzyme ++tt fructose + arsenate + euzyme .
Only for some of the enzymes is xylose needed as an inducer (Natake,
1968). The enzymes have pH optima at 7 and temperature optima at 50°C.
However, due to their requirement for arsenate they are not used in
commercial production processes.
A xylose/glucose isomerase (D-glucose ketol-isomerase, EC 5.3.1.18) has
been characterised which is linked to NAD' and produced by Bacillus
nzeguterium (Takasaki & Tanabe, 1963). Its pH optimum is 7.8 and the
temperature optimum is 35°C. A similar xylose/glucose isomerase activity,
which catalysed the iso~nerisation of both glucose and mannose to fructose ,
was isolated from t-'aracolobacteritrm aerogenoides (Takasaki & Tanabe,
1964 ). Various heterolactic bacteria (Lucrobacillus brevis, L. ,fkrnzenti, L .
pentoaceticu , L.mannitopolu,s, L. guyoni, L. huchneri) produce xylosel
glucose isomerases (Yarnanaka, 1968). These enzymes require D-xylose as
well as manganese ions as inducers and have been thoroughly studied for
their utility in the production of fructose. They however suffer the
disadvantage of relative instability at higher temperatures.
D-glucose
CHO I
H-C-OH I
HO-C-H
I + H-C-OH
I H-C-OH
I CH2 OH
.1
CH2 OH I
C=O I
HO-C-H
I + H-C-OH
I H-C-OH
I CH2 OH
D-fructose
H-C-OH I1 C-OH I
HO-C-H
I + H-C-013
I H-C-OH
I CHI OH
CHI OH I
C-OH II
HO-C
I + H-C-OH
I H-C-OH
I CH2 OH
D-mannose
CHO I
HO-C-H I
HO-C-H
I H-C-OH
I H-C-OH
I CH2 OH
CH2 OH I
C=O I
H-C-OH
I H-C-OH
I H-C-OH
I CH2 OH
D-psicose
Figure 3 : Chemical isomerisation of Glucose
Beets Cane \ 1
Sucrose 4,
biochemical reaction 4
invert syrup - 50150
Starch Artichokes Chicory 3- \ I
biochemical lnulin 3- -1
Glucose Chemical 3- -1
biochemical k 4, fructoselglucose
glucose/fructose - 80120 HFS-60140
Figure 4 : Production of fructose syrup
CHO CH2 OH I I
H-C-OH C=O I I
HO-C-H HO-C-H I I
H-C-OH ++tt H-C-OH I I
H-C-OH H-C-OH I I
CH2 OH CIlz OH
D-glucose D-fructose
Figure 5 : Reaction of glucose isomerase
The only commercially applied enzymes are D-xylose/glucose isomerases
(D-xylose ketol-isomerase, EC.5.3.1 S). Their advantages over the other
isomerases include :
i) low pH optimum (which discourages secondary reactions).
ii) high specific activity.
iii) high temperature optimum (which prevents contamination of reaction
mixture).
iv) non requirement for cofactors (ATP and NAD').
2.5 XYLOSE ISOMERASE PRODUCTION IN BACTERIA
Since discovering the ability of Pseua'omona~s hydrophila to produce an
enzyme that converts glucose to fructose in the absence of arsenate
(Marshall & Kooi, 1957), a large number of other true bacteria and
actinomycetes with similar capability has been reported (Table 1).
Among the heterofermentative lactic acid bacteria, Lac1ohnci1llu.s brevis
produced the highest yield of the enzyme. Reports on extracellular secretion
of xylose/glucose isomerase are not common. Extracellular xylose/glucose
isomerase production has been reported for Streptomyces glauce,scens
(Webcr, 1976) and S. ,finvogviscus (Chen el a/., 1979). This phenomenon
was attributable to a change in the cell wall pelmeability and partial lysis of
the cells. Other reported cases are in respect of Chainiu sp ( Srinivasan ef
al., 1983; Vartak, 1984) and an alkalothemophilic BaciNu.s sp (Chauthaiwale
& Rao, 1994).
Streptomyces spp have been the most extensively studied and it is therefore
not surprising that most commercial xylose/glucose isomerases come from
this group of bacteria . The first Streptomyces associated with xylose/glucose
isomerase production was Streptomyces phaeochron2ogene.r SKI. Since
then, over 26 other species have been reported. Streptomyccs olivaceus
NRRL B-3588 is used commercially by Miles Laboratoy Inc. while a mutant
of this organism, NRRL B-3916, is the source of the enzyme used by Miles-
Cargill Inc. in its productions. Apart from species of Streptomyces,
Actinomyces mi.ssouriensis is a potent producer of xylose/glucose isomerase
(Shieh, 1974), and is indeed, the source of the enzyme used by Anheuser-
Busch Inc. in its commercial operations. Other producers among the
actinomycetes have been found within the genera of Microellobospora,
Micromonospora and Norcadia.
Within the genus Bacillus, a commercial xylose/glucose isomerase has
been developed from Bacillus coagulans (Outtrup, 1974), while Bacillus
steareothermophilus (Suekane et a/., 1978) and Bacillus TX-3 (Kitada el a/.,
1989) have also been reported to produce thennostable enzymes.
From Lehmacher & Bisswanger (1990a) came a description of a highly
thennostable xylose/glucose isomerase from Thermus aquaticus HB 8. A
summary of various microbial sources of xylose/glucose isotnerases, their
respective trade names and manufacturers is given in Table 2.
The cost of enzyme production is an important factor in determining its
suitability for industrial application. Intensive efforts have been made to
Table 1 : Xylose/glucose isomerase producing organisms (Bhosale et al., 1996)
.4ctinomyce.s olivocinereus . A . phaenchromogenes Aclinoplanes missouriensis Aernhacfer aerngenes , A . cloacae , A . Ievanicuni drthrnhacter spp Bacillus .slearn!hermnphilu.s, B . megahocteriunr . H coagulans H~jkiohacterium incertunr , B . pento.soonrinoacidicunr ('hninia spp. (hrynehacferium spp. Cor!ohacterium helvolum Escherichinfieunrlii . E .inlernredia, E . coli Flavobacterium arhorescen.~ , F devornns LnclohnciNus hrevis , L, huchneri , L . firmenti. L .nrannitopoeu.s, L .gayonii , L ,,JErnrenti , L . plantarunr , L . lycoperci , L . pento,sns
Leuconostac me.wttemides :\dicrohi.sporu rosea Micr~~ellohosporin,f/al,en ~Mcromonospora coerula Mycohac!eriunr spp. ~Vorcadia asleroides , N . corallia , N , dmsonvillei Parocolohacterium aeragenoides I'seudonorcadia spp P,seudonronos hydrophila Sarcina .vpp. Staphylococcus hihilo , S.Javovirens , S. echirla1u.s ,Streptococcus achromogene.s ,S.phaeochromogenc.s ,S ,JFncliae , S roseochromogenes S.olivaccu.s, S.cal$)rnicos. Xvenuceus , Svirginial Slrepromyces olivochramagenes, .S.venezaelie , .S.ivedmorensis, Sgri.seoln.s. Sglaucescens ,S hikiniensis , S ruhiginosis . Sachinnlus ,S, cinnanronensis Spadiae , Salhus . S.griseus , Shivens, Smatensis , Snivens .Splatensis Strptospurnngiunr alhum , S.oulgare Theni~opolyspora spp. Thermus spp. Xanfhomonm spp. Zymononas mobilis
optin~ise the fermentation parameters for the production of xylosel glucose
isomerase with a view to developing an economically feasible technology.
Research is focused on h e e major aspects:
i) improvements of yields of glucose isomerase using microorganisms with
GRAS status,
ii) optimisation of the fermentation medium with special reference to
replacement of xylose by a cheaper substitute and elimination of the
requirement for cobalt ions and other toxic cofactors, and
iii) immob~lisation of the enzyme (Cruegcr & Ctueger, 1990; Bhosale
eta[., 1996).
2.6 STRAIN YIELD IMPROVEMENT
The yields of xylose/glucose isomerase from potent producer organisms
are listed in Table 3; they range from 1,000 to 35,000 U litre-'. A 60%
increase in enzyme level by mutagenising Streptomyces ~aedmorensi.~ with
ethyleneimine and N-methyl-N-nitro-N-nitosoguanidine was reported by
Bengston & L a m (1973). Equally, a mutant produced by ethyl
methanesulphonate gave a yield of 1,500U ml" when grown on only glucose
whereas the parent strain produced IOU ml-' under similar conditions (Hafner,
1985). UV irradiation of Streptomyces o1ivoc~hromogene.v resulted in a mutant
strain with 70% increased activity (Suekane & Iizuka, 1982). Lee (1976),
reported the development of constitutive mutants of Bacillm coagulans with
100% increase in activity when cultured on lactose as the carbon source. One
Table 2 : Commercial xylose/glucose isomerase producers (Bhosale et al., 1996)
Organism
Actinoplancs
missouriensis
Bacillus coagulanv
Streptotnyces
rubiginosrrs
Streptomyces
phaeochromogenes
Arthrobacter sp.
Streptomyces olivaceus
Trade name
Maxazyme
Sweetzyme
Optisweet
Spezyme
Sweetase
Manufacturer
Gist Brocades , Anheuser-Busch Inc.
Novo-Nordisk
Miles Kali-Chemie
Finnsugar
Nagase
Reynolds Tobacco
Miles laboratories Inc.
of these mutants produced enzyme with higher affinity for glucose than for
xylose. Also, Bok et al. (1984) reported the isolation of a number of
constitutive and high-yielding xylose/glucose isomerase mutants by applying
multiple UV irradiations to Streptoniyces acidodurans.
Further improvement in yield, and other vital properties of the enzyme
have been achieved by strain improvement, using either conventional or
recombinant DNA technology. More than 50% of industrial enzymes are now
produced from genetically engineered microorganisms (Hodgson, 1994). One
of the ways to increase the production of xylose/glucose isomerase is to
identify the xylose/glucose isomerase gene and clone it on a multicopy
vector containing a strong promoter such as lac, tac, or p ~ . Xylose/glucose
isomerase gene has been cloned from several microorganisms with the
primary aims o f : i) overproduction of the enzyme by gene dosage effect,
ii) direct conversion of xylose to ethanol by yeasts, and iii) engineering of the
protein to alter its properties to suit its biotechnological applications.
Molecular cloning and expression of xylose/glucose isomerase have been
carried out in both homologous and heterologous hosts as well as in yeasts
(Hodgson, 1994).
2.6.1 Cloning in homologous hosts
Homologous hosts offer sevcral advantages for cloning and expression of
exogenous DNA. One of them is the easy recognition of the expression
sibmals by the host RNA polymerase. There are few reports on the
homologous cloning of xylose/glucose isomerase from E,schemhia coli and
Table 3 : Production of xyloselglucose isomerase by various
organisms
Organism Yield ( Ullitre) Temp ('C) pH
mi.ssouriensi.s
Bacillus 10,500 70 NA"
wedmorensis
Streptomyces 4,800 - 11,440 60 7.5
olivochromopnes
"NA, not available
Streptornyces spp . --mHP w .y?jyy ."V
mbx* 2.6.1.1 Homologous cloning in E . coli
The first report on the isolation of the xyloselglucose isomerase gene was
from E.coli by Ho et al. (1983). D-xyloselglucose isomerase and
xylulokinase activities were amplified by transformation of a xylose/glucose
isomerase deficient E.coli strain with plasmid pMB9 bearing a Hind11
restriction fragment of E.coli chromosomal DNA (Wovcha et al., 1983). The
molecular cloning, sequencing, and expression of the xylose1glucose
isomerase gene in E.coli have also been reported by Briggs et al. (1984),
Lawlis et al. (1984) and Ueng et al. (1985). Xylose/glucose isomerase has
been over-produced in E.coli by several workers. Ho and Stevis (1985)
observed that hyperexpression of the gene was not accomplished by merely
cloning it on a high-copy-number plasmid, probably because the expression
of the gene in E. coli is highly regulated through its natural promoter. The
fusion of the structural gene with strong promoters such as lac or tac resulted
in 20-fold over-production of the enzyme. Ligation of a promoterless DNA
fragment containing the E.coli gene into a plasmid downstream of a strong p~
promoter followed by the transformation of an E. coli strain containing a
temperature-sensitive repressor resulted in over-production of xyloselglucose
isomerase (Lastick et al., 1986). Cloning of the xylA gene under control of the
tac promoter produced xylose/glucose isomerase, which accounted for 28% of
the total cell protein. Ecoli carrying the gene was encapsulated in calcium
alginate beads and used in the column for isomerisation of the substrate (Batt
et al., 1986). The properties of the genetically over-produced enzyme were
similar to those of the enzyme purified from the parent organism (Tucker
et a/., 1988).
2.6.1.2 Homologous cloning in Streptomyces species
Homologous cloning of xylose/glucose isomerase from Streptomyces
phaeochrornogenes in Streptonzyces liviu'nns via the S.stI site of pIJ702 with
thiostrepton resistance and insertional inactivation of melanin pigmentation as
markers led to a 50-fold increase in the xylose/glucose isomerase activity of
S. lividans, which was 2.5 times that of the wild type (Kho, 1984).
Another strategy to overexpress thc protein was by integrating the xylA
gene into the chromosome. The Streptomyces promoter (Pl) has been cloned
upstream of the xylA gene, leading to strong and constitutive expression. To
avoid plasmid instability of xylose/glucose isomerase expression, the Pi-xylA
gene has been integrated into the chromosome with the integl-ation vector
pTS55. Integration into the host chromosome resulted in the CBS 1
strain,with about sevenfold-higher xylose/glucose isomerase activity in the
absence of xylose as an inducer compared with the wild-type strain that was
fully induced by xylose (Bejar et al., 1994).
2.6.2 Cloning in heterologous hosts
Xylose/glucose isomerase genes from different organisms have been cloned
in E. coli. Although cloning of genes in homologous hosts is desirable for an
easy recognition of expression signals and efficient secretion of proteins,
E. coli still remains the most popular host of choice in view of the wealth of
information available about this organism. Moreover, several cloning vectors
have been constructed for use with E.coli as a host to meet various specific
requirements. Identification of genes in Ecoli allows their easy sequencing
and manipulating by site-directed mutagenesis to produce tailor-made
proteins.
2.6.2.1 Cloning from Bacillus subtilis to E . coli
A HumHI restriction DNA fragment coding for xylose/glucose isomerase
from Bacillus suh~ilis was isolated by complementation of an isomerase-
defective Ecoli strain. The expression of the gene was shown to be under
control of IS5, which is inserted 195bp upstream from putative ATG initiation
codon of the structural gene for xylose/glucose isomerase ( Wilheim &
Hollenberg, 1984). The ribosome-binding sequence and two hexamer
sequences typical of Bacillus promoter regions were located in the DNA
fragment. EcoRI fragments of chromosomal DNA from Buci1lu.s lichenfbrmis
were ligated to vector plasmid pBR322 and used to transfotm a GI-negative
mutant of E.coli (Shin & Kho, 1985).
The xylose/glucose isomerase gene from a thermophilic Bacillus sp. was
cloned and expressed in E.coli. The xylose/glucose isomerase produced by
the recombinant was active at 85°C and was partially purified to yield
49.02U per mg of protein, which represented the highest ever recorded
specific activity for xylose/glucose isomerase (Wuxiang & Jeyaseelan, 1993).
2.6.2.2 Heterologous cloning into other bacterial hosts
Bacillzrs is generally regarded as a safe microorganism. Therefore, it has
been found attractive to clone the xylose/glucose isomerase gene from Ecoli
into Bacillus species using a bifunctional plasmid. However, the expression
of the gene was initially not obse~ved. Fusion of the E.coli structural gene
downstream of the promoter of the penicillanase gene from BaciNus
lichenfi,rrnis eventually resulted in functional expression of the xylosel
glucose isomerase in Bacillus suhtilis (Huang & Ho, 1985). Xylose/glucose
isomerase gene from Clostridium thermosulfurogenes has been cloned in
HaciNus ,suhtilis using E. coli-Bacillus shuttle plasmid pMGI. The expression
of the xylose/glucose isomerase gene in R.,suhtili,s was constitutive and was
higher (1.54urng-' ) than that produced in CL thermosu~f~rogenes
( 0 . 2 9 ~ m g ' ) (Lee el al., 1990).
2.6.2.3 Heterologous cloning in Yeasts
A wide variety of microorganisms can utilise xylose, but none can fetlnent
it to ethanol. The main bottle neck lies in the conversion of xylose to
xylulose, which is usually an aerobic process, as in (h-ndida utilis (Bhosale
et al., 1996). The pentose-utilising yeasts like E'achysole tannophilus can
ferment xylose anaerobically, but the rate of fermentation is very low and is
accompanied by considerable amounts of side products.
Saccharomycev cevevisiae and Schizo,saccharomycc,s pornbe offer a high
fermentation rate, higher end-product yield, and increased ethanol tolerance.
Transfer of xylose/glucose isomerase genes to these yeasts holds some
promise for developing an organism which can ferment xylose directly to
ethanol. A 2.4-kb DNA fragment containing the xylose/glucose isomerase
gene from E.coli was isolated from the Clarke-Carbon gene bank and
introduced into S. pombe via a shuttle plasmid. The recombinant plasmid
showed complementation with xylose/glucose isomerase-deficient E. toll and
expression of the xyloselglucose isomerase gene in the yeast (Chan ei al.,
1989). The transformed S. pomhe was able to ferment 10% (wlv) xylose to
produce 3% (wlv) ethanol. Investigation of the metabolism of D-xylose in the
transformed yeast showed that xylitol, which is a by-product of xylose
fermentation in yeasts, had no effect on the activity of xylose/glucose
isomerase. The observed low activity of xyloselglucose isomerase in the
yeast was due to its proteolytic degradation by the yeast protease and remains
the limiting step in xylose fermentation by yeast.
2.6.2.4 Heterologous cloning in Plants
The xylose/glucose isomerase gene from E.coli has been cloned on a
plasmid pBR322 derivative downstream of the nopaline synthetase gene (nos)
promoter of Agrohacterium [unwfacien.~ plasmid pTiC58. This construct was
transformed into tobacco leaf discs. The transformants expressed
xyloselglucose isomerase in transgenic tobacco, thus indicating that the
mRNA was successfully translated by plant system (Piruzyan eta/., 1989).
Cloning of the xyloselglucose isomerase gene from E.coli in potato (S'olanum
tuherosum) and in tomato (Lycopersicunr esculentum) has been achieved and
the presence of the xyl gene has been confirmed by the expression of
xylose/glucose isomerase activity (Krashinnikova el a/., 1991; Norova et a/.,
1991).
2.7 OPTIMISATION OF FERMENTATION MEDIUM
Xylose/glucose isomerase is generally produced by submerged
fermentation under aerobic conditions. Optimisation of the fennentation
medium has been extensively studied with a view to developing an
economically viable process for the production of xylose/glucose isomerase.
Research efforts have been directed mainly to:
i) replacement of xylose by another inexpensive inducer; ii) search for
cheaper nitrogen sources; iii) optimisation of pH and temperature for
maximum enzyme production; and iv) substitution of cobalt ions by other
divalent metal ions in the fennentation medium.
2.7.1 Inducer
Most of the xylose/glucose isomerase producing organisms have an
obligate requirement for D-xylose to induce production of the enzyme.
However, xylose being very expensive is impractical for use on a commercial
scale. According to Drazic and his coworkers (1980), starch, glucose, sorbitol
or glycerol could be used at 75% level of substitution in place of xylose.
Takasaki &Tanabe (1966) showed that Streptomyces strain YT-5 was able to
grow on xylan or xylan-containing material such as corn cobs or wheat bran.
