UNIVERSITI PUTRA MALAYSIA ISOLATION, PURIFICATION...
Transcript of UNIVERSITI PUTRA MALAYSIA ISOLATION, PURIFICATION...
UNIVERSITI PUTRA MALAYSIA
ISOLATION, PURIFICATION AND CHARACTERISATION OF PAPAYA PECTINESTERASE
FAYYAZ ASHRAF
FSMB 1993 4
ISOLATION, PURIFICATION AND CHARACTERISATION OF PAPAYA PECTINESTERASE
By
FAYYAZ ASHRAF
Thesis Submitted in Fulfilment of the Requirements for the Degree of Doctor of Philosophy in the Faculty of Food Science and Biotechnology,
Universiti Pertanian Malaysia
April 1993
ACKNOWLEDGEMENTS
I would l ike to exp ress my deep est gratitude, honour and sincere
appreciation to my chief supervisor Dr. Asbi Bin Ali for his invaluable
guidance, encouragement and constructive suggestions throughout the
s tudy p eriod . His enthus iasm and p atience have left a fee ling o f
indebtedness which can not be fully expressed.
My heartfelt app reciation and gratitude go to the member of m y
supervisory committee, Dr. Hasanah Mohd. Ghazali for her invfJ.luable
advice, guidance, encouragement and time throughout the research p roject
and for reviewing the manuscript and making valuable comments from
time to time.
I extend my s incere gratitude to the member of my sup ervisory
committee, Dr. Yaakob Bin Che Man, for his generous guidance, valuable
comments and encouragement throughout this study.
My deep appreciation is accorded to Dr. Jinap Selamat, a member of
my supervisory committee for her generous g if t of t im e, advice ,
encouragement and also for her help with the statistical analysis and
suggestions in the preparation of this thesis.
I also extend m y sincere appreciation to the Dean and all the staff
members of the Faculty of Food Science and Biotechnology for their kind
iii
cooperat ion and warm behaviour . Specia l thank s ar e d ue to
Dr. Gulam Rusul Rahmat Ali and his family for their kind help in op. e way
or another throughout this study.
I am grateful to Malaysian Technical Cooperation Programme
(MTCP) for awarding me the fellowship to pursue this graduate study.
G rat i tude i s expressed to D r. Chaan Ying Kwok o f Malays ian
Agricultural Research and Development Institute (MARDI ) , for providing
the papaya fruits to conduct this study.
I take pleasure to thank all my friends for providing very pleasant
environment during my study at UPM. Siti Arbaisah Misman is cr edited
for the Bahasa Malaysia translation of the abstract. My regards are due to
Mr. Abdul Aziz Bahsir, M s . Fadzlon Yusof and Mdm . Faridah
Shamsuddin of G raduate School for their kind cooperation throughout
this stu dy.
Finally, I would l ike to express my indebtedness to my father,
mother, brothers and s isters for their moral support, affection and
enc our agement in all my undert ak ing s . L ast , but not least , t he patienc e ,
endur ance , love , sacrific es and understanding o f Humaira and Neha,
helped me to complete this work.
iv
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
L I ST OF TABL ES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix L I ST OF F IGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x
L I ST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
ABSTRAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi
CHAPTER
1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
� L ITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . �
Pectic Substances and Pect ic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pect inesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Occurrence . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I solat ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purificat ion of Pect inesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Propert ies of Pect inesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of Inhibitors on Pect inesterase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Specificity of Pect inesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Stability Studies . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . .
Thermostability of Pect inesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . .
pH Stability of Pect inesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Storage Stability of Pect inesterases . . . . . . . . . . . . . . . . . . . . . . . .. . .