This was the landmark in selecting strains capable of growth in cheaper
media. Recently, Inyang et a1.(1995), showed that the thermophilic
Streplomyces sp. (strain PLC) was able to gl-ow on xylan containing materials.
Today several strains are capable of producing xyloseiglucose isomerase with
glucose as the inducer. These include strains of Actinoplanes, mutant strains
of Bacillus coagulans and Slreptomyces olhochromogene,~ (Chen, 1980).
Another approach to the elimination of the requirement of xylose as an
inducer is the generation of mutants able to produce xylose/glucose
isomerase constitutively. One of the wild-type strains of Actinop1ane.s
missouriensis produces xyloselglucose isomerase constitutively and is used
for the comtnercial production of the enzyme by Gist Brocades (Anhauser-
Busch Inc., 1974). Constitutive enzyme production has been shown to be
possible through the cloning of the xylA gene in front of a strong
Rreptomyces promoter. The Pl-xylA gene has beeu integrated into the
chromosome with the aid of the integrative vector pTS55. The resultant
strain (CBSI) gave about sevenfold greater activity in the absence of xylose
compared with the wild-type strain fully induced by xylose (Bejar et al.,
1994).
2.7.2 Nitrogen source
The nitrogen source is a critical factor which needs to be optimised for each
organism. Although complex nitrogen sources are usually used for xylosel
glucose isomerase production, the requirement for a specific nitrogen source
differs from organism to organism. Peptone, yeast extract, or inorganic
ammonium salts can be used by Bacillus coagulans, but urea and nitrate are
unsuitable (Yoshimura et al., 1966). Corn steep liquor was found to be a
cheap and suitable source of nitrogen by some workers (Anhauser-Busch Inc.,
1974; Bucke, 1981; Hafner & Jackson, 1985), but its use is limited by its
seasonal and interbatch variability. Suitable nitrogen substitutes for corn
steep liquor are still being evaluated. Soy flour has been shown to give a
50% higher yield than corn steep liquor (Shieh, 1977) while the addition of
certain amino acids improves the enzyme yield in Streptomyces
wolaceorzrher (Vandamme et al., 198 I ) .
2.7.3 pH and temperature optima
The nature of nitrogen somce affects the pH and consequently the yield of
the enzyme. Most xylose/glucose isomerase fermentations are catried out at
between pH 7.0 and 8.0 without pH control. Fermentations driven by
Streptomyces spp., Arthrohactcr sp. and Actiwoplanes missouriensis are run
at around 30°C (Anhauser-Busch lnc., 1974) but those involving thermophilic
Bacillus spp. run at 50 to 60°C (Diers, 1976; Brownewell, 1982). The period
of fermentation varies from 6 to 48h depending on the culture used.
2.7.4 Metal ion requirement
Divaient cations are required in the fermentation medium for optimum
production of xylose/glucose isomerase. However, the requirement for
specific metal ions depends on the producer organism. Cobalt ions are
essential for xylose/glucose isomerase production by Streptomyces strain
YT-5 (Takasaki & Tanabe, 1966), whereas Hacrllus coagdans requires MnZ'
or Mg2' (Outtlup, 1974; Yoshimura et a/., 1966). Generally, mesophilic
Slrepromyces spp, have a requirement for co2' unlike the thermophilic
species (Bhosale el al. , 1996). From the point of view of public health, CO*'
is undersirable in fermentation media for the production of HFCS. Some
organisms such as Arlhrohacter spp. and S/reptomyces o1n~aceu.s (Reynolds,
1973) as well as some mutants of Streptomyces ol~vochron~ogene,s
(Anhauser-Busch Inc., 1974), have no requirement for coZt for optimal
production.
2.8 IMMOBILISATION OF XYLOSEICLUCOSE ISOMERASE
One of the approaches to cost reduction during HFCS production is the
use of immobilised enzyme. This makes possible the recovery and reuse of
the immobilised xyloselglucose isomerase. The largest market for xylosel
glucose isomerase is for its immobilised fo~m. Development of immobilised
xyloselglucose isomerase has been a subject of great interest (Hemmingsen,
1979; Verhoff el a1.,1985; Pedersen, 1993). Xylosel glucose isomerase is an
expensive inhacellular enzyme which must first be extracted from the cell
before use, and large quantities are needed to compensate for the high Km for
glucose.
Several methods for immobilising xyloselglucose isomerase have been
described (Antrim et al., 1979). However, only a few are economical and
yield enzyme preparations with properties that are suitable for commercial
production of HFCS. Table 4 gives a list of commercially used immobilised
preparations of xyloselglucose isomerase.
Two main methods are used for the immobilisation of xylose/glucose
isomerase: cell-free enzyme immobilisation and whole-cell immobilisation
2.8.1 Cell-free immobilisation
Soluble enzymes that are immobilised to a support structure have excellent
flow characteristics suitable for continuous operations, in contrast to whole
cell immobilised supports, and offer considerable savings in terms of capital
equipment. Xylose/glucose isomerases from Streptomyces phaeochromogenes
and Lactobacillus hrevis were immobilised on DEAE-cellulose (Bucke,
198 1). The Streptomyces enzyme immobilised on DEAE-cellulose is being
used to produce HFCS in a semicontinuous plant by the Clinton Corn
Processing Company. A xylose/glucose isomerase preparation from
Streptomyces sp. immobilised on porous alumina exhibited a half-life of 49
days and was found to be suitable for continuous use in plug-flow reactors.
The use of enzyme immobilised on controlled-pore alumina in the presence
of cobalt ions had the advantage that the cobalt ions could be eliminated from
subsequent operations. Monsato lnc. coimmobilised xylose/glucose
isomerase on large-pore polyethylene discs by permeating the discs with a
solution of polyacrylonitrile in dimethyl sulphoxide and finally fixing it with
glutaraldehyde. An elegant procedure involving entrapment of Streptoniyces
xylose/glucose isomerase in a filament of cellulose acetate was described and
a similar strategy was used to immobilise xylose/glucose isomerase and
amyloglucosidase together (Bucke, 198 1).
2.8.2 Whole-cell immobilisation
Because xylose/glucose isomerase is an intracellular enzyme, whole-cell
immobilisation is the method of choice for most of the commercially available
immobilised xyloselglucose isomerases. Whole cells containing xylosel
glucose isomerase were spray-dried and used in the first industrial process to
produce HFCS by Clinton Corn Processing Co. Addition of inorganic salts
such as magnesium hydroxide to the fermentation broths of Streptomyces or
Arthrohacter species followed by filtration and drying of the cake provided a
straight forward method to immobilise cells containing glucose isomerase
(Reynolds, 1973). Physical entrapment of whole cells in polymeric materials
was used as an immobilisation method by Novo Industries, whereas chemical
entrapment of cells in a membrane followed by cross-linking with
glutaraldehyde was used on a commercial scale (Miles Laboratories Inc.,
1972). Xylosel glucose isomerase from SI~.eptomyce,s sp. NCIM 2730
has been immobilised on Indion 48-R, leading to an improvement in its pH
and temperature stability (Feldman et al., 1992).
The details of the present technology used by various manufacturers in the
production of HFCS are documented in the form of patents (Armb$ster et
al., 1973; Barker et a[ . , 1973; Bengston & L a m , 1973; Reynolds, h973;
Barker, 1976). In a broader sense, modem technology uses immob"1ised
xyloselglucose isomerase preparation in a continuous system at hig er
feed syrup.
1 temperature (65°C) and higher pH without the requirement of c o Z , in the
I
2.9 PURIFICATION OF XYLOSEIGLUCOSE ISOMERASE
A number of reports regarding the purification of xylose/glucos
from various microorganisms are available. However, a few of th'
the purification of xylose/glucose isomerase to homogeneous stat
commercial use of xylose/glucose isomerase involves the immobil
the enzyme, which is cheap and effective and does not require thc
purification and concentration of the enzyme (Bhosale ef al., 199f
purification of xylose/glucose isomerase is important for academic
considerations involving basic studies on chemical modification, r
function relationships and properties, et cetera.
Xylose/glucose isomerase is generally an intracellular enzyme
few cases when the enzyme production is extracellular (Chauthai~
1994). The enzyme is extracted from the microbial cells by mecha
disruption (such as sonication, grinding or homogenisation) or by
cells with lysosyme, cationic detergents, toluene, et cetera (Chen,
Purification of xylose/gllucose isomerase from microbial sources 1:
purification methods, such as heat treatment, precipitation by alml
sulphate-acetone-~g~' or ~ n ' + salts, ion exchange chromatograpl
gel filtration, has been reported (Chen, 1980).
,omerase
describe
The
d form of
The
cture-
:ept in a
: & Rao,
al
is of the
80).
lassical
ium
and /or
Table 4 : Immobilised xylose/glucose isomerase of commercial importance (Bhosale et a / . , 1996)
Source organisnl(s)
Cell-free enzyme
S. oliiwchromopnes
5. rrrhiginosus
Whole cells
Actinoplanes missouriensis
Flavohac/erium arhore.scen.s
.S.murinus and Bacillus coagulans
Trade name
G-zyme G-994
Spezyme
Optisweet 11
Ketomax 100
Maxazyme
Takasweet
Sweetase
Sweetzyme T
Manufacturer
CPC (enzyme biosystems) Genencor International
Solvay
UOP
IBIS
Solvay
Godo-Shusei
Nagase
Novo-Nordisk
Immobilisation method
Adsorption on an aniun- exchange resin DEAE-cellulose agglonierated with polystyrene and Ti02 Adsorption of specific Si02 particles followed by cross linking with glutaraldehyde . Polyethyleneimine-treated alumina with glutaralde- hyde crosslinked glucose isomerase .
Cells occluded in gelatin followed by glutaraldehyde
Polyamine glutaraldehyde cross linked cells extruded and granulated
Chitosan -treated glutaraldehyde cross linked cells Heat treated cells bound to anion exchange resin Glutaraldehyde cross- linked cells extruded .
2.10 PROPERTIES OF XYLOSEIGLUCOSE ISOMERASE
The enzymatic and physicochemical properties of xylose/glucose isomerase
from several organisms have been extensively studied. The knowledge of
specific properties of the enzyme, such as its stability, substrate specificity,
and metal ion requirement, is important to prevent its inactivation and to
assess its suitability for application in HFCS production (Bhosale et al., 1996).
2.10.1 Substrate specificity
The ability of the enzyme to isomerise a wide variety of substrates such as
pentoses, hexoses, sugar alcohols and sugar phosphates has been reported.
Some of these reports have shown that no one enzyme can isomerise all the
listed substrates. Substrate specificity is therefore strain dependent. In
addition to the traditional substrates - xylose and glucose, such other
substrates as D-ribose, L-arabinose, L-rhamnose, D-allose and 2-deoxy
glucose are known to be subject to isomerisation by xylose/glucose isomerase.
Maximum isomerisation was obtained with the substrates having hydroxyl
groups at carbons 3 and 4 in the equatorial position, as in glucose and xylose.
The conversion ratios of D-glucose to D-fructose catalysed by xylose/glucose
isomerase from various organisms in soluble or immobilised form range
from 26 to 56 % while the Km values for D-glucose and D-xylose as
substrates range from 0.086 to 0.920 M and 0.005 to 0.093M, respectively
(Chen, 1980).
2.10.2 Metal ion requirement and inhibitors
Xylose/glucose isomerase requires a divalent cation such as M ~ ~ ' , co2',
or Mn2+ or a combination of these cations, for maximum activity. Although
both both ~ g ~ ' and co2' are essential for activity, they play different roles.
While Mg2' is superior to co2' as an activator, co2' is responsible for the
stabilisation of the enzyme by holding the ordered conformation, especially
the quatenlay structure of the enzytne (Callens el al., 1986; Callens et al.,
1988; Gaikwad et al., 1992). Kasulni el al. (1982) have reported the presence
of four CO*' ions per tetramer of glucose isomerase from Streptomyces
gr~se~fuscus. The catalytic activity of glucose isomerase was inhibited by
metals such as A ~ ~ ' , ~ g ~ + , cu2+, zn2', and ~ i ~ ' and to some extent by ca2+.
Other known inhibitors of glucose isomerase are xylitol, arabitol, sorbitol,
mannitol, lyxose and Tris (Bucke, 1983; Smith et al., 1991).
2.10.3 Subunit structure
The sedimentation constants and molecular weights of xylose/glucose
isomerase vary from 7.55 to 11.45 and from 52,000 to 191,000, respectively
(Chen, 1980). The subunit structure and amino acid composition of
xylose/glucose isomerase reveal that it is a tehamer, himer or dimer of
similar or identical subunits associated with noncovalent bonds and is devoid
of interchain disulfide bonds. The extracellul~ xylosel glucose isomerase
from Bacrllus sp. is a tetramer (Chauthawaile & Rao, 1994).
Basuki et a1 (1992) have reported the existence of isoetizymes of
xyloselglucose isomerase from S'trep~omyces phac.ochromo:~.nes. The
isoenzymes differ in their N-terminal amino acids and in the ~ept ide patterns
of trypsin and cyanogen bromide generated digests.
The effects of denaturants such as urea, guanidine hydrochloride, sodium
dodecyl sulphate and heat on the activity of xylose/glucose isomerase from
Arthrobacter and Streptomyces spp. were reported by Gaikwad et a/. (1992)
and Rangarajan e/ al. (1992). These reports revealed that the denaturants led
to the dissociation and unfolding of the tetrameric xylose/glucose isomerase
from Sfreptomyces sp strain NClM 2730. Furthermore, it was revealed that
the tetrarner and dimer are the active species whereas the monomer is
inactive. Intact tertiaty rather than secondary structure was shown to be
responsible for the biological activity of xylose/glucose isomerase (Ghatge
et al., 1994).
2.10.4 Optimum temperature and pH
The optimum temperature of xylose/glucose isomerase for activity ranges
from 60 to 80°C and increases in the presence of co2'. The optimum pH
range of xyloselglucose isomerase is generally between pH 7.0 and 9.0. The
enzyme from Lactohacillus hrevis has a lower pH optimum (between 6 & 7),
which is desirable for commercial applications of xylose/glucose isomerase.
The enzyme from Streplomyces spp., Bacillus spp., Actinoplanes
missouriensis, and Thermus spp. is stable at high temperatures, but that from
Lacfobacillus and E,scherichia spp is less stable.
2.10.5 Active-site studies
The identities of amino acids involved at or near the active site of xylosei
glucose isomerase have been deciphered with group-specific chemical
modifiers and by X-ray crystallography. There is strong evidence for
essential histidine and carboxylate residues at the active site of xylosel
glucose isomerase (Callens el al., 1988; Gaikwad et al.. 1988; Ghatge &
Deshpande, 1993). Although it has long been recognised that xylose/glucose
isomerase catalyses the isomerisation of both glucose and xylose, it was
however not immediately clear whether the reactions occur at onc site or at
two different sites. The presence of a single active site for the isomerisation
of both glucose and xylose was demonstrated by Gaikwad et al (1989) and
Deshmukh & Shankar (1996) using the kinetic method elaborated by Keleti
et aL(l987).
2.11 MECHANISM OF ACTION OF XYLOSE ISOMERASE
Despite its commercial importance, vety little information is available
about the structural and mechanistic properties of xylose/glucose isomerase.
The catalytic mechanism of glucose isomerase has been a subject of great
interest to researchers. Earlier, xylose/glucose isomerase was assumed to
function in a manner similar to sugar phosphate isomerases and to follow the
ene-diol mechanism (Rose et al., 1969) (Figure 6). Recent studies have
attributed the action of xylose/glucose isomerase to a hydride shift ruechanism
(Collyer et al., 1990; Nargorski & Richard, 1996) (Figure 6).
Different approaches have been used to study the active site of glucose
isomerase and to delineate its mechanism of action. These include :
i) chemical modification, ii) X-ray c~ystallogaphy, and iii) isotope
exchange. The features of the mechanism proposed for xyloselglucose
isomerase are ring opening of the substrate, isomerisation via a hydride shift
from C- 2 to C- 1, and the ring closure of the product (Bhosale et a/., 1996).
Nagorski & Richard (1996) reported that the aldose-ketose isomerisation of
D, L-glyceraldehyde to give dihydroxyacetone in dilute alkaline solution by
proton and hydride transfer proceeds at similar rates and that there is no
strong mechanistic imperative for enzyme catalysis of this isomerisation by
either reaction mechanism.
2.1 1 . Chemical modification of xyloselglucose isomerase
Chemical modification of amino acid residues with specific chemical
reagents serves as a simple means of probing the active site of the enzyme.
The possible involvement of histidine in the active site of xyloselglucose
isomerase was postulated by studying the effect of dlethylpyrocarbonate on
the inactivation of xylose/glucose isomerase (Kume &Takahisa, 1983). Later,
evidence for the presence of an essential histidine residue at the active site of
xyloselglucose isomerase from different Lactobacillus spp. and ,Streptomyces
spp. (Gaikwad et al., 1988; Vangrysperre el al., 1988) was provided.
Inhibition by diethylpyrocarbonate was remedied by hydroxylamine. Total
protection of enzyme activity was afforded by the substrate and substrate
analogue xylitol during chemical modification. Histidine is known to function
as a proton-abstracting base and to assist in hydrogen transfers (Figure 6).
The presence of an aspartate or glutamate residue in xylose/glucose isomerase
was documented by its inactivation by Woodward's reagent K or guanidine
hydrochloride (Vangryspelre el a/ , 1989; Ghatge & Deshpande, 1993).
Involvement of carboxylate residues is implicated in the binding of metal ion
cofactors (Callens et at, 1988). Chemical modification of protected
andunprotected xylose/glucose isomerase and subsequent peptide mapping
allowed the identification of an active-site region with a consensus sequence
consisting of Phe-His-Xaa-Asp-Xaa-Xaa-Pro-Xaa-Gly (Vanglysperre el al,
1990). The results of studies on the chemical modification of xylose/glucose
isomerase complement the conclusions drawn on the basis of X-ray
crystallographic studies.
2.11.2 X-ray Crystallography
X-ray crystallography gives a detailed picture of the three-dimensional
structure of the protein and allows actual visualisation of the enzyme-
substrate or enzyme-inhibitor complexes. Xylose/glucose isomerase from
different bacterial species such as Aclinomyces, Arthrohacter,
Actinoplaner, and Racill~rs species has been been studied by X-ray
crystallography at different levels of resolution, in the presence and absence
of inhibitors and metal ions, to understand and explain the mechanism of
action. Since xylose/glucose isomerase is a single-substrate-single-product
enzyme, it is possible to obsewe the Michaelis complex directly at a substrate
concentration higher than its Km. The structures of xylose/glucose isomerase
from several Streptomyces spp. are accurately kno'pn. They are all very
similar, especially at the active site. The structure bf xylose/glucose
isomerase from Streptomyces rubiginosus as dete med at 4A0 (lAO=O. lnm) .e. resolution (Carell et al, 1984) has shown that the ebzyme consists of eight
P-strand-a-helix [(df3)8] units as found in triose-phosphate isomerase. The
smaller domain forms a loop away from the larger domain but overlaps the
larger domain of another subunit, so that a tightly bbund dimer is formed. The
tetramer is thus considered to be a dimer of active dimen ( B h o d e et al,,
1996). Resolution of the crystal structure from Striptomyces
olivochromogcnes at 3A0 showed that the xylose/@~cose isomerase barrel is
30A" long and 40A0 in diameter (Farber et al., 1989). The n/p barrel fold is
stable and is useft11 as scaffolding for the constructii)~ of an active site.
Characterisation of crystals of xylose/glucose i s o m e i ~ e from Slreptovtyces
violaceoniger at 2.2A0 resolution revealed a variatiob in the quaternary
structure from that of Streptomyce,~ olivochro~nogcbe~s xylose/glucose
isomerase in solution (Glasfeld et al.. 1988). The sttjucture of c~ystalline
xylose/glucose isomerase from Streptomyce.~ ruhiginpsus has been dete~mined
in the presence of substrate and an active site-directeb inhibitor at 1 .9A0
resolution. These studies have led to the identificaticin of the active-site
region and two metal-binding sites. One of the metal! ions binds to C-3-0
and C-5-0 of the substrate, while there is a close cohtact between histidine
and C-l of the substrate. The results indicate that the hechanism involves an
open-chain conformation of substrate and probably a fo~tnation of a cis-
aldose cationic form ketose
Figure 6 : Mechanism of action of glucose isomerase a) cis-enediol
I b) proton shift c) hydride shift. Boxes indicate the hydrogen
1 ,, atoms that are transferred stereospecifically (Bhosale et a1 , 1996).
enediol intennidiate. Recent studies on X-ray crys allographic structures of
the metal activated xylose/glucose isomerase from Y.olivochromogenes show t that the isomerisation is catalysed by two metal colactors and their bridging
through a glutamate residue to promote a hydride shift. Of the two essential
magnesium ions per active site, M? was observe4 to occupy two alternate
positions separated by 1.8A0 (Carell el al., 1989). $he obsreved movement of
the metal ions in the presence of substrate was attibbted to a step following
substrate binding but prior to isomerisation (Lavie q / al., 1994). The substrate,
in their linear extended forms, were observed to intdract with the enzyme and
the metal cofactor. Carell et a1.(1994) have shown (hat the xylose/glucose
isomerase from S. ruhiginoszrs can bind substrates and inhibitors in a variety
of binding modes depending on the size of the suga~t. D-Threonohydroxamic
acid resembles the putative transition state in the isoberisation step of xylose
by xylose/glucose isomerase and is a potent inhibitoi. of the enzyme. Studies
on the high resolution X-ray crystallographic structuke of a complex between
the xylose/glucose isomerase from S. 01ivochromogme.s and D-threono
hydroxamic acid provides evidence for the metal mo'pement during catalysis
on deprotonation, which is followed by the fonnatidn of a bridging ligand
(Allen et a/., 1995). These results confirm the earlier; observations that
protonation of the hydroxyl group occurs after ring opening (Allen st al,
1994).
The crystal structure of xylose/glucose isomerase from Arthrohacter strain
B3728 containing the inhibitors xylitol and D-sorbitol has been studied at
2.5 and 2.3A0 resolution, re' spectively (Hemick, 1989). The molecule is a
tetramer, and the assymetric unit of the c~ystal contains a dimer. Each subunit
contains two domains. he main domain is a parallel-stranded alp barrel.
The C-terminal domain is a loop structure consisting of five helical segments
and is involved in intermol+cular contacts between subunits. The requirement
for two metal ions per monpmer has also been substantiated by spectroscopic
analysis and by electron paramagnetic resonance (EPR) studies (Sudfelt er al.,
1990; Begumil el a/., 1993). The metal ion is complexed at the high affinity
site by four carboxylate sidq chains of the conserved residues. The inhibitors
are bound to the active site in their extended open-chain conformation and
cotnplete an octahedral cooidination shell for the magnesium cation via their
oxygen atoms 0 -2 and 0-4. i ~ h e active site lies in a deep pocket near the
C-terminal ends of the p-sMands of the barrel domain and includes residues
from a second subunit. Several internal salt linkages that stabilise tertia~y and
quaternary structure of the enzyme were detected. Collyer el al.(1990) and
other investigators (Blow & Collyer, 1990; Blow el al., 1992) have shown
further that binding at a second cation site (site 2) is also necessary for
catalysis. The site binds co2' more strongly than site 1 does, and it is
octahedrally coordinated to three carboxylate groups, an imidazole and a
solvent molecule. During the hydride shift, the '2-0-1 and C-0-2 bonds of
the substrate are polarised by the close approach of the site 2 cation. After
isomerisation, ring closure is catalysed as the the reverse of the ring-opening
step. The anomerism and sterkospecificity of the enzyme are shown to be
fully consistent with the propbsed hydride shift mechanism (Collyer & Blow,
1990). Crystallisation and bharacterisation of isomerase from
Hacillus coagulans (~asmdssen, 1994 ) and
(Jenkins et al. , 1992) are also reported.
isomerase was prompted by $he absence of solvent e change during i
I
investigations on the incorporation of tritiated water nto the product (Rose et
al., 1969). However, the possibility of a fast proton ansfer in a shielded
activity could not be suled oqt. Allen el al . (1994) h ve carried out isotope l1 exchange experiments at higier temperature, extreme pHs, and in the I
2.1 1.3 Isotope exchange
The available crystallogtaphic data for xylose/gl
a proton transfer mechanism and suggest a hydride
structural data alone are lnspfficient to conclude the
presence of guanidine hydrodhloride to investigate th possibihty of shielded i
cose Isomerase rule out
chift mechanism. However
mecllanisrn of action of
proton transfer. Their nuclear magnetic resonance stu ies, coupled with the 1 studies on fluorine-substitute4 substrate analogues, d not support a proton 9 transfer mechanism for xylos&/glucose isomerase. 1
an enzyme. Uncertainty about a proton transfer mec anlsm in xylose/glucose t ' '
I Recent studies of the wild-type and mutant D-xylos /glucose isomerases I" from Act~noplanes mrssourremr.c support the role of the water
molecule, Trp-690, Asp-255, And the adjacent in proton transfer fiom
2-OH to 0-1 of the open and extended aldose (Van Bastelaere
et al., 1995).
2.12 GENETIC REGULATION OF XYLOSE/(;LUCOSE ISOMERASE
BIOSYNTHESIS
D-xylose, though not ascommon a sugar as gluc'~se, is a major component
of plant hemicelluloses. The microorganisms that {urvive on decaying plant
materials have evolved effikient biochemical pathwbys to assimilate
D-xylose. D-xylose as an herby source is utilised)by bacteria through a
pathway involving transport across the cytoplasmic imembrane and
isomerisation to D-xylulose. The pentulose residue is phosphotylated by
xylulokinase to yield D-xylblose-Sphosphate, which is further metabolised
through the pentose phosphate and Embden-Meyerhoff pathways. A xylose-
H' proton symporter and a binding protein-dependent system are responsible
for the transport of xylose idto E .colr K-12 (Tiraby et al., 1989).
Investigations on the organidation of genes involved in the xylose metabolism
pathway are useful in underitanding the molecular mechanism of gene
regulation. Considerable information on the biochemical and genetic aspects
of xylose utilisaton in variou(s microorganisms has emerged in the recent past.
2.12.1 Genetic qanisatiod of xyl genes
Genetic studies on Salmo lyphinwium provided evidence for the
existence of four clustered (xyl operon) that are responsible for
xylose catabolism; these a gene specifying the transport of xylose
across the cell membrane; xy{ A, the glucose/xylose isomerase gene; xyl B,
the xylulokinase gene; and x41 K, a regulatory element essential for
transcription of xyl genes (Sh b ana & Sanderson, 1979). The transduction
2.12.2 Divergent promoter
Studies on the xylA gene ~volaceonrger have indicated that
xylA and xylB promote directions (Tiraby er ai.,
1989). The existence Streptonzyces spp and other
procayotes was Sequence analysis has
indicated the encodes a regulatory
analysis of S . typhimuriurjl genes indicated the order to be xylT-xyR-xylB-
! xyl-A. Studies on E . coli enorne revealed an analogous genetic organisation b and similar xylose ntilisati n pathway (Maleszka et a/ . , 1982). These results
strongly support a repress0 -operator mechanism for the regulation of xylAB
expression and postulate a model for coordinate (positive) control of the
xylA, xyM, xylT genes by t ~ e xylR gene product (Rosenfeld e ta / . , 1984). In
the absence of xylose the
activator in the presence of
araC gene product of arabinose
Mutants of Streptomyew
xylose/glucose isomerase
et a/., 1982). Chromosomal
three different classes of xy
x:dR product acts as a repressor, while it acts as an
xylose , which is analogous to the action of the
regulon (Ogden el a/., 1980).
violaceoniger that are deficient in either
&or xylulose kinase were isolated (Maleszka
fragments with the ability to complement all
-negative mutants were cloned on a plasmid.
Localisation of the genes in icated that the putative xylulose kinasc gene 1 resides near the xylose/gluc$se isomerase gene , which is consistent with the
d organisation of the locus in . almonella typhirntwium , E .coli and Bacillus 1 .ruhrilis (Bhosale et al., 1996).
protein. It is suggested tha a regulatory molecnle may act within the I divergent transcription uni/ to control the expression of opposite genes and
also regulates its own syn+esis (Bhosale rt 01,. 1996).
2.12.3 Catabolite repress on
The expression of the I operons in Salmonella wphimurium and E . coli
seems to be regulated by a ositive control mechanism (Sharnana &
Sanderson, 1979) and by c tabolite repression exerted by glucose (David &
Weissmeyer, 1970). In E 1 . ,011, catabolite repression is mediated via
transcriptional activation b gene activator protein and cyclic AMP (CAMP).
In conclusion, the ation of xy[A and xylB seems to be greatly
conserved in all bacteria. T ese two genes are always adjacent to each other,
but on closer inspection rev als marked differences in their organisation. The 1 analysis of xyl genes fiom v iety of organisms will help to form a consensus k opinion about genetic organ$ation and regulation of xyl genes.
2.13 GENETIC IMPROSEMENT OF XYLOSEIGLUCOSE
BY SITE DIRECTE b MUTAGENESIS
Advances in recombinant NA technology have led to the successful
isolation of the genes of a1 ost any protein. Protein engineering by
manipulation of genes is at p esent a viable approach which complements
shucture-function studies pe \ ormed by already existing methods and allows
production of tailol-made with desirable properties to give a
complete insight into the of the enzyme. Site-directed mutagenesis
knowledge about the mech ism of action of the enzyme and has produced an
enzyme with improved are as follows :
(SDM) of xyloselglucose isomerase has been canied out with several
objectives, such as i) inc 1 easing the thermal stability, ii) lowering of the pH I optimum , iii) changing of the substrate preference, iv) deducing the
2.13.1 Thermal stabilisatidjn
Most of the commercial p .eparations of xylose/glucose isomerase have a
temperature optimum of GO t 6S°C. The activity of xylose/glucose isomerase
declines as a result of its the al inactivation. This confers a limitation on
the operating time of the rea 1 tor. Several mechanisms are known to be I
involved in the irreversible i activation of xyloselglucose isomerase, such as 't irreversible unfolding, glycat'on, and/or deamidation of Asn or Gln (Volkin &
Hibanov, 1989). Under pract'cal conditions, xylosel glucose isomerase is
exposed to high sugar concen ations (3M), which may lead to non- 1 enzymatic glycation of and subsequent inactivation of glucose
isomerase. Elegantly engineering experiments on
xyloselglucose missouriensi.~ have shown that a
functional role of essential amino acid residues, and v) studying the subunit
interactions. These studies ave contributed substantially to our knowledge
about the ~nolecular mecha ism of xylose/glucose isomerase and have
created new possibilities o producing an enzyme with properties that are
better suited for biotechnol gical applications. i A few examples of how site directed mutagenesis has helped to increase our
xylose/glucose isomerase utant containing a substitution of arginine for "r lysine at position 253 at th dimel--dimer interface increases the half-life of
the enzyme by 30% (Quax s f al., 1991). The largest stability gain was
achieved in a triple mutant (G70SlA73S IG74T) of the enzyme, for both
soluble and immobilised p parations. The hymophobic interaction among
the aromatic amino acid re idues present in the active site of xyloselglucose I I isomerase is postulated to be one of the important factors that help to maintain
the association of monome I p into active dimers. An increase in thestno-
stability may therefore be aihieved by strengthening the interactions at the
interface of the active dime s. Enhancement of the thermostability of
xylose/glucose isomerase fr m Thermonnasvobacteriunt thernzosulfurigenes i was obtained as a conseque ce of the reduction of the water-accessible
hydrophobic surface by site' lrected mutagnesis of aromatic amino acids in f ' the active site. ~e~lacemend,of W139 with F, M, or A resulted in increased
catalytic efficiency proportid/nal to the decrease in hydrophobocity of the side
chain of the substituted amin acid (Meng ct al., 1993).
The effect of changing the residues at the subunit interfaces on the activity
and thermostability of xylose glucose isomerase from Arlhrohackr spp. was I studied by Varsani eta/. (1913). Introduction of one or two disulfide linkages
or salt bridges at the subunit i terfaces does not result in any change in
enzyme activity or stability. analysis of the results indicates that subunit
dissociation is not a pathway of thermal inactivation but that movements of
active-site groups may trigger I conformational changes which may be
responsible for the initiation df the unfolding of the protein. Attempts were
made to study the effect o altering the metal ion at the M-2 site on the f thermostability of the D-xyloselglucose isomerase of S. ruhiginosus. Sh~dies
on SDM-generated positio a1 analogues of His-220 mutants of S. ruhiginosus
have confirmed the role of the geometry and the binding affinity of the metal
ion at site 2 in the stability of D-xyloseiglucose isomerase. Even a subtle
difference in the co-ordina 'on of the M-2 site metal ion affects the catalytic I activity in the case of His-220 mutants, indicating the possible role of site 2 in
isomerisation (Cha el a/., 1 4 94). i
2.13.2 Deciphering the
Deciphering the role respect to themostability and
catalysis is difficult. of xylose/glucose isomerase
from Aclinoplanes missotiri nsis were investigated after the side chains
involved in metal binding w re substituted by site-directed mutagenesis
(Jenkins et al., 1992). The r sults demonstrate that the two metal ions play an
essential role in binding and stabilising the open forms of the substrate and in
catalysing hydride transfer b tween the C-1 and C-2 positions. The distinct
role of two magnesium ions ssential for the xyloselglucose isomerase activity
of Streptomyces olivochrom )gene,s was determined by neutron activation I analysis and site-directed mu+genesis (Allen eta[., 1994). One of the metal-
binding sites, M-1, was remoled by substitution of Glu-180 by Lys. Ring-
opening assays with the muta t E180K and with 1 thioglucose as the substrate I showed that Glu-180 is for isomerisation but not for ring opening.
The wild type and the no other significant structural differences
, . 2.13.3 Alteration of subs' rate specificity f Xylosefglucose isomer se displays higher affinity for xylose than for i glucose. However, increaqed affinity toward glucose is desirable in view of
its application in the produ tion of HFCS. Attempts to alter the substrate
preference of the thermoph'lic xylose/glucose isomerase from iYos/ridium
sulfurogenes were made b 1 redesigning the amino acids situated in the
substrate-binding pocket ( eng et a/., 1991). The W-139 -+ F substitution
reduced the Km and increa i ed the K,,, of the mutant towards glucose, while
the reverse effect towasd &re war observed. Double mutants (W-139 -t
F/V-186 -+T and W-139 -+ F/V-186 -+ S) had five- and two-fold higher
catalytic efficiency, respecti ely, than did the wild type.
These results provide evi ence that the substrate specificity can be altered I by reducing the steric ints and enhancing the hydrogen-binding
capacity for glucose pocket of the active site.
I 2.13.4 Functional role of essential amino acids residues
The essential active-site h'lstidine residue in the xylose/glucose isomerase 'I from Clostridium thermosulf rogenes was identified by substituting histidine i; residues at four different pos'tions. Substitution of His- 101 by phenylalanine 'i abolished the enzyme activid, whereas substitution of other hlstidine residues
had no effect (Lee et a/., 199 ) His-101 and His-271 were shown to be
essential components of the a tive site of xylose/glucose isomerase from i' E, coli by selective substitutiop of each amino acid (Batt et al, 1990). It was
speculated that His-101 is thd catalytic base mediating the reaction whereas !
His-271 behaves as a liga d for one of the metal ions in the active site of
xylose/glucose isomerase. Site-directed mutagenesis was used to assess the
sbucturd and functional r les of specific amino acid residues in the xylosel
glucose isomerase from Ac inoplanes mis.sozrricn.si,u. His-220 and His-54
were important but not ess ntial for catalysis (Lamheir et al., 1992). His-54 :: was implied to govern the yomeric specificity. Lys-183 was assumed to play
a crucial role in the isomeri ation step by assisting the proton shuttle. Lys- i 294 is indirectly involved ill binding the activating cations, whereas Trp-16
and Trp-137 contribute to 'aintenance of the general architechre of the
substrate-binding site.
Site-directed mutagenesis of the conserved tryptophan residues in the
E.coli enzyme (Trp-49 and 1 rp-188) reveals that fluorescence quenching of
these residues occurs binding of xylose by the wild-type enzyme.
Additional active-site at His- 10 1, which result in inactivation of
enzyme, show altered (Jamieson & Batt, 1992).
2.13.5 Alteration of pH optimum
Commercial application of xyloselglucose isomerase demands an acidic
pH optimum to enable starch liquefaction and glucose isomerisation to be
camed out in a single step. Glu-186 is a conserved residue which is situated
near the active site of xylose/glucose isomerase from A. missouriensis but
does not participate in the substrate or metal ion binding. The negative charge
from this group was removed by its mutation to glutaxnine, which resulted in
lowering its pH optimum to 6.25 and in changing its preference from ~ g ~ ' to
~ n " (Tilbeurgh et al., 1992). This study adds new informaton on the
catalytic mechanism of aldose-ketose isomerisation by xylose/glucose
isomerase and demonstrates that a single amino ac id substitution is able to
shift the pH optimum by more than 1 pH unit.
2.14 IDENTIFIED PROBLEMS AND POSSIBLE SOLUTIONS
Introduction of enzymatic xylose/glucose isomerisation for the production
of HFCS is beset by several problems. Among the major problems are the
inactivation of xylose/gluoose isomerase at higher temperatures, the high pH
optima of many of the xylose/glucose isomerase operations, the requirement
of cobalt for enzyme activity, the lower affinity of xylose/glucose isomerase
for glucose than xylose and the suboptimal concentsations of the product.
Intensive research into ways of overcoming these problems has resulted in the
development of substantially improved processes. Nevertheless, there is scope
for further improvement iq all the above mentioned areas to evolve an
economically feasible c o h e r c i a l process to substitute glucose totally by
HFCS. Some of the impo ant problems faced in industrial applications of I xylose/glucose isomerase $nd the plausible solutions thereof are discussed
I
below.
2.14.1 Enhancement of tbermostability
The equilibrium conver$ion of glucose to fructose under industrial process
conditions is around 50%, and the enthalpy of the reaction is 5KJImol. The
commercial application of HFCS requires the use of high fructose concen-
. . trations. The concentration of fiuctose desired for many applications in the
industry is higher than 50%. Higher isomerisation yields may be achieved by
increasing the reaction temperature. The effect of temp- erature on the
concentration of fructose at equilibrium is shown in Table 5. Use of higher
concentrations of feed syrup and increased temperatures of operation keep the
reaction times required for the isomerisation processes from becoming
excessive. Lower temperatures lead to an increased risk of microbial
contamination.