v
�
1�
1�
15
� 1
� 8
35
37
38
38
� 1
� 2
3 PECTINESTERASE EXTRACTION FROM PAPAyA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 43
Introduct ion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Pect inesterase Extract ion at Varying NaCI Concentrat ions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Enzyme Extract ion at Different pH Levels . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . " ' ,' 45
Enzyme Extract ion at Different Incubat ion Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Enzyme Extract ion According to Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Enzyme Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
4 PURIFICATION AND MOLECULAR PROPERTIES OF PAPAYA PECTINESTERASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Enzyme Extract ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Ammonium Sulphate Fract ionat ion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
CM-Sephadex C-50 Column Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Sephadex G-IOO Column Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Enzyme Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 0
Determinat ion of Enzyme Activity in Chromatographic Fract ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Protein Determinat ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1
vi
Analysis of Purity and MOlecular Weight (M. W.) Determinat ion by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1
Analysis of Purity and Molecular Weight (M. W.) Determinat ion by Gel Filtrat ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 62
Non-Denaturing Polyacrylamide Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Results and Discussion . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
5 KINETICS OF PAPAYA PECTINESTERASE . . . . . . . . . . . . . . . . . . . . . . 76
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 76
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Enzyme Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Enzyme Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Effect of Incubat ion Time and Enz>,�e Concentrat ion on Enzyme ACt ivIty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Effect of NaCI on Enzyme Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Opt imum pH for Enzyme Act ivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Effect of Temperature on the Enzyme Act ivity . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Determination of Energy of Acti vatio n a nd QlO Val ues.......................................... 79
Kinet ics Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Result s and Discussion . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 80
6 STABILITY STUDIES OF PAPAYA PECTINESTERASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . 102
Introduct ion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
vii
Enzyme Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 103
Enzyme Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Storage Stability Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Thermost ability Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 04
D and Z Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
Inactivat ion Energy and Q 10 Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
pH Stability Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 105
Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .. . 106
7 SUMMARY AND CONCLUSIONS . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 124
BIBLIOGRAPHy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
V 111
LIST OF TABLES
Table
1 Technological Roles and Applicat ions
Page
of Pectic Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Chain-Splitt ing Pect ic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1
3 Occurrence of Pect inesterase in Higher Plants .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3
4 Purificat ion of Plant Pect inesterases from Different Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
5 Some Properties of Plant Pect inesterases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
6 Some Optimum Values for Plant Pect inesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
7 Comparison of Different Procedures for Extract ion of Pect inesterase from Papaya Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 56
8 Purificat ion of Pect inesterase from Papaya Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 63
9 A Comparison between Pect inesterase Purificat ion from Different Variet ies of Papaya Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 75
10 Rate Const ant s for the Deesterificat ion of Citrus Pectin by Papaya Pect inesterase .. . . . . .. . . . . . . . . . . . . . . . 9 1
1 1 A Comparison of Pect inesterase Properties from Different Variet ies of the Papaya Fruit . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 101
12 D Values (Rates of Inact ivation) in Minutes at pH 4 and pH 7.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 12
13 Rate Constants at Different Temperatures for the Calculat ion of Inact ivat ion Energy at pH 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 15
14 Rate Const ant s at Different Temperatures for the Calculation of Inactivat ion Energy at pH 7.5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 16
1 5 Effect of pH on the Rate Constant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
ix
LIST OF FIGURES
Figure
1 Fragment of a Pectin Molecule and
Page
Points of Attack of Pectic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2 Reaction Catalysed by Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3 Splitting of Glycosidic Bonds in Pectin by Hydrolysis (Polygalacturonase) and by B-E limination (Pec tate Lyase and Pectin Lyase) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . , 10
4 Effect of NaCI Concentration on Pectinesterase (PE) Extraction from Papaya Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
5 Effect of Various pH Levels on Pectinesterase (PE) Extraction from Papaya Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
6 Effect of Various Incubation Times on Pectinesterase (PE) Extraction from Papaya Fruit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
7 Chromatography of Papaya Pectinesterase (PE) on CM-Sephadex C-50 Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
8 Chromatography of Papaya Pec tinesterase (PE) on Sephadex G-I00 Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
9 Elution Pattern of Papaya Pectinesterase (PE) on Sephadex G-I00 Column and Spec ific Activity in the Fractions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
10 Non-Denaturing Polyacrylamide Gel E lectrophoresis of Purified Papaya Pect ineste rase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
1 1 SDS-Polyacrylamide Gel Electrophoresis of Purified Papaya Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1
12 Molecular Weight Determination of Papaya Pectinesterase by Gel Filtration on Sephadex G-100 Column. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 72
13 Molecular Weight Determination of Papaya Pectinesterase by SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
x
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
Effect of Time Course on Purified Papaya P ' A .
. ectInesterase CtIVl ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of Enzyme Concentration on Purified Papaya Pectinesterase Activity . . . . . . . . . .. . . . . .. . . . . . . . . .
Effect of Sodiun Chloride Concentration on Papaya Pectinesterase Activity . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .
Effect of pH on Papaya Pectinesterase ACtIVIty . . . . . . . .. . . . . .. . . . . .. . .. .. . ... . . . . .. .... .. . .. ... .... .. ... .. . .. .
Effect of Temperature on Papaya P ' A
. . ectInesterase C tlVlty . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .
Arrhenius Plot for Papaya Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . .
Arrhenius Plot for the Reaction of Papaya Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lineweaver-Burk Plot of Papaya Pectinesterase Activity as a Function of Substrate Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lineweaver-Burk Plot of Papaya Pectinesterase Activity with Polygalacturonic Acid Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lineweaver-Burk Plot of Papaya Pectinesterase Activity with Alginic Acid Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lineweaver-Burk Plot of Papaya Pectinesterase Activity with Sucrose Inhibitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effect of Temperature on the Stability of Papaya Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermal IQactivation Rates of Purified Papaya pectinesterase at pH 4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermal Inactivation Rates of Purified Papaya Pectinesterase at pH 7 .5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Thermal Destruction Curves for Purified Papaya Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
xi
81
82
83
86
87
89
90
93
96
97
99
108
1 10
1 1 1
1 13
29 Graphic Method for the Calculation of Inactivation Energy of Papaya Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 1 17
30 Effect of pH on the Stability of Papaya Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . 1 18
3 1 pH Inactivation Rates of Purified Papaya Pectinesterase at 30°C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 120
32 pH Inactivation Rates of Purified Papaya pectinesterase at 30°C. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 121
33 Schematic Representation of Effect of pH on the Stability of Purified Papaya Pectinesterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122
xii
LIST OF ABBREVIATIONS
cm centimeter
g gram
xg times gravity
M Molar
mM millimolar
mg milligram
min minute
ml milliliter
mmol millimole
M.W. molecular weight
var. variety
v/v volume per volume
v/w volume per weight
w/v weight per volume
w/w weight per weight
Ilmo1e micromole
Abstract of the Thesis Presented to the Senate of Universiti Pertanian Malaysia in Fulfilment of the Requirements
for the Degree of Doctor of P hilosophy.
ISOLATION, PURIFICATION AND CHARACTERISATION OF PAPAYA PECTINESTERASE
Supervisor
Faculty
BY
FAYYAZ ASHRAF
April, 1993
Asbi Bin Ali , Ph.D.
Food Science and Biotechnology
P ec tinesterase (EC. 3 . 1 . 1 . 1 1 ) is a pectic enzyme that can have a
great impact in the fruit and vegetable processing technology because of its
potential effect on the quality of the finished products . This project w as
designed to extract, purify and study the properties of the pectinesterase
found in the papaya (Carica papaya L. var. Exotica) fruit. Studies
carried out showed that the incubation time, pH and NaCI concentration
influenced the extrac tion of the enzyme from the papaya fruit . A
maximum activity of 7.0 p.mole of carboxyl groups/minlml (7.0 units/ml)
was o bta ined whe n 2M NaCI solut io n (pH 8) a nd a n incu bat io n t ime of
five hours were used. The procedure adopted for purification resulted in a
250-fold purification (784 units/mg protein) w ith a 45 % recovery of the
enzyme ac tiv ity. The enzyme preparatio n w as confirmed to be
homogeneous by gel filtration and non-denaturing polyacrylamide gel
electrophoresis and it has an apparent molecular weight of approximately
32,000 Daltons . The purified enzyme was further charac terised as a
xiv
function of NaCI concentration, pH and temperature. I ts kinetic properties
were also studied . The ac tivity was found to be linear up to 20 minutes
w ith an enzyme concentration of up to 6.42 p.g protein. Maximum activity
was obtained using 0.25M NaCl solution, pH 8.0 and 65 °C. The energy
of activation of the enzyme was calculated to be 5690 cal mot-i. A QiO of
1 .29 was obtained for the temperature range of 30 - 50°C at pH 7.0. The
K value of the enzyme for c itrus pectin as a substrate was 0. 1 125 mg/ml, m
c orresponding to a V max value of 730 p. mole/min/mg proteiri . The
Turnover number was c alculated as 23360 mole/(mole.min) . Inhibition
studies showed that polygalacturonic ac id acted as a c ompetitive inhibitor.
On the other hand, alginic ac id exhibited a c ompetitive-non-c ompetitive
type of inhibition w hile suc rose displayed an unc ompetitive type of
inhibition. The D values (time to inactivate 90% of the enzyme) at 55, 60,
65 and 70°C at pH 4.0 were estimated to be 1 12. 14, 23 .78, 8 . 33 and 1 .7 1
minutes, respec tively. Lower inactivation rates were observed for pH 7 .5 ,
w ith the D values ranging from 143 .27 to 1 .67 minutes for temperatures
between 60 to 75 °C. The Z values, which indicate the rise in temperature
nec essary to observe a 1 0 time faster rate of heat inac tivation, w ere
estimated to be 7 .8°C and 8 .38°C at pH 7 .5 and pH 4.0, respec tively. The
inact ivat io n e ne rg ies a nd QI0 values we re calculated as 256.9 kJimole and
15 .59 at pH 4.0 and 284.76 kl/mole and 19 .21 at pH 7 .5 , respectively. pH
stability studies showed that the enzyme was stable from pH 4-1 1 after
exposure of the enzyme to these pH for 24 hours at 30°C. More than 85 %
of the ac tivity was retained in all of these c ases . However, at pH 1 and
12 , the enzyme was unstable and it c ompletely lost its activity after 24
hours of incubation at 30°C.