2.14.2 Enrichment of fructose
The major application of HFCS is in the sweetening of soft drinks. A
55% HFCS concentration matches the sweetness of sucrose and allow 100%
substitution. Its price is 10 to 20% lower than the price of sucrose, based on
sweetening power. A 42% HFCS concentration is used in the baking, dairy,
and confectionery industries and for preparing canned food, jann, jelly and
ketchup. However, its application in these industries is limited by some
drawbacks inherent in HFCS, namely, its hygroscopic and viscous nature,
browning tendency, and inability to crystallise. In the commercial processes,
42% fructose is generally produced in the equilibrium mixture; this needs to
be enriched for its major applications. The earliest method to enrich fiuctose
involved the complexatrion of fructose by addition of borate compounds
during isomerisation (Takasaki, 1971). The degree of enrichment depended
on the glucose concentration and the amount of borate added. This method
resulted in the production of syrups containing 80% fructose. However, the
cost of removal and recovely of borate prevented the economic success of this
process. The most straightforward complete conversion of glucose to fructose
has forever been the dream of corn-milling and -refining industries.
Another route to increase the fructose yield by using D-g1uc;ose was to
produce a transient overshoot equilibrium concentration of products as
described by Schray & Rose (1971). Another approach to make 55% fructose
is to increase the isomerisation temperature (Antrim, 1979). Increasing the
temperature to more than 70°C leads to increase in the HFCS concentration
by 50% or more. Resinous molecular exclusions have been used to increase
the fructose concentration. A syrup containing more than
90% fnlctose was obtained by forming fn~ctose-oxyanion complexes with
germanate (Barker et a[., 1983). Modem chromatographic techniques with
ion-exchange resins are the best for seperating fructose from glucose. A syrup
containing 95% fructose is on the market in France and is sold in crystalline
form (Bhosale et a/., 1996).
2.14.3 Lowering of isomerisation pH
The optimum pH for isomerisation is 7.0 to 9.0. The activity of the
enzyme decreases rapidly at lower pH values. Low pH is preferable for the
sake of monosaccharide stability and for the compatibility of the process with
sacchaification of starch by a-amylase. The most common raw material used
for HFCS production is corn starch. Liquefaction and saccharification of
starch involve participation of a-amylase, glucoamylase and debranching ~. .
enzyme, all of which have pH optima in the range of 4.4 to 6.2, whereas that
for isomerase is between pH 7.0 and 9.0. A big saving in cost will be
possible if the two processes can be carried out simultaneously at the same
pH in a single reactor. Isomerisation at low pH is advantageous, because it
reduces the formation of the colored carbonyl compounds at higher
temperatures and may lead to lower costs of ion-exchange and carbon
purification. The xylose/glucose isomerase from Thermus aqua8ticus
(Lehmacher & Bisswanger, 1990a) is reported to be active at pH 3.5 and to be
fully active at 5.5. The term "uni-process" implies a process in which
liquefaction, saccharification and isomerisation are carried out at the same
pH, preferably at pH 4.5 to 5.0, which is the pH optimum for amylase and
glucoamylase. The presence of ca2' is a prerequisite for the the action of
amylase, whereas ca2' is inhibitory to xyloselglucose isomerase. Acid stable
xylose/glucose isomerases which are resistant to inhibition by ~ a " are
useful in a uni-pH process. A xylose/glucose isomerase from
Thermoanaerohacter sp. was characterised with a view to developing a
single-step process for sweetener production (Lee el ul., 1990).
The combination of saccharification and isomerisation is an ideal
development in the progress of HFCS production and it is likely to be in
operation once an acid-stable, thermostable and Cia2' tolerant xylosc/glucose
isomerase is discovered. Such xyloseiglucose isomerases could be found
either by screening or by protein engineering of the existing enzymes used for
commercial production of HFCS.
Table 5 : Effect of isomerisation temperature on the concentration of fructose
(Bhosale el ul, 1996)
Temperature ( "C ) Fructose concentration (%)
2.14.4 Simultaneous isomerisation and fermentation of xylose
The current shortage of petroleum and natural gas has prompted renewed
interest in the microbial conversion of pentose-containing renewable biomass
resources to ethanol and other useful feedstocks (Rosenberg, 1980). Many
yeasts can grow on xylose but they are ineffictent in fermenting the sugar
anaerobically and have very low ethanol tolerance (Jeffries, 1985).
Schizo,saccharomyces pombe, Saccharomyces cerevisiae, and Chndida
tropicalis are able to ferment xylulose derived from isomerisation of xylose
with xylose/glucose isomerase in totally anaerobic fermentations (Lastick e/
al., 1989). Simultaneous isomerisation and fermentation of xylose (SIFX) is
preferred to isomerisation prior to fementation, because the ratio of xylulose
to xylose ( 1 5 ) is low at equilibrium. Removal of xylulose from the mixture
facilitates conversion of xylose to xylulose, which is simultaneously
converted to ethanol by the yeast. The optimum pH for fermentation is 5.0
whereas xylose/glucose isomerase is most stable at neutral pH. Both
isomerisation and fermentation can occur at a compromise pH of 5.5 or 6.0
(Lastick, 1990). Despite the difference in the rates of fermentation of glucose
and xylose, final yields of ethanol in SIFX were impressive. Low enzyme
levels or inhibition of the enzymes by xylose, xylulose or ethanol may be
responsible for the inefficiency of SIFX. Nevertheless, SIFX provides a
significant improvement over existing systems for fermentation of xylose to
ethanol. Use of immobilised xylose/glucose isomerase and yeasts may lower
the cost of SIFX and the use of acid-stable xylose/glucose isomerase will
contribute to the greater efficiency of SIFX.
2.15 FUTURE SCOPE I
I The ideal xylose/glucose isomerase should possess a lower pH optimum ,
a higher temperature optimum, a resistance to inhib~tion by ca2', and a higher
affinity for glucose than do presently used enzymes. Introduction of all these 1 properties into a single protein is a herculean task, which if overcome, would
I greatly improve the efficiency of the commercial process for enzymatic
isomerisation of glucose to fructose. Advances in recombinant DNA ,
1 technology and protein engineering have opened new and encouraging
I I
possibilities for combining the above desirable properties in a single
organ~sm to produce a tailor-made protein (Bhosale et al., 1996).
CHAPTER 3
i 3.0 MATERIALS AND METHODS
3.1 MATERIALS I !
All chemicals used were of the highest available purity. Tryptone,
I phenylmethylsulphonyl fluoride (PMSF), 3-(N-morpholino) propanesulfonic
I acid (MOPS), cysteine-hydrochloride, yeast extract, L-histidine base, N-tris
i (hydroxymet11yl)methyl-2-amino ethanesulphonic acid (TES), ammonium
1 persulphate (APS), acrylamide, N, N' methylene - bis-actylamide, N, N, N', I i N' - tetra-methyl ethylene diamiue (TEMED), L-cysteine hydrochloride, I I ammonium sulphate, Coomassie brilliant blue R250, Coomassie brilliant blue I G250, Sodium dodecyl sulphate (SDS) and all other materials for SDS-PAGE I
' were from Sewa (Heidelberg, Germany) with the exception of CribcoBRL
1 10-kDa protein ladder from Life Technologies. Nutrient agar, peptone a d I I nutrient broth were from Difco. D-fructose, D-xylulose, D-glucose and
D-xylose were also from Se~va. Protein markers for the determination of the
molecular masses of the native enzymes (bovine serum albumin, Fraction V,
1 Mr.= 67,000; catalase from beef heart, Mr.= 240,000; hexokinase,
I Mr.=100,000; alcohol dehydrogenase from yeast, Mr.=150,000; myoglobin, i
Mr.= 17,000) were from Boehringer (Mannheim, Germany). Carbazole was
from Aldrich. The cyanogen bromide activated Sepharose 4B, Sephacryl
S200 HR powder and the fast protein liquid chromatography (f.p.1.c)
prepacked columns of phenyl superose (HR 515), Superose 6""' and I
I
i Superose l T M were from Pharmacia (Uppsala, Sweden). The controlled pore
i glass, BIORAN~-CPG, was from Schott Geraete (Hofheim, Germany).
I DEAE-cellulose (DE52) was from Whatman (Kent, England) and I I ethylenediamine-tetra acetic acid (EDTA) was from Roth (Karlsruhe, I
Germany). Glucose oxidase, peroxidase and benzidine were from Sigma.
I D-xylitol, D-lyxosc, glycerol , L-asparagine, MgSOd .7H20! MnS04 .H20,
I CaS04 .2H20, CoSOJ .7H20, CuCI2.2H20, VOS04 .5H20, NiSOr .7H20,
I I ZnS04 .7H20, sodium metaperiodate, p-phenylenediamine. 2HC1, sodium I I borohydride, sodium nitrite, glycine, NaOH, NaCl and all other laboratory i I chemicals were from Merck (Darmstadt, Germany).
3.2 COLLECTION OF SAMPLES AND ISOLATION OF I I MICROORGANISMS
Soil samples were collected from refuse dumps, self-healing compost and
rhizospheres of soy-bean, sugar cane and tomato plants in Nsukka, Enugu
State, Nigeria.
To collect soil samples from the rllizosphere of the plants, the soil in the
immediate vicinity of the roots of plants uprooted from farms around the
university community was collected in fresh cellophane bags. Compost
samples were collected from self-heating compost heaps in the Animal farm
of the university in fresh cellophane bags while refuse-dump samples came
from 5cm soil layer beneath the soil surface at Nsukka market.
, ' . 3.2.1 Samples preparation
The soil samples collected from the various sources were each (log) I
! dispensed into 250ml Erlenmeyer flasks containing 90ml of sterile saline.
! Each suspension was vigorously agitated manually for 5 minutes to detach I
spores and vegetative cells from soil particles. Heavier particles were allowed
I to sediment for fifteen seconds before serially diluting the supernatant to 10".
3.2.2 Isolation of microorganisms
Each diluted supernatant from the samples was enriched in xylose synthetic
medium (Tsumura & Sato, 1961) prepared as follows :
Solution A - D-xylose (20g), MgSOj .7H20 (0.25g), distilled water 500ml.
Solution B - (NH4)2HPO4 (6g), KH2P04 (0.2g), distilled water 500ml, pH
adjusted to 6.8-7.0 with H3PO4. After sterilisation, solutions A and B were
mixed aseptically before use. The solid medium was formed by addition of
2% Agar to the synthetic medium. The medium after inoculation was
incubated for 4-5 days at 30°C and 55°C respectively for the mesophiles and
thelmophiles. Using the glass-spread method, the resultant isolates were
subcultured on glycerol-aspwine agar (Pridham & Lyons, 196 1) of
composition : L-asparagine (anhydrous), 1 Og; glycerol, log; K2HP04
(anhydrous), 1.0g; distilled water, 1 litre; trace salts solution, lml; pH
7.0 -7.4. Agar was added and liquefied by steaming at 100nC for 15 minutes
before sterilisation by autoclaving at 121°C for 15 minutes. The subcultures
were incubated at 30°C and 55°C respectively until visible microbial colonies
appeared (up to 7 days). For the incubation at 55"C, a bowl of water was
placed in the incubator and wet filter papers were placed on the petri dishes to
prevent the medium from drying up.
All resultant colonies were purified by repeated streaking on nutrient agar
and glycerol-asparagine agar plates. The isolated colonies were preserved on
agar slants at 4'C until needed .
3.3 SCREENING TEST FOR XYLOSEIGLUCOSE ISOMERASE
PRODUCTION
Pure isolates were screened for xylose/glucose isomerase production by two
procedures :
i) In the first, screening agar plates were prepared by pouring a base layer of
about 25ml of nutrient agar (for regular bacteria) or glycerol-asparagine agar
(for actinomycetes) unto sterile petri dishes and after hardening, the plates
were dried in a sterile oven at 50°C prior to inoculation. Following
inoculation at 30°C (mesophiles) and 55°C (tbestnophiles) for 36 hours, the
resultant growths were overlaid with 5ml of xylose synthetic agar and after
solidification of the overlay the plates were again incubated until the
apperance of gowth on the overlay. Such apperance is suggestive of ability
to produce xylose/glucose isomerase.
ii)The second screening procedure followed was as described by Lee er a/.
(1990). In this test the differential medium containing fructose (2%),
MgS04. 7Hz0 (5mM), CoClz (OSmM), glucose oxidase (20pg/ml),
peroxidase (4Ulml) and benzidine (0.4mg/ml) in 100 mM 3- (N-mor- pholino)
propanesulfonic acid (MOPS) buffer pH 7.0 was mixed with 0.7% agar at
the appropriate temperature and thereafter poured over colonies on the xylose
synthetic agar plates of the first screening step. After solidification of the soft
agar, the plates were incubated again at the appropriate temperatures. Xylosel
glucose isomerase positive cultures appeared with dark brown halo around the
colonies.
3.3.1 Confirmation of xylose/glucose isomerase production
All positive isolates were subjected to submerged fermentation in the
medium of Chou et d(1976) of composition: hyptone (I'XO), yeast extract
(0.7%), xylose (0.5%), MgS04 .7H20(0. 1%); pH, 7.0-7.2. The seed culture
was prepared by inoculating 50ml of the medium contained in 250 ml
Erlenmeyer flask with the isolate from an agar slant and incubating at 30°C
(for mesophiles) and 55°C (for thermophiles) for 24 - 36 hours. Some 2 ml of
this culture was transfered to a fresh flask containing 100 ml of culture
medium and incubated at the appropriate temperature in a cot~trolled
environment incubator shaker for approximately 48 hours at 160 r.p.m. The
cells were harvested with the aid of Beckman refrigerated centrifuge
(20,00Og, 20 minutes, 4°C). Thereafter the cells were washed twice with
lOmM histidineJHC1 buffer, pH 6.0, 0.5tnM PMSF before resuspending in
minimal amount of the same buffer. The cells were then lysed in an ice bath
using a type B- 12 sonicator (Branson ultrasonics) for three minutes. Cell
debris was removed by centrifugation (35,00Og, lominutes, 4°C) and the
resultant supernatant assayed for xylose/glucose isomerase activity.
3.4 ENZYME ASSAYS
D-xylose isomerase activity was determined by the folmation of D-
xylulose from xylose using the colorimetric assay method of Dische and
Borenfreund (1951). A test mixture (20~1) containing 0. lml TES/NaOH
buffer, pH 7.0, 0.2 M D-xylose and 0.4 mM MnSOJ .7H2 0 was incubated
together with 20pl of the appropriately diluted enzyme solution (crude
extract) for 10 minutes at 70' C, using a dty heating block (Techne D.B.2A).
The samples were immediately cooled to O°C on ice chips to stop the enzyme
reaction before adding 80pI of a I : 1 mixture of 1.5% cysteine-HCl in water
and 0.12% carbazole in ethanol and then 1.2ml 70% sulfuric acid. The
mixture was allowed to stand for 10 minutes for colour development after
which absorbance was measured at 546nnl. The concentration of D-xylulose
was determined from a standard curve of D-xylulose. One unit of enzyme
activity was defined as the amount of enzytne which converted one
micromole of xylose to xylose xylulose per minute under the given assay
conditions.
Where necessary, D-glucose isomerase activity was also measured. It was
basically the same method of assay except that xylose was replaced by 1M
D-glucose and MnS04 .H20 by CoCI2. The incubation period for the colour
development with carbazole was extended to 20 minutes and absorbance
measured at 560nm. The enzyme activity was calculated from a D-fructose
standard curve. One unit of the enzyme was defined as the amount of enzyme
which converted one micromole of glucose to fructose per minute under I
specified assay conditions.
3.5 DETERMINATION OF PROTEIN CONCENTRATION
This was carried out according to the method of Bradford (1976) using
bovine serum albumin as standard.
1 3.6 IDENTIFICATION OF ISOLATES
3.6.1 Identification of actinomycetes I
I The three actinomycete isolates (X-1, SHC-1 and SHC-5) which were
~ positive for xylose/glucose isomerase were identified on the basis of their
morphological and structural characteristics (Gottlieb, 1959). These
1 characteristics were established by cover-slip culture technique on inorganic
starch agar (Kuster, 1963) and glycerol-asparagine agar plates. The
I composition of the inorganic starch agar was as follows :
i Solution 1: Starch (log) made into paste with small amount of cold distilled
I water and thereafter made up to 500ml.
i Solution 2: Prepared by dissolving anhydrous KzHPOJ (lg) , CaC03 (2g) ,
I MgS04.7H20 (Ig), NaCl (lg), (NH4)2 SO4 (2g) in 500ml distilled water, and
I then adding with 1 ml of trace salts solution. The trace salts solution was
made up of FeSO4.7HzO (0. lg), MnC12.4H20 (0. lg), ZnS04.7H20 (0. lg) in
lOOml of distilled water (pH 7-7.4). Solutions 1 and 2 were mixed, 20g of 1 agar added, liquefied by steaming at 100°C for 15 minutes and then
1 ,, autoclaved at 121°C for 15 minutes. Thereafter, the mixture was dispensed in
sterile Petri dishes and allowed to set. Sterile cover slips were aseptically
inserted at an angle of 45" into the medium and pure cultures of each isolate
were then inoculated along the line where the agar surface met the un-
submerged portion of the cover slip (the equatorial region of the cover slip).
The plates were incubated at 30°C and 55°C respectively for 48 hours. Each
cover slip was then carefully removed and its orientation in the medium
noted thus facilitating distinction between aerial and substrate mycelia. The
cover slips were placed on slides and the growth fixed with few drops of
absolute methanol for 15minutes, washed with tap water and blotted dry. The
slides were thereafter stained with 0.5% crystal violet for one minute, again
washed with tap water and blotted dry. The stained preparations were
observed under oil immersion.
3.6.2 Identification of true bacteria
The morphological characteristics of the bacterial isolates were investigated
using the methods described in Bergey's Manual of Determinative
Bacteriology (9th edition). Tests carried out included: Gram stain, motility,
spore formation, citrate utilisation, starch hydrolysis, casein hydrolysis,
catalase, urease, Voges-Proskauer, indole formation, hydrogen sulphite
formation, pH growth optimum, temperature growth optimum,
G + C content. In addition the cultural characteristics were studied in nutrient
broth, nutrient agar slants, glucose nutrient broth, glucose nutrient agar and
peptone water.
C The mesophilic bacterial isolates- X-3 and X-4, which had the highest
activities for xylose isomerase were selected for further studies, and were
subsequently confirmed as Paenihacillzrs sp. and Alcaligenes ruhlanclii
respectively by "Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH" (DSM), Braunschweig, Gennany.
3.7 PRELIMINARY PRODUCTlQN OF THE XYLOSE ISOMERASE
IN SUBMERGED CULTURE
Pure isolates of the two mesophilic bacteria, Pucnihacillzrs sp and
Alcaligenes rzlhlandii, were each grown aerobically in a bacto nutrient broth
medium in a shake flask culture for 24 hours at 30°C. A 2ml amount of each
culture was then used to seed 100ml of the medium of Chou el a/. (1976)
containing 0.7% yeast extract, 1% tryptone, 0.1% MgSOJ 7H20 and 0.5%
D-xylose, and the pH was adjusted to 7.0. The cultures were grown
aerobically at 30°C for 48 hours in a rotaiy water bath shaker (GFL Bachofer)
at 160 r.p.m. The cells of each culture were harvested by centrifugation
(2O7000g, 20 minutes, 4OC) using the Soi-val RCS superspeed refrigerated
centrifuge (Dupoint Instruments). The cells were washed twice and
resuspended in minimal volume of 1 OmM HistidineIHCI buffer, pH 6.0;
0.5mM PMSF and stored at 0°C.
3.8 ANALYSIS OF DATA
All experiments were carried out in triplicate or duplicate. Where
appropriate the data were subjected to statistical analysis (t-test) according to
the method of Spiegel (1972). Where there was significant difference existing
between treatments, the levels of significance were indicated as P > 0.05
(95% confidence limit) and as P < 0.05 if the means were not significant.
3.9 EXTRACTION AND PURIFICATION O F THE TWO XYLOSE
ISOMERASE ENZYMES
All operations were caxied out at 4°C under aerobic conditions unless
otherwise stated.