xv
Abstrak tesis yang dikemukakan Kepada Senat U niversiti Pertanian Malaysia Sebagai Memenuhi Syarat Keperluan Untuk Ijazah
Penyelia
Fakulti
Doktor Falsafah .
PEMENCILAN, PENULINAN DAN PENCIRIAN PEKTINESTERASE BUAH BETIK
Oleh
FAYYAZ ASHRAF
April, 1993
Asbi Bin Ali, Ph .D.
Sains Makanan dan Bioteknologi
Pektinesterase (EC.3 . 1 . 1 . 1 1) adalah sejenis enzim yang penting
dalam teknologi pemprosesan sayuran dan buah -buah an kerana ianya
memberi kesan kepada kualiti produk akhir. P rojek ini dijalankan untuk
memencilkan, menulinkan dan mengkaj i ciri- ciri pektinesterase yang
didapati daripada buah betik (Carica papaya L. var. Exotica) . Kaj ian
yang telah dijalankan menunjukkan mas a inkubasi, pH dan kepekatan
NaCI mempengaruhi proses ekstraksi enzim daripada buah betik. Aktiviti
yang maksimum iaitu 7 .0 /L mol kumpulan karboksil/minlml (7 units/ml)
diperoleh i deng an menggunakan larutan NaCI 2M (pH 8) dan mas a
inkubasi 5 jam . Melalui kaedah penulinan yang dijalankan, didapati
aktiviti pektinesterase (784 unit/mg protein) adalah mengh ampiri 250
ganda penulinan iaitu 45 % pemulihan. Penyediaan enzim dipastikan
dalam keadaan homogen melalui kaedah penurasan gel dan elektroforesis
gel tidak nyahasli dan mempunyai berat molekul yang ketara menghampiri
32,000 Daltons . Kaj ian selanjutnya dijalankan bagi menentukan kesan
xvi
NaCl, pH dan suhu. Ciri-ciri kinetik juga dikaji . Didapati aktivitinya
linear sehingga 20 minit pada kepekatan enzim 6 .42 p.g protein. Aktiviti
maksimum didapati menggunakan 0.25M larutan NaCl, pH 8.0 pada suhu
65 °C . P engaktifan enzim. ialah 5690 kal mol-I . QIO sebanyak 1.29
diperolehi pada julat suhu 30-50°C pada pH 7 .0 . Nilai Km bagi enzim
dengan pektin sitrus sebagai substrat adalah 0. 1 125 mg/ml bersamaC;ln nilai
V sebanyak 730 p.mollmin/mg protein. Nombor pemulangan adalah max
2 3 3 60 mol l (mol . min) . Kaj ian perencatan menunj ukkan a s id
poligalakturonik bertindak sebagai perencat bersaing, sebaliknya asid
alginik sebagai perencat bukan bersaing dan sukrosa pula perencat tidak
bersaing. Nilai-nilai D (masa untuk menyahaktifkan 90 % enzim) pada
55 , 60, 65 dan 70°C pad a pH 4 .0 dianggarkan selama 1 12 . 14 , 23 . 78 ,
8 . 33 dan 1 .7 1 minit masing-masing . Kadar penyahaktifan didapati lebih
rendah pada pH 7 .5 dengan nilai D adalah pada julat 143 .27 hingga 1.67
minit pada julat suhu antara 60 hingga 75 °C. Nilai-nilai Z iaitu nilai
yang menunjukkan kenaikan suhu yang diperlukan untuk mendapatkan
kadar 10 kali ganda penyahaktifan haba, dianggarkan menjadi 7 .8°C dan
8 . 38 °C pada pH 7 . 5 dan pH 4 .0 masing-masing . Tenaga penyahaktifan
dan nilai QIO adalah 256 .9 kJ/mol dan 15 . 59 pada pH 4 .0 dan 284 .76
kJ/mol 19.21 pada pH 7 .5 mas ing-mas ing. Ka jia n kesta bila n me nu njukka n
bahawa enzim stabil pada julat pH 4-11 selepas didedahkan kepada pH ini
selama 24 jam pada suhu 30°C. Lebih daripada 85 % daripada aktiviti
dapat dikekalkan dalam semua keadaan ini . Walaubagaimanapun, pada pH
1 dan 12, enzim ini tidak stabil dan hilang keaktifannya selepas diinkubat
pada suhu 30°C selama 24 jam.