3.9.1 Preparation of crude extract
Cells of PaenibaciNus sp and Alcaligenes ruhlandii were grown as
previously described in the preliminary production of the enzyme. Wet cells
(14g) of each organism were halvested from 1-litre broth culture by
centrifugation at 20,000g for 20 minutes and washed twice with lOmM
histidineIHC1 pH 6.0,OSmM PMSF. After resuspending in 70 ml of the
same buffer, the cells were disrupted in a sonicator (Branson ultrasonics
type B12) at O°C for 3 minutes and centrifuged at 35,000g for 10 minutes.
The resultant supernatant was kept at 4°C as the crude enzyme.
3.9.2 Ion exchange chroatography on Whatman DE 52 column
The DEAE-cellulose (Di-ethyl-amino-ethyl cellulose) column (Whatman
DE52) was prepared by mixing lOOg of Whatman DE52 powder in I litre of
0.5M HCI and stirring with glass rod intermittently for 1 hour to polarise the
powder. The mixture was then carefully washed with deionised water until a
pH of about 4.0 was attained, without allowing the gel to dry. The gel was
then neutralised by the addition of 1 litre of 0.5 M NaOH with intermittent
stirring with glass rod for 1 hour. After washing with de-ionised water till the
attainment of a pH of 7 - 7.5, the gel was preequilibrated with lOmM
histidineIHC1 buffer, pH 6, 0.5mM PMSF and poured into the column and
allowed to settle. The column was continuously washed with the lOmM
histidineIHC1 buffer until a constant height was achieved. The crude enzyme
preparation (35ml) was then applied unto the DEAE-cellulose (Whatman
DE52) column (2.2 x 25cm) pre-equilibrated with lOmM 11istidineIHCI
buffer, pH 6, 0.5mM PMSF (henceforth called the histidine buffer). The
column was washed with 200ml of the histidine buffer and eluted with a
450ml linear gradient of 0.05M NaCl in the same buffer at a flow rate of
OSmlImin. The fraction size was 6.5 ml per cup. The protein profile was
obtained with the aid of a UV detector (Pharmacia) at 280nm. The fractions
were assayed for xylose isomerase activity and those containing significant
activity were pooled (52ml for Paenihacillus sp. and 58.5m1 for Alcaligenes
ruhlandii).
3.9.3 Ammonium sulphate fractionation
The pooled active fractions were treated in stepwise manner with very fine
crystalline ammonium sulphate first to a saturation of 35%. The suspension
was centrifuged at 35,000g for 15 minutes and the precipitate discarded. More
crystalline ammonium sulphate was added to the supernatant up to a final
saturation of 75% with continuous stirring on ice for 30 minutes. The
resultant suspension was centrifuged at 35,000g for 15 minutes and the
precipitate which contains the enzyme was dissolvcd in minimal volume of
the histidine buffer (5ml for each of the enzymes), The enzyme solution was
dialysed against 500ml of the histidine buffer for 12 hours.
3.9.4 Gel filtration on Sephacryl S-200 HR
The dialysate from the ammonium sulphate fractionation was applied to a
1.0 x 48cm Sephacryl S200 HR column (Pharmacia) preequilibrated with
2-bed volumes (76ml) of histidine buffer. Elution was done with the same
histidine buffer at a flow rate of 0.36ml/min and fraction size of 5ml.
Fractions with significant amount of xylose/glucose isomerase activity were
pooled and stored at 4'C until use (25ml for f'uenibucillus sp and 30ml for the
A . ruhlandii).
3.9.5 Hydrophobic interaction chromatography on Phenyl Superose
column (HR 515, FPLC system, Pharmacia)
To the pooled fractions from the gel filtration was added ammonium
sulphate to a final concentration of 1.3M. Hydrophobic interaction
chromatography (H.I.C.) was carried out on the phenyl superose column
(5 x 50mrn) by first equilibrating the colu~nn with 1.3M ammonium sulphate
in histidine buffer, and then applying the enzyme solution. The columr~ was
then washed with 1.3M ammonium sulphate in histidine buffer to remove the
unbound proteins. All the solutions (enzymes and buffers) used in this system
were filtered through a memhrane filter before being applied to the column.
The bound enzyme was eluted with 30ml of the histidine buffer with a linear
gradient of 1.3 to O.OM ammonium sulphate. The flow rate was 0.2mllmin.
Elution was effected by a decrease of the ammonium sulphate concentration
from 1.3 to O.OM. Fractions (lml) were collected and assayed for xylosel
glucose isomerase activity. The pooled fractions from this column (3mI for
Pamibacillus sp and 2ml for the Alcaligene~ ruhlandii) were dialysed against
500ml of histidine buffer for 12 hours and stored at 4°C until use.
3.9.6 Gel filtration on Superose 6T" ( Pharmacia; f.p.1.c.)
A second gel filtration chromatogaphy was cauied out on Superose G ' ~
column with the aid of Phatmacia f.p.l.c.system (Uppsala, Sweden). The
column (24ml) was equilibrated with 72ml of histidine buffer and the pooled
enzyme samples from the H.I.C. charged unto the column. Elution was
carried out with 150ml of the histidine buffer at a flow rate of O.Zmllmin, and
fraction size of 6ml. Each fraction was assayed for xylose/glucose isomerase
activity and protein concentration. All active fractions were pooled and
stored at 4°C (6ml for both xylose/glucose isomerases).
3.10 HOMOGENEITY O F PURIFIED XYLOSE/GLUCOSE
ISOMERASES
The xylose isomerases from the last purification step were subjected to
homogeneity test based on electrophoresis on 10% (wlv) polyacrylamide gel
containing sodium dodecyl sulphate (SDS-PAGE ). To do this, 2.5pdml of
each of the purified enzymes was first dissociated by boiling for 3 minutes in
the presence of 0.2M TrisIHC1 buffer, pH 6.8, 0.4% sodium dodecyl sulphate,
20% glycerine, 10% dithiothreitol (DTT) and O.lmg/ml bromophenol blue.
The SDS-PAGE was performed according to the method of Laemmli (1970)
at 8 mA and 20°C on 7% stacking and 10% seperation gels. The protein
banding patterns were revealed by staining with 2% Coomassie Brilliant blue
R250 solution for about 30 minutes. The gels were destained using a solution
containing 525m1 ethanol, 200ml concentrated acetic acid and 1.275 litres of
water. The gels were then preserved in 7% acetic acid solution.
3.1 1 DETERMINATION OF MOLECULAR MASS OF THE
PURIFIED XYLOSEIGLUCOSE ISOMERASES
The molecular mass of the native enzymes were estimated by the gel
filtration method of Andrews (1964) using a Superose 12TM column with the
aid of Pharmacia FPLC system. The column (24ml) was equilibrated with
lOmM histidineMC1 buffer, pH 6,OSmM PMSF. Five standard protein
markers were used (catalase -Mr 240,000; alcohol dehydrogenase from yeast
-Mr 150,000; hexokinase - Mr 100,000; bovine serum albumin, fraction V - Mr 67,000; myoglobin - Mr 17,000). A 5&ml amount of each protein in
the histidine buffer was applied seperately to the column. The protein was
then eluted at a flow rate of 0.4mllmin with the same buffer. The volume
needed to elute each protein was noted. The two xylose isomerase enzymes
were treated in the same manner. Each sample was run twice and an average
elution volume was calculated. The molecular masses of the xylose/glucose
isomerase enzymes were extrapolated from the points of intersection of their
elution volumes on the straight line obtained by plotting the log molecular
masses against elution volumes of known protein markers. The elution
volumes of the xylose/glucose isomerase enzymes were determined by
assaying for enzyme activity and those for the protein markers by monitoring
absorbance at 280nrn.
The SDS-PAGE was also used to calculate the subunit masses of the two
xylose/glucose isomerase enzymes. The GibcoBRL lOkDa Protein ladder
was used for the standards. The protein ladder contained 13 bands (protein
markers) of molecular masses (kDa) 200, 120, 110, 100, 90, 80, 70, 60, 50,
40, 30, 20, and 10 respectively. The distances migrated by the proteins were
calculated and their molecular masses extrapolated from the points of
intersection of their migration distance values on the straight line by plotting
the molecular masses against migration distances of the proteins of the 1 OkDa
protein ladder
3.12 ENZYME CHARACTERISATION
3.12.1 Effect of temperature on enzyme activity
The temperature activity profiles of the D-xylose/glucose isomerases of
Paenihacillus sp. and Alcaligenes rtrhlandii were determined by adding 20pl
of the enzyme samples to sealed vials containing 20p1 test mixture and
incubating for I0 minutes at the test temperatures. The test temperatures
ranged from 20 - 90°C. The vials were then cooled on ice before enzyme
activity was assayed. The test mixture contained 0.1M TES/NaOH buffer,
pH 7; 0.2M D-xylose and 0.4mM MnS04.
Arrhenius plots of the enzyme activities over the temperature range 20-70°C
were used to calculate the activation energies.
3.12.2 Effect of temperature on enzyme stability
Each purified enzyme (1.5pglml) was preincubated at various
temperatures (4 -90°C) for 1 horn after which the sample was promptly
chilled on ice and the residual activity measured under normal assay
conditions.
3.12.3 Enzyme Decay
The decay rate of each D-xylose/glucose isomerase was determined by
preincubating the purified enzyme samples (1.5ygIml) with 0.4mM MnS04
at 55°C and aliquots removed for enzyme assay at different time intervals.
The half-life was estimated from a plot of logarithm of enzyme activity
against time (day).
3.12.4 Effect of pH on enzyme activity
The effect of pH on enzyme activity was examined by incorporating
different buffer systems of differing pH values into the test mixture for
normal isomerisation reaction. Buffer systems used were 50mM acetic
acidhodium acetate buffer for pH 3.5-5.5; 50mM TESINaOH buffer for
pH 5.6-7.9 and 50mM glycine1NaOH buffer for pH 8.0-10.
3.12.5 Effect of pH on enzyme stability
For the pH stability, the enzymes were preincubated in the various buffer
systems for I hour at 25'C. Thereafter, the enzyme activities were measured
under normal test conditions at pH 7.
3.12.6 Effect of substrate concentration on D-xyloselglucose isomerase
activities
D-xylose and D-glucose were used for these assays. For the effect of
D-xylose concentration on enzyme activity, different concentrations of
D-xylose (5- 100mM) in TESINaOH buffer pH 7 containing 0.4mM MnC12
were reacted with 1.5pdml of each of the xylose/glucose isomerase
enzymes. These were incubated for 10 minutes at 70°C and the reaction
stopped by placing the samples on ice bath. The activities of the enzymes
were determined as earlier described. The kinetic parameters (Km and Vmax)
were calculated by measuring the initial rate of reaction at various D-xylose
concentrations and plotting a Lineweaver-Burke diagram.
With D-glucose as the substrate, various concentrations of D-glucose
(0.2 - 2M) were used. The reaction mixture contained 5pdml of each
enzyme, 2mM CoCI2 in place of MnClz and was incubated for 20 minutes at
70°C. The absorbance was measured at 560nm. The concentration of the
product was extrapolated from a standard curve of D-fructose. The kinetic
parameters were calculated by measuring the initial rate of reaction at various
D-glucose concentrations and plotting a Lineweaver-Burke diagram.
3.12.7 Effect of divalent metals on D-xylose isomerases
The metal ions examined were: ~ g ~ ' , ~ n ~ + , Co2+, zn2', vo2+,ca2.', ~ i ~ +
and cu2'. Prior to the tests it was necessary to obtain metal-free enzymes.
To this end, each of the purified xyloselglucose isomerases was incubated for
12 hours with 0.5mM EDTA at 4°C and dialysed for 24 hours at the same
temperature against 1 litre of 0. IM TESINaOH buffer, pH 7. The dialysis
buffer solution was changed every 8 hours. Thereafter, the samples were
assumed to be metal-free. All the buffers used were treated with Chelex 100
and stored in acid-washed plastic containers. The metal-free enzymes were
used for the following tests:
i) incubating the metal-free enzyme with the test mixture containing no metal
and assaying for xylose isomerase activity.
1 ii) incubating the metal-free enzyme with test mixtures containing one of the
underlisted divalent metals: Mg2' (MgS04.7H2 0 ) ; Mn2' (MnS04.H2 0 ) ; cu2'
(CuSO4.2H2 0 ) ; CaZ' (CaSO4.2H2 0 ) ; ~ 0 ~ ' (VOSO., .SH2 0 ) ; co2'
(CoSO4.7H2 0 ) ; zn2 ' (ZnS04.7H2 0 ) and ~ i ~ ' (NiS04.7H2 0 ) . The enzyme
activity was measured thereafter.
iii) kinetic studies of the xylose isomerase enzymes were cmied out using
various concentrations (2 - 50mM ) of Mg2', Mn2' and co2' ions which were
found to be activators of the two xylose isomerases. The enzyme assay was
carried out as described earlier except that the concentrations of the divalent
metal ions being studied were varied. A 1.5pdrnl concentration of each metal
free enzyme was used. The kinetic constants for these divalent metal ions
were deduced from the Lineweaver-Burke plot.
.. 3.13 INHIBITION OF D-XYLOSE ISOMERASE ACTIVITY
3.13.1 Inhibition by EDTA
Each enzyme sample (4mdml) was incubated with varying
concentrations of EDTA (0 -200pM) using the normal test mixture for
enzyme assay. The activity of the enzyme was measured as previously
described.
3.13.2 Inhibition by D-xylitol ( sugar alcohol ) and D-lyxose
( 2-epimer analogue)
Each enzyme sample was incubated with test mixtures containing 40mM
D-xylose, 0.4mM MnSOJ in 0.1M TES/NaOH buffer, pH 7 and the different
concentrations of D-xylitol and D-lyxose (0 - 0.2M) respectively. Activities
of the enzymes were measured as previously described. Thereafter, the
inhibition constants of D-xylitol and D-lyxose were determined by canying
out the enzyme assays with different fixed inhibitory concentrations (0, 80,
120, 240mM) of D-xylitol and D-lyxose. The slopes of the lines from
Lineweaver-Burke diagrams were used to derive the inhibition constants
when plotted against their corresponding inhibitory concentrations.
3.13.3 Inhibition of the xylose/glucose isomerase enzymes by CU" in the
presence of ~ n "
The competition of CU'' with ~ n ~ ' in the enzymic assay of each of the two
xylose/glucose isomerases was detenuined. The test mixture containing
different concentrations of MnS04 (2 - 150mM) was assayed for xylose
isomerase activity in the presence of different concentrations of cu2' (0, 5,
15,25mM).
3.14 IMMOBILISATION OF THE XYLOSEIGLUCOSE
ISOMERASE ENZYMES
3.14.1 Polyacrylamide gel entrapment
The method of Hicks & Updike (1966) was used with the exception that
polymerisation was initiated using ammonium persulfate (APS) and
tetramethylethylenediamine (TEMED). Stock solutions were prepared by
dissolving 40g of acrylamide monomer in lO0ml of 0.1M phosphate buffer
pH 7.4 and 2.3g of N,N-methylenebisaclylamide (Bis) in 100ml of the same
buffer. Solutions are stored at 0°C and reactions carried out at 0 - 4°C. lml of
the monomer solution was then mixed with 4ml of the cross-linker solution
and then lml of the enzyme solution (2mglml). To this mixture was added
30p1 of the initiator solution containing lop1 of TEMED and 20p1 of 20%
APS. The initiator and monomer solutions were mixed and the reaction
vessel closed. After 15 minutes, the polymerisation was essentially
completed and the gel was fragmented into small particles (about 0.3cm3).
The gel particles were washed several times with lOmM histidineIHC1 buffer,
pH 6.
3.14.2 Covalent bonding to controlled pore glass (CPG)
BIORAN~ controlled pore glass beads with particle size of 60 -100pm,
pore diameter of 46.4nm, surface area of 85m2g-' and pore volume of
0.83mlg -' were used. Using the method of Messing & Weetal (1970), 1 g of
the clean controlled pore glass beads was added to 70ml of distilled water
containing 91mg of sodium metaperiodate (NaJO,,). The mixture was stirred
for lh at a pressure between 300 and 400mbars. The glass beads were then
washed five times with 350ml of distilled water. The washed glass beads
were mixed with 70ml of distilled water and 77mg of p-phenylene diamine
.2HC1 and stirred for l h at 300 - 400mbars. Thereafter, 70mg of sodium
borohydride was added and stirred for 20 minutes. This step was repeated
three times and the mixture stirred for 20 minutes on each addition. The glass
beads were then washed with 10 - l51nl distilled water. This was repeated
three times. Thereafter, 88ml of 2M HCL and 1120mg of NaNOz were added
and stirred for l h in the coldroom (4°C). The glass beads were washed three
times with adequate volume of ice-cold water (0 - 4°C) followed by the
addition of lml of buffer containing 2mg of the enzyme. After preliminary
mixing, the mixture was kept in the cold-room for 18h with gentle stirring.
The glass beads were thereafter washed with I OmM histidineIHC1 buffer, pH
6 and stored at 4OC until use.
3.14.3 Fixation on cyanogen bromide activated Sepharose 4B
Each purified enzyme (2mg /ml) was immobilised on cyanogen bromide
activated Sepharose 4B according to the method described in Lehrnacher &
Bisswanger (1990a). The gel powder (Ig) was washed with 200ml of 1mM
HCl and suspended in lml of 0.1M sodium hydrogen carbonate, pH 8.3 that
contained 0.5M NaCL and 2mg of purified enzyme. The suspension was
shaken at room temperature for 5 hour. The supernatant was discarded and
then 20ml of 0.2M glycinel NaOH buffer, pH 8.0 was added and the mixture
shaken for another 211 in order to block free cyanogen bromide groups.
3.15 ASSAY OF THE IMMOBILISED XYLOSE ISOMERASES
The xylose isomerase activity of each immobilised enzyme was measured in
a reaction mixture similar to that used for the soluble enzyme. The
immobilised enzyme was added to the test mixture and incubated for one hour
at 70°C in a water bath shaker at 160 rpm. The reaction was stopped by
decanting the reaction mixture from the imnobilised enzyme and chilling
immediately. The xylulose formed was measured by the cysteine-carbazole
reaction method used for the soluble enzyme.
3.16 PROTEIN ASSAY OF IMMOBILISED ENZYMES
The protein content was measured by the method of Bradford (1976) and
specific activity expressed in units per mg protein.
3.17 CHARACTERISATION OF IMMOBILISED ENZYMES
3.17.1 Temperature stability of the immobilised enzymes
The stability of each immobilised xylose/glucose isomerase was tested by
shaking the bound enzyme in the test mixture at different temperatures (4,25,
55, 70°C ) using the same method as described for the soluble enzyme.
3.17.2 pH stability of immobilised enzymes
The pH stability of each immobilised enzyme was tested by incubation at
different pH values (4.5 - 9.5) using the same method as described for the
soluble enzyme.
3.17.3 Half-life study a t 5S°C
The decay rate of each immobilised enzyme was determined at 55°C as
described for the soluble enzyme.
3.17.4 Activity Yield of immobilised enzymes
The activity yield ( O h ) of the immobilised enzyme was calculated as a ratio
) of the determined activity of an aliquot of an immobilised enzyme to the same
aliquot of initial enzyme solution.
Activity Yield (%) = overall activity of imrnobilised enzyme x 100
overall activity of initial enzyme solution
CHAPTER 4
4.0 RESULTS
4.1 Isolation and identification of microorgansms with xylose/glucose
isomerase activity
Using the various isolation and screening techniques described earlier,
eleven microbial isolates (six mesophiles and five thermophiles) capable of
utilising xylose as sole carbon source were isolated from the soil samples.
Eight of these were true bacteria and three were actinomycetes (Tables
6 & 7). There were no fungal isolates.
Six of the bacterial isolates were Bacrllus spp. Among these, isolate RP- 1
was the most active for D-xylose/glucose isomerase (1.3U k 0.03) while
isolates RP-2 and SHC-4 showed the least activity (0.6U f 0.02). The other
two bacterial isolates, Paenrhacrllus sp (X-3) and Alcalrgenes sp (X-4),
isolated from refuse dumps showed the highest activities (2.6U f 0.03 and
2.4U f 0.02 respectively).
Among the actinomycetes, the mesophilic isolate, X- 1 from the refuse
dump had an activity of 1.2U rt 0.02 and was presumptively identified as a
Streptomyces sp. The other two isolates, SHC-1 and SHC-5, were
thermophilic Streptomyces spp with activities of 0.9U rt 0.03 and
0.7U f 0.03 respectively.
Most of the isolates with high xylose isomerase activity were found to be
mesophiles (X-3, X-4, RP-I, X-1).
8 6
Table 6: Isolated Bacteria and their xylose isonierase activilies' - Isohte
Code N o
X - 2
x - 3
X - 4
IZI' - 1
KI'- 2
'SHC - 2
'StIC - 3
'51 1C - 4
Source of Organism
lZefuse Dump
[+fuse Dump
liefuse Dump
Khizosphere
(tomato) plant
Khizosphere
(bean plant)
Self-heating compost
Self-heating compost
Self-heating compost
Suspected
Organism
Bacillus sp ---
t'aenibacillus sp -
Alcal~aencs sp
Bacillus sp --
Bacillus sp
Bacillus sp --
Bxil lus sp
Bacillus sp ---
Cylose
somerase
rctivity (U)"
0.8 + 0.02 11
I Lnzyme activity determined by the cysteine carbazole method of
Disclie and Borenfrelrnd (1951).
' V,ilues represent mean staiiclard deviation of triplicate
dctemi~iations. I I l - ~ r r ~ i i o p l ~ i l i r slrains.
8 7
Tablc 7: Isolated Actinon~~cetes and their xylose isornerase activities'
Isolate
Code No
Source of Organism
Kefuse Dump
Self-heating compost
Self-heating compost
Suspected
Organism
Streptomyces s p
Streptomvces sp
Stre~tomvces sp
Xy lose
Isomerase
activity (U)' -
1.2 * 0.02
I Enzyme activity determined by the cysteine carbazole method of
Dische and Borenft-eund (1 951).
"Vaes represent mean + standard deviation of triplicate
delel-minnlions. I l 'h~l-mopti i l ic strains.
4.2 Selection of strains
X-3 (Paenihacillus sp.) and X- 4 (Alcaligenes ruhlandii) organisms were
found to be the most stable isolates that gave the best yields of the xylosel
glucose isomerase enzyme. The two organisms were mesophiles. The
identities of the X-3 and X- 4 isolates were confinned by the Gennan
Collection of Microorganisms and Cell Cultures "Deutsche Sarnmlung von
Mikroorganismen und Zellkulturen GmbH (DSM)", Braunschweig,
Germany. Based on DNAIRNA homology tests the isolates were confirmed
to be a new strain of Paenihacillus and a strain of Alcaligenes ruhlandii
respectively.
4.3 Purification of the enzymes of Paenibacillus sp and
Alcaligenes ruhlandii
Figures 7 and 8 show the elution profiles of the enzymes of Paenihacillus
sp and Alcaligenes ruhlandii respectively on Whatman DE52 ion-exchange
chromatography. The highest xylose isomerase activity peak for each of the
enzymes corresponded to one of the protein peaks and the enzymes were
eluted between 0.3 and 0.52M NaCl linear gradient with most of the
activities appearing between 0.2 and 0.3M NaCI. The elution profiles of the
two xylose isomerases on Sephacryl S200 HR gel filtration are shown in
Figures 9 and 10. Both xylose isomerase activities corresponded to the most
prominent protein peak. Elution profiles of the two enzymes on phenyl-
Superose hydrophobic interaction chromatography are shown in Figures 1 I
and 12. The PaenibaciNus and Alcaligenes ruhlandii enzymes were not
homogenous after the phenyl-Superose H.1.C step as confirmed by SDS-
PAGE. Homogeneity and satisfactory purification were only achieved for
Alcaligenes ruhlandii and Paenibacillus respectively after the second gel
filtration on Superose 6TM (Figure 15). The elution profiles of the enzymes on
the second gel filtration on Superose 6TM column are shown in Figures 13 and
14. About one sixth and one seventh of the original activities of the
Paenibacillus sp and Alcaligenes ruhlandii enzymes respectively were
recovered. A 6.74 purification factor and a yield of 18.69% were achieved
for Paenibacillus sp enzyme. For the Alcaligenes ruhlandii enzyme, a
purification factor of 10.11 and yield of 13.53% were achieved (Tables
8 & 9).
4.4 Molecular mass determination
The molecular mass of the native enzymes as estimated by the gel filtration
method of Andrews (1967) on Superose 12TM column gave values of 181,000
for Paenibacillus and 199,000 for A1caligenc.s ruhlandii (Figure 16) while
those of the subunits as determined by SDS-PAGE were 45,000 for
Paenibacillus and 53,000 for Alcaligencs ruhlandii. This suggested that each
of the enzymes consists of four subunits per molecule (Figure 17).
4.5 Effect of temperature on enzyme activity
The temperature activity profiles of the two enzymes were studied at 20'
to 90°C. The temperature for maximum activity for Paenibacillus was
Fraction Number
Figure 7: Elution prolila of Paenibacillus sp. enzyme on Whatman DE52
Kay: 0-a - Protoln Absorbance
k--* = Enlyrno Acilviiy
- - - = NaCl solutlon grudlerrf
Fraction Number
Figure 8: Elution profile of Alcaliqenes ruhiandii enzymo on Whalman DE52
C---* : Enzyme Act lv l ly
- - - = NaCl solutlon gradient
0.0 4 8 12 16 20 24 28 32 36 38 40 44 48
Fraction Number
Figure 9: Elution prolile o f h m h a d h sp. enzyme on Sephacryl SPOO HR.
Key: e- 0 = Protein Absorbance 6 *--Enzyme Aclivity
Fraction Number
Figure 10: Elution profile of Alcaligenes rulilandii enzyrne on SephacrylS200 t iR.
Key: e = Protaln Absorbance
Fraction Number
Figure 11: Elution profile of Paer~ibacillus sp. enzyme on Phenyl Superose 515 HR
.Key: = Protein Absorbonce
h--* = Fnzymo Acllvlfy
- - - = (NtlA)2S04 soiutlon gradlont
Fraclion Number
Figure 1 2 Elution profile of Alcaligenes ruhlandii enzyme on Phenyl Superose 515 i i R
Key: e = Pmteln Absotbonce
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 Fraction Number
Figure 13: Elution profile of Paenibacms sp. enzyme on Superose 6 TM
Key: .-q = Protein Absorbclncn
1M Figure 14: Elutlon profile of Alcaligenes ruhlandii enzyme on Superose 6
Table 8: Purification Summary of Paen ibac i l l~ sp xylose isotxerase . --
Purification
Crude extrac
Whatman
DE52
Ammonium
sulphate
fractionation
Volume
(ml)
35.0
52.0
5.0
25.0
3.0
6.0
Total
activity
(mglml)
128.69
100.34
54.1 1
41.70
31.25
24.05
Protein
(mg)
196.00
43.65
22.10
14.89
8.60
5.40
--
Specific
activity
0.66
2.30
2.58
2.80
3.63
4.45
Purification
factor
Table 9: Purification Surnmary of Alcaliaenes ruhlandc xylose
Volume
(lnl)
35.0
58.5
5.0
30.0
2.0
6.0
Purification
Step
Crude extract
Whatmarl
DE52
Ammonium
sulphate
fractionation
Sepliacryl
S-200tlK
Phenyl
Superose
Superose 6
TM
-- Total
activity
(~ngllnl)
11 5.30
85.50
40.60
20.65
18.43
15.60
I
Specific
activity
0.54
1.68
2.18
3.06
5.36
5.46
Purification
factor
-- I I I
10,5 19,O 19,5 20.0 20.5 Elution volume [rnll
Figure 16: Molecular weight determination on Superose 121M gel filtrstion column
Key : X3 = Pacnihacillus sp. enzyme X4 = Alcaligenes ruhlandii enzyme
65°C. Over 60% of this maximal activity was at temperatures between
50" and 75°C. For the enzyme of Alcaligenes ruhland~i, the temperature for
maximum activity was 65 - 70°C and over 55% of this maximal activity
was recorded between 45"and 75°C (Figure 18).
Arrhenius plots of the data of the linear pa ts of the curve (20" to 70°C)
using the Arrhenius equation, K = Ae - EdRT (In K = In A - EdRT) where R
is gas constant and Ea is activation energy, revealed an activation energy of
about 39.61kJmol-' for the Paenibacillus enzyme and42.15kJmol-' for
Alcaligenes ruhlandii enzyme (Figures I9 & 20).
4.6 Thermal stability profiles of the enzymes
Thermal stability studies after one hour incubation period at 4 - 90°C
showed that the enzyme of Pa~aenihacillus retained over 80% of the initial
activity up to a temperature of 60°C while that of Alcaligenes ruhlandii
retained over 80% up to a temperature of 65°C. At 80°C, the enzymes-of
Paenibacillus and Alcaligenes ruhlandii retained 37.5% and 43.44% of
their initial activities respectively (Figure 21).
Half-life studies at 55'C showed that the of Paenibacillus had a
half-life of about 4 days while that of Alcaligenes ruhlandii had a
half-life of about 6 days (Figures 22 & 23). The two enzymes exhibited
exponential decay as a function of time.
1,5 2 0 2 3
Migration distance (crn)
Figure 17: Molecular weight determinatior~ by SDS-PAGE
n n y : ~3 = Pacnik~aciIIus sp. enzyme 1-1 - Alc Ii cnes ruhlarldii enzyme X - a - g
Figure 18: Effect of lemperature on the activity of the enzymes
Figure 19 : Arrhenius plvt for Paenibacilus sp. xylose isomerase
Figure 20: Arrhenius plot for -- Alca- - ruhlandii xylose isomerase
0,o 0,5 1.0 1.5 2 0 2,5 30 3,:
Migration distance [cml
Figure 17: Molecular weight determination by SDS-PAGE
K ~ Y : ~ 3 ' = YceniLmciIIus sp. enzyme X4 = Alcalig~n$~ rutt la~~dii enzyme
Fiyllrtt 21: Effect of temperature on the stability of the enzymes
Key : m-83 = Paenibocillrrs sp. xylose isomerase
Q--4 = Alcoliyerros rrritlantlii xylose isomerase --
I I I I I I I I I I I I r-- 0 2 4 6 8 10 12
Time (days)
0 Figure 22: Enzyme decay at 55 C for Paenibacillus sp. xylose isomerhse
--I-- 0 2 4
Time ( 8
Figure 23: Enzyme decay at 55'C for A_ aliaenes ruhlandii xylose isomerase
4.7 pH activity and stability profiles of the enzymes
The pH activity and stability profiles for each of the two enzymes
followed basically the same pattern (Figures 24 & 25). The optimum pH
for the activity and stability of the etlzyme ofPaenihaciNu.v was 7 and
that of Alcaligenes ruhlandii was 6.5. The enzyme of Paenibacillus retained
over 50% d the initial activity between pH 6 and 8 while that of
Alcaligenes ruhlundii retained over 60% of the initial activity between pH
6 and 8.
4.8 Dependence of enzyme activities on substrate concentration
The concentration of xylose in the test mixture was varied and
Lineweaver-Burk diagrams of the activities at the various concentrations
plotted. From these plots the Km and Vtnax values for the enzyme of
Paenibacillus were calculated to be 26.22mM and 6.07pmolmin-'
respectively while those for the enzyme of Alcaligenes ruhlandii were
62.21mM and 12.87pmolmin" respectively (Figure 26).
Using D-glucose as substrate, the Km values were 590mM and
780mM for the enzymes of lJaaenihacillus and A1caligene.s rzrhlandii
enzyme respectively while the Vmax values were 2. 14pmolmin~' and
6.98pm0lmin-~ respectively (Figure 27).
Figure 24: Effect of pH on Activity and Stability of Paenibacillus sp. enzyme
Kay : C- 9 = Aclivily
= Slabilily
Figure 25: Effect of pl-l on the activity and stability of Alcal~g_encs rulilandii enzyme
Key:
C - - * = Aclivity
a-@ = Slabil i ly
Figure 26: Lineweaver-Burk diagram for the enzymes with xylose as substrate
Figure 27: Lineweaver-Burk diagram for the enzymes with glucose as substrate
Key:
0-0 = Paet~ibacillus sp. enzynie
(B-tp = - Alcaliqsnes ruhlandii enzyme
4.9 Effect of divalent metals
Each of the enzymes after treatment with EDTA showed very neglihle
relative activity in the case of E'aenihacillzrs (0.03%) and no activity in the
case of Alcaligenes ruhlandii. Reactivation of the activities of the enzymes
by addition of various divalent metal ions was studied using xylose as
substrate. Reactivation to various degrees was recorded with ~ g ~ ' , Mn2.',
and co2' . With cazt, ~ i ' ~ , and zn2' reactivation was neglible while cuZi
and VO~' were totally ineffective. M$' ions were the most effective
reactivators for the enzyme of PmnihaciNtrs while Mg2+ ions were the most
effective reactivators for that of Alcali;yene.s ruhlandii (Table 10).
4.10 Effect of various concentrations of divalent metals on
enzyme activity
The activities of the enzymes were studied using various concentrations of
~ g ~ ' , ~ n ~ ' and CO" ions (2 -50mM). Eadie-Hofstee plots of these activities
showed that thc dependence of the reaction velocity on the metal ion
concentration did not follow the normal hyperbolic saturation rule, but
demonstrated a clear biphasic kinetics (Figures 28 & 29). While the first
phase in the lower saturation range (<lOmM) is difficult to visualise because
of very slow reaction rates, the second phase in the higher concentration range
(15-50mM) was quite linear for all active metal ions so that binding constants
(Kd) and the maximum velocities could be estimated
From the Lineweaver-Burk plots of the higher saturation ranges of the
activities for these divalent metals (Figures 30 & 3 I) the values for the
kinetic constants for the metals were calculated as indicated in Table 1 1.
4.1 1 INHIBITION STUDIES
4.11.1 EDTA
A direct dependence of the activities of the enzymes on the concentration
of EDTA was observed. Using 4 nig/ml of each of the two enzymes,
about 200yM of EDTA was needed for complete inhibition of the enzyme
of Paenihacillus while about 165yM was needed for that of Alcaligenes
ruhlandii (Figures 32 & 33).
4.11.2 D-xylitol
D-Xylitol (a sugar alcohol of xylose) was found to be a very strong inhibitor
for both xyloselglucose iso~nerases. At xylitol concentrations of 15mM (for
Pacnibacillus) and 20mM (for Alcaligenes ruhlandii), over 50% of their
activities were inhibited (Figures 34 & 35). The Lineweaver-Burk plots of
the inhibition of both enzymes by xylitol at different fixed concentrations
in the presence of varying amounts of xylose showed that inhibition was
competitive (Figures 36 & 37). A replot of the slopes against the fixed
amounts of xylitol gave inhibition constants (Ki) of 13.8mM for
Paenibacillus and 38.75mM for A1caligene.s uuhlandii (Figures 38 & 39).
4.11.3 D-Lyxose
D-Lyxose (a 2-epimer analogue of xylose) was also shown to be a
competitive inhibitor of the two enzymes though not as strong as xylitol.
About 3001nM (for Paenibacillus) and 450mM (for Alcaligenes nlhlandii) of
lyxose were needed to inhibit over 50% of their respective activities
(Figures 40 & 41). A replot of the slopes of the Lineweaver-Burk diagrams
for the two enzymes which showed competitive inhibition (Figures 42 & 43)
gave the D-lyxose inhibition constants (Ki) as 133mM for Paenibacillus and
2001nM for Alcaligenes ruhlandii (Figures 44 & 45).
4.11.4 Competitive inhibition by cu2+ ions in the presence of ~n'' ions
Studies on the effects of various fixed concentrations of copper ions
(in the presence of varying concentrations of manganese ions) on the activity
of each enzyme showed that cu2' was a strong inhibitor. It tended to
displace the ~ n * ' ions from the enzyme molecules. The biphasic nature of
the isomerisation reaction in the presence of divalent metal ions became less
noticeable with higher CU*' concentrations and the lines tended to meet at
higher ~ g ~ ' ions concentrations (Figures 46 & 47).
118
Table 10: Effect of divalenl melais on metal-free PaenibaciIIus sp. and
A l c a l i g g ~ rul~landii enzyliies - ---
M e l d ion
Iklative Activity % 11 'I
enzyme
Figure 28: Eadie-Hofstee diagram for dependence of
V/(S) X lov
s c s sp. enzyme on divalent met&
Figure 29: Eadie-liofstee diagram for dependence of Alcaligenes rulilandii enzyme on divalent metals.
Key: H.= ~ ~ 2 +
@-a= Mn2-1- O Q = c 0 2 + -
Figure 30: Lineweaver-Burk diagram for dependence of Paenibacillus sp. enzyme on divalent metals.
Key:
Figure 31: Cineweaver-Burk diagram for dependence of Alcaligenes rulilartdli enzyme on divalent metals.
123
Table 11: I<inetic Constnnts*fol the divalent niel<~l ions
- Paenibacillus sp. enzyme --
Kni (mM) Vrnax (prnollmin.)
M$' 11.94 25.38
M I 0.74 34.69
(* Valurs wwc calculakl Crorn figures 30 and 3 1 ) 1
. .. .
Alcalirenes rulilandii enzyme
Km (mM) Vmax @mol/rniii.) -.
Mg" 23.72 36.93 ii
Mn" 39.10 29.28
Co" 12.93 29.10 Co2' 37.80
EDTA conc. (/,{.MI
Figure 32: E f fec t of EDTA on r-'aenibacilk~s sp. enzyme
1V-,-I 0 50 100 150 200
€0-rA conc IIU M I
F iy~~r -e 33: Effect of EO'T'A o n ~ I c a l i ~ e n e s ruhlanclii enzyme
r I I I I -r.- 0 5 10 15 20 25
0-xylitol concentration [mMI
Figure 34: Inhibition of Paenibacillus - sp, enzyme by 0-xyliEol
Figure 36: Lineweaver-Burk diagram of the inhibition of Paenibacillus sp. enzyme by D-xylitol at various xylose concentrations.
Key:
, w = 240rnM D-xylitol =120n1M D-xylitol
g-e = 80mM D-xylitol
0-0 = OmM D- xylilol
Figure 37: Lineweaver-Burk diagram of the inhibition of Alcaliaene? &LI&ILK!! enzyme by D-xylitol at various xylose concentrations.
Key: o--o = 0 mM D-xylitol a- = 80 rnlvl D -xylitol cb-8 = 120 mM U-xylitol 0-0 = 240 rnM D-xylitol
50 100 150 200 250
D-xylitol Concentration [mMl
Figure 38: Replot of the slopes of D-xylitol inhibition of Paenibacillus sp. enzyme.
Figure 39: Replot of the slopes of I
I I I I I
100 150 200 250
ylitol Concentration [mMl
:ylitol inhibition of Alcaligenes ruhlandii enzyme.
Figure 40: Inhibition of Paenib; -
ose conc. [MI
llus - sp. enzyme by 0-lyxose
Figure 42: Lineweaver-Burk diagram o the inhibition of Paenlbacillus sp. enzyme by D-lyxose at various xylo 1 e concentrations.
Key:
0-0 = 0 rnM D-lyxose 0-0 = 200 mM D-lyxose w = 260 mM D-lyxose =-B = 320 rnM D-lyxose
Figure 43: Lineweaver-Burk dia by D-lyxose at vario~
Key: o-a = 320 mM D-lyxose =-I = 260 mM D-lyxose t e = 200 mM D-lyxose
0-0. = 0 mM D-lyxose
rn of the inhibition of Alcaligenes ruhlandii enzyl cylose concentrations.
-50 0 50
Figure 44: Replot of the slop of D-lyxose inhibition of Paenibacillus sp. E
7
-- I
3:
ryme.
Figure 45: Replot of the slopes
-- 150 200 250 300
xose conc. [MI
D-lyxose inhibition o'f Alcaligenes ruhlandii enz:
Figure 46: Eadie-Hofstee plot of the Paenibacillus sp. enzyme
2t . npetitive inhibition of MP by Cu in the ~ction
Figure 47: Eadie-Hofstee plot of tl in the
Key: 0-0 =OmM CL?+ e m = 5mM CL?+ w =15rn~ct?+ m-m = 25mM cu2+
om~eti t ive inhibition of M ~ ~ + by CU*'
zyme reaction.
4.12 Immobilisation of the enzymes
The results of the three immobilisat'on methods are suminarized in Table
12. Both enzymes following immobili ation by the contrOlled pore glass
and cyanogen bromide activated Seph ose 4B methods wsre stable at 4 and
25°C for 28 days. Immobilisation by 1 olyacrylamide gel eptmpment method
showed a steady daily decrease in of both enzymes stored at
4 and 25°C. The enzyme of activity on the 12th day
at 4°C and on the 10th day mhlandii lost
all activity on the 17th day
The highest activity yield
enzyme was achieved by
4B while that for
immobilisation on controlled pore glass
4.12.1 pH stability studies
The pH stability studies showed that aenibacillus enzyt$e retained over
50% of the activity at the pH range 5.5 to 8 after the polyacrylamide gel I immobilisation and pH 5.5 to 8.5 afte immobilisation on aontrolled pore
glass as well as cyanogen-bromide activ ted Sepharose 4B (Figure 48). For
the Alcaligcnes mhlandii enzyme, 50% f the activity was retained at the 'I pH range 5.5 to 8.5 after polyacrylamid gel immobilisation: pH 5.5 to 8.0
after controlled pore glass irnmobilisatio and pH 6 to 8.5 after cyanogen-
bromide activated Sepharose 4B irnmo ilisation (Figure 49). 6
Table 12: Summary o f results of i 4 mobilisation expdriments:
Immobilisation
method
Polyxryl,mide gel entrdpnlent
Controlled pore glass (CPG)
Cyanogen bromide activated sepharose 4 B
specific
activity
2 hours
1 hour
4 hours
4 hauls
3 lho~lrs
5 hours
_i_
Half life at 55OC
3 days
5 days
7 days
G days
7 days
12 days
- P H stabi
optir -
Key: X3 = Paenibacillus sp. enzyme X4 = Alcaliaenes ruhlandi enzyme
Figure 48: Effect of pH on the stabili y of irnmobilised Pa$nibacillus sp. xylose isom t Key:
0-0 =.~olyacrylamide gel entrapmen 0-0 = lmmobillsation on controlled p re glass
0-0 = lmmob~lisation on cyanogen br mide activated sepharose 4B I
4 5 7 8 9
Figure 4 9 : Ef fect of pH on the stability Alca1iE)enes ruhlandii xylose ison
Key:
0-0 = Polyacrylarnide gel u-0 = lrnmobilisation on 0-0 = lmrnobilisation on sepharQse 4B
1'43
1
-I 10
ase
5.0 DISCUSSION
Using the xylose enrichment & sat4 (1961), eleven
microbial isolates consisting of three actinomycete
isolates were obtained. This is notidn that xylosel
glucose isomerase producing in prokqotes
(Bhosale eta / . , 1996).
The true bacteria and actinomycetes nerally employ an lsomerase to
convert D-xylose to D-xylulose The mesoph~lic bact$~ial strain of
Paemhacrllus sp and the mesophilic Alcalrgenes ruhlandr produced
the h~ghest amount of the xylose se enzyme. This IS expected since
bacteria together with fungi ale widely distributed m
natural environment where they contrib to nutrient recyclipg and
humification (Ball & McCarthy, 1988). also agrees with e&lier reports that
xyloseiglucose isomerase production to mesoph~l~c temperatures
(Takasaki, 1966; Chou et al., 1976; These organisms are
therefore potential sources of plant degra ing enzymes and adtivity against
ihe major components of plants has been in most btpteria and
actinomycetes (McCarthy, 1987). I The xylose/glucose isomerase from A1 .aligcnes ruhlandri has completely i
lromogeneous after the fifth purification as reflected by aisingle protein
band in the SDS-PAGE. This agrees wi report of Calleds e ta / . (1985)
on the purification of the of SIreptolmyces
violaceous-ruher using ion exchange
chromatography, gel filtration and hy obic interaction ~hromatography.
The purification factor of the Alcal hfandii enzyme/ (I 0.11) was close
to those reported by Inyang er a/. ( Strepromyces st, (strain PLC)
with a value of 9.3, and by Kwon
alkolophilic Bacillus No.KX-6 wh due of 10.4. hor the xyloset
glucose isomerase of Paenihacillu artial purificaiion was achieved
as evidenced by the presence of a
purification factor (6.74) was hi
(1981) for the xylose isomerase
Chen (1980) only very few xyl erase enzymbs have actually
been purified to homogeneity.
reported a purification factor o
Lehmacher & Bisswanger (19
xy1ose/glucose isomerase of B8. ~ r o m l ~ a w a i el al.
(1994) came a report of pu 1 for the ehzyme of
H$dohacterium adolescent tor (233) $0 far reported
came from Pawar ct al. (19 rase from Chainia sp.
The molecular masses of
method of Andrews (1964) 0 for Paenihacillus sp
and Alcaligenes ruhlandii respectively. Near similar data to 181,000 have
been reported by various workers - 183,000 (Inyang et al, 1995; Yamanaka &
'Tdahara, 1977) and 185,000 (Kasumi et a/, 1981). Equally, other workers
have presented data close to the molecular mass of A1caligene.v ruhlandii
(199,000) for example 196,000 (Lehmacher &
200,000 (Lee & Zeikus, 1991). Those of the
SDS-PAGE method of Laemmli (1970) were
Paenrbac11lu.s sp and Alcalrgenes ruhlatkh
results are confirmatory of the
two enzymes. Various subunit
different xylose/glucose isomerases, for lexample one subu it (Suekane et a[,
1978), two subunits (Kwon el a/, 1987); three subunits (KI ada et a!, 1989; 1 Chauthaiwale & Rao,1994) and four subunits (Callens el a 1985; Lehmacher
& Bisswanger, 1990a; Inyang et al, 1995). T h ~ s study seen1 1 to confilm the
report by Callens et a1 (1985) that xylose/glucose isomeras enzymes with
molecular weights in the lange of 157,000 to 230,000 have tetxameric subunit ! structure and the existence of great variations in their moledular weights of
I depending on the source (Mr. 52,000 to 230,000).
With respect to their subunit masses, each of the enzyme belongs to one I of two different classes, with the enzyme of Parnrbac~llus Isp belonging to
the class with lower (45,000) subunit molecular mass and th j enzyme of
Alcalrgenes mhland~~ belonging to the class with higher (49, 00) subunit
molecular mass. Yamanaka & Takahara (1977) have reporte 8 a subunit
molecular weight of 45,000 for the enzyme of Lactohac~llus #ylosus while
Inyang ct a1 (1995) reported a subunit molecular weight of 44,100 for the
enzyme of Streptomyces sp (strain PLC). Chauthaiwale & R ~ O (1994) and
Lehmacher & Bisswanger (l990a) reported a subunit moleculktr weight of
50,000 for the enzymes of a thennophilic Hacrllus sp and T+rmus aquaticus
respectively while Kawai et a1 (1994) reported a subunit r/lolecular mass of
53,000 for the euzy~ne of H~jdohacterium adolescenti,~. quekane el a1 (1978)
also reported a subunit molecular weight of 56,000 for th$ enzyme of
Streptomyccs 01ivochromogene.s.
The activity of the enzymes increased in a linear mannei with xylose as
substrate up to a temperature of 70°C, remained nearly co stant up to 80°C 4 and declined thereafter, probably due to thermal inactivati4n. From the slopes
of these plots activation energies of 39.61kJmol-I and 42. )5kJmol-' were
calculated for the Pac.nihacil1u.s sp and Alcalrgcnes ruhla$Yii enzymes
respectively. Inyang et a1 (1995) reported an activation en rgy of 32k~mol-' i for the enzyme of Streptonyces sp (strain PLC) while ~ a n & e z & Smiley
d (1975) reported a value of 47.3k~mol" for the enzyme o f , treptomyces albus. i
Lehmacher & Bisswanger (1990b) reported an activation e ergy of
64.8kJmol-' for the xylose/glucose isomerase of Thermus a traticus while Y Smith et a1 (1991) reported a value of 75k~mol" for ~rthrd?hac~er strain
N.R.R.L. B3728.
The temperatures for maximum activity of the xylose/glu~ose isomerases
from Paenihacillus sp and Alcaligenes ruhlandii were 65'C/ and 65 -70°C
respectively. These values differ somewhat from the maxir&m activity
temperature of 60°C recorded for the enzyme of RrJidohacte~iu~n adolescentis
and 75°C for the xylose isomerase of Bacillus coagulans strain HN 68. They
are however, in strong agreement with the report of ~ h o s a l i el al(1996) that
the maximum activity temperature for most xylose/glucose ilsomerases ranges
from 60 to 80°C. Higher maximum activity temperatures have been recorded
for some other micro-organisms. lnyang el a1 (1995) recorded a maximum
activity temperature of 80 - 85OC for the enzyme from Streptomyces sp (strain
PLC) while a maximum activity temperature of 85°C was reported for the
xylose isomerases of Streptomyces griseofufuscu.~ (Kasumi et al, 198 l),
Thermus aqualicus HB8 (Lehmacher & Bisswanger, 1990b) and Clostridi~rm
thermosu[furogen~.s strain 4B (Lee & Zeikus, 1991). A maximum activity
temperature of 105 - 110°C has also been reported for the xylose isomerase of
7'hermotoga maritima (Brown et al, 1993).
Thermostability studies showed that the residual activities of the
xyloselglucose isomerases of Paenihacillus sp and Alca11gene.s ruhlandii
measured at pH 7 after lh incubation in the presence of M ~ ~ ' remained high
at temperatures under 60°C. Enzyme activities were totally lost at
temperatures beyond 80°C. These properties of the purified enzymes from
the two organisms appear similar to those of the enzymes from Lactobacillus
xylosus (Yamanaka & Takahara, 1977), B@Johacteriurn adolescentis (Kawai
el al, 1994) and Bacillus coagulans strain HN-68 (Danno et a / , 1967). The
xylose/glucose isomerase from Streytomyces sp (strain PLC) (Inyang et al,
1995) lost only 12% of its original activity at 90°C. In general, enzyme
thermostability is an intrinsic property determined by the primary structure of
the protein. However, external environmental factors including cations,
substrates, co-enzymes, modulators, polyols and proteins often increase
thermostability (Ward & Moo-Young, 1988). With some exceptions, enzymes
present in theimophiles are more stable than their mesoph{lic counterparts and i xylose/glucose isomerases are known to be generally ther4ostable. It has
Z+ . been reported that M ~ ~ ' and Co either singly or collectiJelY inhibited I thermal denaturation of xylose isomerase from S/reptomycqs ulhus (Sanchez
& Smiley, 1975). I
Enzyme decay studies in this work showed that loss of '1 activity followed
a first order kinetic, exhibiting an exponential decay as a f&tion of time .
! The Puenibucillus sp enzyme had a half-life of about 4 day; at 55'C and that
I ofAlcaligenes ruhlandii had n half-life of about 6 days at s/'c. Studies
carried out on enzyme decay for enzymes from various orgabisms at d~fferent I
I
tempemtures showed that at 70°C, the D-xyloselglucose isjmerase of I Thermus ayuatms HB8 had a half-life of 96h (~ehrnachel& Bisswanger,
1990a) while the enzyme from Slreptomyces sp had a half-11qe of 120h I I ( Chou et al, 1976). Inyang et a1 (1995) recorded a half-life if 18 m~nutes at !
98°C for the enzyme of Streptonzyces sp (strain PLC). The hllf-life report at ,
55°C is of relevance since it approximates the temperature otmost industrial
operations.
The optimum pH for activity and stability of the purified etizymes from I
Paenihacillus sp and Alcaligenes ruhlandii were 7.0 and 6.5 wkspectively. The
pH activity and stability profiles for each of the enzymes fo~dwed basically
the same pattern. The Paenihacillus enzyme retained over 50% of its maximal
activity between pH 6 and 8 whereas the Alcaligenes ruhlandiit enzyme
i retained over 60% of its maximal activity between pH 6 and 8 . ! ~ o t h enzymes I
I were unstable under pH 4.5 and over pH 9.0. This indicates that in the low pH
range (<pH 4.5) and in the high pH range > pH 9), the &cline in activity is
caused essentially by irreversible processes. Callens et all(1986) suggested
that the decrease in activity of D-xyloseiglucose isomerake from I
Streptomycts violaceoruber below pH 7.5 and above pH 9.5 could be I attributed to at least one deprotonated and one protonated iatalytic group.
!
Also evidence for a deprotonated carboxyl (du or Asp) anb a protonated Lys
I has been obtained from modification studies (Vangryspen-e e f al, 1990). In
general, optimum pH for xylose isomerase activity will depend on enzyme !
I
I source and is usually alkaline. Under alkaline conditions, ainon-metabolisable
I sugar D-psicose is produced in hot solution of glucose and kuctose (Bucke, !
1977). Hence, low pH optimum is an attractive property f ~ r enzyme I
i application since there is limited production of D-psicose ad neutral and lower !
I ! pH values. The two enzymes from Paenibacil1u.s sp and ~l&aligenes !
ruhlandii are therefore well suited for industrial application.' A pH optimum
of 7.0 has been recorded for the xylose isomerase enzyme frbm Actino~plunes
missouriensi.~ (Gong el a/, 1980), B&/obacterrum au'ole.sceritis (Kawai et a/,
1994) and S'treptoniyces sp (Iuyang et al, 1995). For C.'lostri&ium
thermosulfurogenes and Thcrmoanaerc~bacfer strain B6A the optimum pH
lies between 7.0 and 7.5 (Lee & Zeikus, 1'991). Other reportid pH optima are
7.5 - 8 for the enzyme from Racillu.s .stearothcmiop~iilu,s (SuQkane et al,
1978); 5.5 - 8.5 for lhermus aquaticus HB8 (Lehmacher & iisswanger,
1990a); 6.5 - 7.5 for i'hermotoga marilinia (Brown et 01, 1943); and 7.5 for
l,actobaci/lus xylo.sus (Yamanaka & Yakahara, 1977). Most Strepromyces
spp xyloselglucose isomerase have higher pH optima as examplified by pH
8.5 for S . ,flavovircns (Vaheri & Kauppinen, 1977) and pH 8 - 10 for
Streptomyces olivochromogene,~ enzyme (Suekane et al, 1978). Other
reports show a pH optimum of 7.5 - 9.5 for the enzyme from Strepornyces
violuceoruber (Callens et al, 1986) and 7 - 9 for the S . albus enzyme
(Sanchey & Smiley, 1975).
The variations in pH optimum may be due to the different buffers used
(Vaheri & Kauppinen, 1977). Yamanaka & Takahara (1977) reported a pH
stability optimum of 6.5 - 11.0 for Lactobacillus xylo.~us and 5.7 - 7.0 for the
enzyme of Lactobacillu,~ brevis enzyme. The neutral to slightly acidic
optimum pH for stability agrees with the values reported in the present study
namely 7 for Paenibacilltrs sp enzyme and 6.5 for the Alcaligcxes ruhlundii
enzyme.
Studies on the catalytic properties of the xylose/glucose isomerases
revealed that the enzymes from both organisms were able to utilise xylose and
glucose as substrates. This is because xylose/glucose isomerase is known to
transfer a proton from the 2-position of D-xylose or D-glucose to the 1-pro-R
position of D-xylulose or D-fructose (Schray & Rose, 1971; Whitlow ef al,
199 1) . Micliaelis-Menten behaviour was observed for both substrates. The
enzyme of Paenihacillus sp had a Km of 26.22mM and Vrnax of
6.07pmol/min for xylose and Km of 590mM and Vmax of 2.14pmol/min for
glucose. For the Alcaligenes ruhlandii enzyme, the Krn and Vmax were
62.21mM and 12.87pmol/min respectively for xylose and 780mM and
6.98pmolhnin respectively for glucose. The Km value for 0aenibacillu.s sp
for glucose is about 23-fold higher than the Km value for xylose and the
Vmax for glucose is about 3-fold less than the value observed for xylose. For
the Alcaligenes nrhlanu'li enzyme, Km for glucose is about 13-fold higher
than that for xylose and the Vmax is about 2-fold less than that observed for
xylose. This shows that for both enzymes, xylose is a prefered substrate to
glucose with respect to kinetic constants.
Comparative analyses of the kinetic properties of the enzymes of
Paenihacillus sp and Alca1igene.s rtrhlandii show that for xylose the Km of
Paenibacillu,s sp is about 3-fold smaller than the Km of Alcaligenes ruhlandii
while the Vmax is about 2-fold smaller than that of Alcaligencs vzrhlandir.
Using glucose as substrate, the Km of the tJaenihacillus sp is about 1.5-fold
less than the Km of the Alculigenes ruhlandi enzyme while the Vmax is about
3-fold less than that of Alcaligen~..~ ruhlandii From these kinetic parameters
one can conclude that the enzyme of Pamibacillu.~ sp is prefered to that of
Alcaligenes ruhlandii.
Various Km values have been reported for xylose isomerases from different
microbial sources. Kawai el a1 (1994) reported Km values of 4mM and
398mM for xylose and glucose respectively for the R@hbucieriurn
adolescentis enzyme. Other Km values iu-e 5mM (xylose) and 920mM
(glucose) for Laclobacillirs brevis enzyme (Yamanaka, 1968); 20mM
(xylose) and 140mM (glucose) for the (~los~rrdiurri fheumo.szrlfurogencs
enzyme (Dekker et al, 1991); 54mM (xylose) and 220mM (glucose) for the
Strtpromyces gr ise~f~~scus enzyme (Kasumi el al, 1981); ImM (xylose) and
90mM (glucose) for the BaciNus cocryulans enzyme ( ~ m & 1970); lODmM
(xylose) and 222mM (glucose) for the enzyme of Hacillrt,~ $~amthcrnwphlphilus
(Suckane et al, 1978); 3SmM (xylose) and 3MlrnM (glucosk) for the
Shvptomyccs sp PLC enzyme (Inyang et a!, 1995). It is inteiesting to note that
from previously reported Km values, the xylose isomerase $nzylrle has
always shown greater affinity for D-xylose than D-glucose. k s should be
expected since D-xylose alone induces xylosc isomerase fo&ation and is
therefore presumed to be the natural substrate. It also reflects, the presumed
physiological function of the enzyme, which acts in other or&nisms to 5 I
produce xyldose, which is subsequently metabolised via the bentose
phosphate pathway or the phosphoketolase pathway {Chen, I 4 ,801. The
preference for xylose as a substrate as observed in the huge differences in the
apparent Michaelis constants can also be explained by reports bf Schray &
Rose (197 I ) and Young er al(1975) who deposed that the a-p)ranose forms
of D-xylose and D-j$cosr are the reactive aldose species (fo4ard reaction).
Makkee ef a1 (1984) used nuclear magnetic resonance (NMR) spectroscopy to
establish the reactive aldose species of both forward and reversel reactions and
concluded that a-D-glucopysanose is the reactive aldose fonn in,accordance
with earlier reports. They also observed that the a-furanose form of
D-fnrctose is initial1 y formed. D-xylose isomerase thus specifical'ly catalyses
the isomerisation of the a-D-ddopyranose foim in the fo~wad reaction
(a-D-xyfopyranose, a-D-ghcopyranose) and of the a-D-ketofurahose form
in the reverse reaction (a-D-xylulose, a-D-fructofuranase) (Van 13astclaere el
I
I a/., 1991). It is known that there is relative abundance ofthe a-anomers (the
prefered forms) in solution (Rangarajan & Hartley, 1992). According to
Angya1(1984), this a-anomer concentration is as follows: k-D-xylopyranose,
36.5% a-D-glucopyranose, 38% and a-D-fructofuranose, 6.5%. Another
I explanation for the lower Km values for xylose is that the $carbon substrate I
a-D-xylose does not experience stearic hinderance due to i&eraction between
0 -6 and Thr 89 (Collyer et al, 1990). Therefore, the observed low Km values
of 26.22mM and 62.21mM for D-xylose for the enzymes of; Paenihacilltrs sp
and Alcaligenes rtrhlandii respectively in comparison to the high Km values
observed for the 6-carbon D-glucose of 59OmM and 7 8 0 m ~ i r e s ~ e c t i v e l ~
should be expected due to lack of this stearic hindrance. Hoiever, Sanchez
& Smiley (1975) have reported that the xylose isomerase fro* Streptonzyces
I albus showed greater affinity for D-glucose (Km 86mM) thad for D-xylose
(Km 93mM) and the significance of this is not yet understood\ I
Like all known D-xylose/glncose isomerases from other organisms , the
activities of the enzymes of Parnihacillus sp and Alcaligenes 4huhlandii were
I strictly dependent on the divalent cations Mg2-, co2' or ~ n ~ ' 'for their
activities and these cations have also been shown to stabilize the proteins
(Chen, 1980). After extensive dialysis against buffer solutions tontaining
EDTA, the two xylose isomerases showed linear decline in enzyme activities
and absolute dependence on these cations. The dependence of the reaction
velocity on the metal ion concentration did not obey hyperbolicisattuation
behaviour but showed remarkable biphasic kinetics. The striking non-linearity
observed in the Eadie-Hofstee diagams for the two enzymes m& be a
reflection of two different binding sites for the divalent cations as observed
already with various other xylose isomerases (Lehmacher & Bisswanger,
1990b; Marg & Clark, 1990; Sudfeldt el al, 1990; Inyang el ul, 1995). In the
higher saturation range the curves approach linearity. This linear range can
be taken as the saturation behaviour of the second low -affinity binding site
after occupation of the first site with higher affinity . Thus the kinetic
constants of this second site were determined for the different metal ions .
There were no significant differences between the efficiencies of Mg2", Mn2'
and Co2" . While the enzyme of Puerribacilkrs sp clearly prefered Mn" ions,
the enzyme of Alculigenc~s ruhlandii prefered M ~ ~ ' ions. Diverse results
were obtained by different workers with respect to the kinetics of the xylose
isomerase reaction. Callens e/ a/. (1986) reported a rapid equilibrium random
mechanism for the enzyme from ,S/rep/omyccs ~~ioluceoruber whel-e the
catalytic step is rate determining in comparison to the fast binding steps .
Their prediction from the effects of different metal ions on the enzyme
activity shows Mg2' to be superior to co2' for catalysis. Rangarajan &
Hartley (1992) also repolted M$' to be the best activator for the xylose
isomerase of Arthrobucter while co2+ and MnZT which bound more stmngly
showed less activity . A mechanism which considers different effects of the
metal ions on the enzyme from Slrepfomyces grisec?jir.scus was reported by
Kasumi et a1 (1982) who showed an independent non-competitive binding for
M$+ and glucose and a synergistic behaviour of Co2' with respect to the
substrate. They also observed a competition of both metal ions for the same
metal binding site on their enzyme. They therefore proposed a rapid
156
! . ~ equilibrium mechanism where the subswate binds to the f h e enzyme while
previous binding of metal ions to the enzyme increases the affinity for the I
substrate. However, two non-identical binding sites per piotein monomer for
the activating cations were established by ctystallographic Studies of the
xylose isomerase from Arthrohacter (Henrick el a/, 1989) 4s well as by
binding studies from Strepto~nyc~.~ vrolaceoruher (Callens 41 a/, 1988). From
the biphasic kinetics observed for divalent cations, it can be~concluded that
the enzyme must be active after binding of one metal ion to ihe high affinity
site , while binding of a second metal ion to the low affinity bite influences
the enzymatic activity. One can assume that only one of the two metal
binding sites is directly involved in the catalytic process as was confirmed by
Carrel1 et a1 (1989) who showed that only one metal ion is in contact with the I
substrate at the active site. Marg & Clark (1990) also provideb evidence for
two distinct metal-binding sites in the enzyme from Racilllrs c+agulans. They
found out that each site binds either MI?' or co2', but simultaDeous addition
of ~ n * ' or CO'' to the apoenzyne indicated that one site pre'ferred the
~ n ~ " (site 1 or A) and the other, CO*' (site 2 or B). The enw& activity
towards glucose for the Bacillus coagdans was highest when both sites
were filled with co2+, whereas the activity towards xylose was highest when
site I was filled with ~ n ' ~ . The presence of metal in site 2 did mot affect the
activity towards xylose. The co-ordination sphere of the two metpl-binding
sites/subunits of the homotetrameric xylose isomerase from Sfrefitonzyces
ruhig~nosics was investigated by Sudfeldt et a1 (1990) who confirmed that the
spectrum of the site (site B) indicated a distorted octahedral
complex geometry and that the spectrum of the low-af5nity site (site A)
showed a distorted tetrahedral or penta co-ordinated complex structure as was
described by Callens el 91 (1988) for the enzyme from S~repiomyces
violaceomrbcr. Two metal binding sites have been identified by Jenkins et al
(1992) who observed that metal site 1 is four-coordinated aid tetrahedral in
the absence of substrate and is &coordinated and octahedral in the presence
of substrate, while the metal site 2 is octahedral in all cases, The work of
Collyer ct al(1992) described the binding of two Bivalent metal cations to the
active site of tlie enzyme and both were believed to be essential for activity.
The cation at site 1 binds and orients the sugars while the cation at site 2 is
directly involved in the isomerisation reaction. A basic solvent rnolecuIe
(Wat 5 19) in the coordination sphere of divalent cation 2 is within hydrogen-
bonding distance of the sugar oxygens and is implicated in the reaction
mechanism.
Crystallographic studies have showtl that keto or aldose ring fonns of the
substrates are bound to the active site of the enzyme (Collyer ef a!, 1990;
Whitlow el a!, 14911, indicating that they are reactive species or interrnidiates
in the reaction (Collyer & Blow, 1990). Nan-linear biphasic saturation curves
have been observed in the case of negative cooperativity or half-site reactivity
for the enzyme from Sirepomyces /hsrmovuigancus (Neet, 1980). These
mechanisms are based on subunit-subunt interactions which have been
observed with both glucose and xylose as substrates for the Srreptoniyces
thrrmovtdgurrcus enzyme and may suggest that such inter- actions are the
reason for this phenomenon.
A direct dependence of the activities of the two xylose isomerases on
EDTA was observed. The straight line extrapolates to abobt 200pM EDTA
being needed for total inhibition of 4mg Iml of the Paenihuci1lu.s sp enzyme
and about 165pM EDTA for the enzyme of Alcaligenes dhiandii. This
means that equivalent amounts of the cation-activators must have been
withdrawn from each of the enzymes for total inhibition tooccur. Since
lpmol of the PaenibaciNus sp enzyme has a molecular mas$ of 45,00Opg, !
therefore 200pM (0.2pmol Iml) of the enzyme will have a Inass of 9mglml
while 165bM (0.165pmol /nil) of the enzyme of Alca1igenc:v rzthlandii
enzyme in which l p o l has a molecular mass of 53,OOOpg dill have a mass
of 8.745 mdml. Since only 4 mg Iml of each of the purified enzyme was
used, this extrapolates for both enzymes to a ratio of approxir/lately 2.2mol
EDTA per mol D-xylose isomerase subunit for complete inhibition thereby
comfirming that two metal ions are bound to each subunit.
D-xylitol, a sugar alcohol of xylose and D-lyxose, a 2-epimer analogue of I xylose were expected to act as competitive inhibitor. The results show that
both analogues were h'uely competitive, with xylitol being a lot stronger
inhibitor than lyxose for the two xylose isotnerases. Lehmacher &
Bisswanger (1990b) showed that the best inhibitors of the xyloselglucose
isotnerases are the C1 and C2 analogues of D-xylose like D-lyxose and D-
xylitol. Therefore, D-xylitol can be regarded as an analogue of the cis-enediol
inte~midiate. In addition to the configumtion at C1 and C2, the position of the
hydroxyl groups at C3 seems to be important for binding. Thus Bentoses with
the same configuration at C3 as D-xylose like D-lyxose aie potent inhibitors. I
The xylitol inhibition constant (Ki ) of l3.8mM for the emzyme of
I F'aenihaciNus sp and Ki of 38.75mM for the enzyme of Alca1igene.s
I ruhlandii were much higher than the Ki of 0.3mM reporl:&d for the i
Arthrohacter B3728 xylose isomerase (Smith eta/ , 1991)and Ki of 1.6mM I 1 for the Streptomyces sp (strain PLC) enzyme (Inyang el al; 1995). The lyxose
I inhibition constant (Ki) of 300mM for PaenihaciNus sp enzyme and Ki of
450mM for the enzyme of Alcaltgenes ruhlandii enzyme are much higher
than the Ki of 20mM reported for the enzyme of Thermus &uaticus I ! (Lehmacher & Bisswanger, 1990b) and Ki of 86 mM for tde enzyme of
Arthrohacrer B3728 (Smith et a/, 1991). Callens et a1 (1916) in their
I inhibition studies on the xylose isomerase of S/repfon?yce.s iwlaceoruher by
I xylitol and sorbitol observed that the inhibition patterns rcs:embled a linear I ~,
mixed type inhibition according to the rapid equilibrium michanism where
i I
substrate and metal randomly combine with the active site a d where only the
ternary enzyme-metal-substrate complex is catalytically active, and that the
inhibition affected either the binding of the substrate or the binding of ~ g ~ ' . i I An important question is whether the enzyme distinguishes between the I
i straight chain or the cyclic hemiacetal f o ~ m of the substrate:. Since I
i carbohydrates exist preferentially in the cyclic form it may be suggested that
I this is the form that is recognised by the enzyme. With X-ray studies Cart-ell
I et a1 (1989) demonstrated the binding of the open chain corhguration of the
sugar to the xylose isomerase from S/reptomyces ruhiginosr~~s. It should be
noted that D-xylose, D-glucose and their analogues exhibit a 1,3-syn-
conformation, which forces their acyclic sugar alcohols inio a sickel-shaped I
conformation. This special form could be of importance ih distinguishing the
substrate. I i
The inhibition of the enzymes by CuZ' was shown to belin competition
with the ~ n " . The biphasic behaviour of the ~ n * ' dread+, shown in figures
is evident in the absence of the CU" inhibitor. With increasing amounts of
Cu2' the low affinity region became more prominent and thk high affinity
I rcgion was reduced. At high concentrations of hln2' the lines appear to
intersect at a point on the ordinate. This is an indication of a competition
between the inhibitoly CU" and the essential ~ n ~ ' at high ~pncentrations. To
make sure that the inhibitoly effect of cu2' is not caused by \a reaction of the
heavy metal ions with essential thiol groups, Lehmacher & disswanger
(1990b) treated their purified enzyme from Thermus aqzraticus with some
I thiol reagents and found no detectable loss of enzymatic activity. They
concluded that their enzyme carries no essential thiol groups b d that the 1 I metal ions interact directly with the catalytic site of the enzyme.
Of the three inunobilisation methods used for the two purified xylose
isomerase enzymes, immobilisation on controlled pore glass gFve the highest
yield of 76.45% for the Paenibucillus sp enzyme whle a yield of 63.6% was
achieved for the Alcaligenes vuhlandii enzytne with the same support. The I I highest yield of 82.6% for the Alcaligenes ruhlandii enzyme was achieved
with the cyanogen bromide activated sepharose 4B while a yield of 69% was
achieved for the Paenibucilius sp enzyme with the same suppoit. The half-life
I at 55°C for the immohilised enzymes werc greatly increased to 7 days fol
Paenihacillus sp enzyme and 12 days for the Alcaligcnes nthlandikir enzyme
and agree with the finding of Strandberg & Smiley (1971) for the immablised
xyIose/glucose isomerase of Streptomyces phueoc~hromogcncs. However, the
stability of the two imtnobifised enzymes at higher temperatures and lower pH
only increased marginally unlike what was observed far the Sfrqtcmyces sp
NCIM 2730 iininobilised on lndion 48-R (Gaikwad & Deshpande, 1992)
There are two cost factors involved in the economic feasibility of using
immobilised xylose isomernse: cost of production of the enzyme, and cost of
inunobilisation, which depends mainly on the type of support and method
employed for immobilsation. The controlled pore glass beads are very
effective and cheap as support material and therefore hold a tremendous
potential fat use in imobilisation of xylose/glucose isomerase.
CHAPTER 6
6.0 CONCLUSION
I . Two mesophilic microorganisms, Pacnihacillus sp and Alcdigenes
ruhlmdii, capable of producing xylose/glucose isomerase &nzymes were
isolated from soil samples in Nsukka, Nigeria. T h ~ s result along wit11 an
earlier one by hyang t.1 n/ (1995) suggest that Nsrikka soiIs\contain
xylose/glucose isomerase producing true bacteria and n ~ t i n o ~ ~ c e t e s .
2. Going by the temperatures for tnaxiinum activity, 65°C and 65 - 70°C for
the enzymes of Pt~enihacillz~s sp and A lcaiigenes ruhlanh reSpectivel y, and
their high relative stability at 60°C, along with other favowable temperature
stability indices, the two enzymes satisfy ttle temperature reqdremcnts for
most industrial enzymes.
3. With optimum pH for activity and stability at 7 (PaembcrciBhs sp ) and 6.5
(Alcnlrgcnes ruhhndii ), both enzymes again seem to satisfy the desired pH
requirements for that class of industrial enzymes.
4. Imrnobilisation of both enzymes on controlled pore glass beads appears to
be the best of the tested immobilisation procedures based on enzyme yield,
conferment of measured stability at 7Q0C and cheapness of the support
material.
5. Both xylose/glucose isomerases were able to utilise D-xylose and D-
glucose as substrates and Michaelis-Menten kinetic patterns were observed
for both enzymes. Enzyme activities were strictly dependent on the divalent
cations ~ n ~ + , ~ g " and co2+. The exact mechanism of binding of these
enzymes to the metal ions is not known. It is therefore recommended that
more electron paramagnetic resonance (EPR) studies be carried out with the
enzymes to elucidate the structures of the enzymes, the temperature
dependence, the binding sites and to reveal the exact mode of dependence of
the enzymes on divalent cations. * v&?#
6 . Since the xylose/glucose Isomerases of the Paenrhacrllus sp and
Alcalrgenes ruhlandri have desirable industrial properties, they are
recommended for use 111 the production of h ~ g h fructose corn syrup (HFCS)
However, this is predicated on the organisms achieving GRAS status.
7. It is further recommended that the cultural characteristics of these two
organisms be subjected to further detailed, studies with a view to optimising
fermentation parameters. In this regard, efforts should also be geared towards
finding a cheaper replacement for the highly expensive xylose. This could
come from rice straw, corn cobs, yam peels, cassava peels and other xylan
containing hemicelluloses. Furthermore, the utility of locally available
nitrogen sources for the formulation of fermentation media should also be a
point in focus.
8. The most potential tool available for us to use in achieving these desired
properties is genetic engineering. We can clone the genes responsible for
these desired properties from several microorganisms with the sole aim of
overproduction of the enzyme by gene dosage effect and engineering of the
xylose isomerase to alter its properties to suit its biotechnological
applications. The other very tedious approach of achieving this is by
continuous screening of our soil environment for microorganisms with the
desired properties.
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APPENDICES
APPENDIX 1
Statistical validation o f treatment effects
The means and standard deviations of replicates of the investigated factors
and controls were computed. The probability values of the t-scores of
differences in means of the factorand control measurements were generated
and used to decide the significance or otherwise of such differences . The
relevant formulae were drawn from Spiegel (1972).
In summary , if - XF , SF and n~ as well as z c , Sc and nc are the means ,
standard deviations and sample sizes of the factor and control measurements
respectively ; - - X - Xc -L--
J n F - 1 ) s 2 F + ( n c - 1 ) s 2 c
s 2 p = nF + n c - 2
6 where
t" F = the observed t-score for factor F
t = the t-statistic on df = nF + nc - 1
S* P = the pooled variance of the factor and control
measurements
p = the prob-value of the score t "F
df = the degree of freedom of the test
Decision Rule :
Significance of factor effect was confirmed at 5% level of error, if
p < 0.05 and non-significant if p> 0.05 .
A,' APPENDIX 2
Stock solutions for SDS-PAGE
1. Acrylamide solution (30%T, 2.7%C ): dissolve 146 g of acrylamide and
4 g of Bis acrylamide and make up to 500 ml. This solution is stable foe
at least 1 month at 4'C .
2 . Separating gel buffer : 1.5 M Tris-HC1 , pH 8.8. This solution is stable for
at least 1 month at 4'C.
3 . Stacking gel buffer : 1 M Tris-HC1, pH 6.8. This solution is stable for at
least I month at 4OC.
4 . Electrode reservoir solution : 0.192 M glycine (free acid), 0.025 M Tris
base, 0.1% wlv SDS. This should be prepared fresh for each
electrophoretic run.
5 . SDS solution : 10% w/v SDS in distilled water. This solution should be
prepared fresh weekly.
6 . Ammonium persulphate : 10% vlv in distilled water. This solution should
be prepared fresh daily.
7 . Sample solubilisation solution (double strength ) : mix 1 g of SDS,
2 ml of glycerol, 2 ml of bromophenol blue tracking dye (0.1% wlv
solution in distilled water), 1.25 ml of 1 M Tris-HC1 , pH 6.8, 2 1111 of
2-mercapto ethanol and make up to 10 ml with distilled water. When ths
solution is diluted to single strength, samples will contain 5% w/v SDS,
10% v/v glycerol , 5% vlv 2- mercaptoethanol and 0.0625 M Tris-HC1 pH 1' k. 6.8. This solution should be prepared fresh each week and stored at 4OC .
Concentration [Prnoll I !
Standard Curve of D-Xylulose
+ Appendix 4
Concentration (Pmol]
Standard curve of D - fructose dissolved in U.1M TES/NaOli pH 7.0
Appendix 5 d
.I .2 .3 .4 .5 .6 .7 .8 .9 1.0 1.1 1.2 1.3 1.4 1.5 ? ,%.,
Concentration [mg/rnl]
Standard curve of Bovine Serum Albumin [BSA) dissolved in water for protein determination using absorbance a t 578 nm af ter 4 minutes