xvii
CHAPTER 1
INTRODUCTION
Pectinesterase (EC.3 . 1 . 1 . 1 1) is a pectic enz yme and is present in
abundance in many fruits and vegetables (Versteeg, 1 979; Voragen and
P ilnik, 1 989) . The enz yme de-esterifies pectin, producing pectate and
methanol (Rombouts et ai. , 1979) .
P ectinesterase h as a great impact on th e fruit and vegetable
processing technology, because of its potential effect on the quality of the
finished products (Pilnik and Rombouts, 1978a) . Native pectinesterase in
many fruits and vegetables may produce desirable and undesirable effects
before, during or after processing (Versteeg, 1 979) . Endogenous
pectinesterase can be used for protecting and improving the texture and
firmness of several processed fruits and vegetables as well as in th e
extraction and clarification of fruit juices (Voragen and P ilnik, 1989) . On
th e oth er h and, native pectinesterase i s h igh ly undes irable in th e
production of cloudy juices and it must be destroyed in order to maintain
the desired stable cloud and viscosity (Underkofler, 1 972) . Gelation of
papaya puree is also attributed to the enzymatic action of pectinesterase
(Yamamoto and Inouye, 1963) .
Pectinesterase has been purified and characterised from various
p lant sources s uc,h as from tomato (Lee a nd Macmi l lan, 1 96 8;
Nakagawa et al. , 1970a; Pressey and Avants, 1972), Ficus awkeotsang
(Komae et al. , 1989; Lin et al. , 1989), apple (Castaldo et al. , 1989;
1
2
King, 1991), potato (Puri et aI., 1982) , citrus (Manabe, 1973a; Evan and
McHale, 1978; Versteeg et al. , 1978; Korner et al. , 1980; Seymour et al. ,
1991) and it has been found that pectinesterases from different sources are
different in terms of their molecular weight, specific activity, Km values,
stability to heat and pH and their response to pH, cation concentration and
temperature for the enzyme activity. In fact pectinesterase from different
varieties of th e same fruit also differ in th eir properties (Pressey and
Avants, 1972).
Reports available on pectinesterase from different varieties of papaya
fruit have also shown a remarkable difference in their properties (Chang et
al. , 1965; Lourenco and Catutani, 1 984; Lim and Chung, 1989) . Due to
th is variation in properties it seems necessary to purify and characterise
th e enzyme from oth e r variet ies of th e papaya frui t wh i ch are
commercially important. A Malaysian variety, exotica, also belongs to this
group. This variety was introduced in 1987 by the Malaysian Agricultural
Research and Development Institute (MARDI) , Serdang . There has been
much interest shown by entrepreneurs in planting the exotica on a large
scale and 3,240 hectares have been grown in Peninsular Malaysia in 1988
(Ahmad, 1989) .
A number of products can be processed from the papaya fruit and a
large segment of the market of processed papaya is puree (Nordin and
Adinan, 1989) . The puree itself often serves as a source of raw material
from wh ich oth er papaya products are manufactured . Examples of
products wh ich may be made from papaya puree are juices , nectars ,
j am s , j e l l ie s , syrups, toppings and dried fru i t rol l s or l ea th ers
3
(Jagtiani et al. , 1 988) . The other products that can be processed from
papaya are pickled products , deh ydrated products , canned slic�s and
frozen chunks (Nordin and Adinan, 1989).
Presently, th ere is no report available on th e properties of the
p ectinesterase from the exotica variety. Therefore , the objectives of this
study were:
1 . To develop a procedure to extract the pectinesterase from the papaya (variety exotica) fruits .
2. To purify the enzyme and study its physical and chemical properties .
3 . To characterise the purified enzyme and carry out kinetic studies.
4. To carry out stability studies on purified enzyme.
CHAPTER 2
LITERATURE REVIEW
Pectic Substances and Pectic Enzymes
Several literature reviews have been published on the pectic enzymes
(Rombouts and P ilnik, 1 972; Macmillan and Sheiman, 1 974; Rexova
Benkova and Markovic , 1 976; Rombouts and P ilnik, 1 980; P ilnik and
Rombouts , 1978b, 198 1; Whitaker, 1 972; Voragen and P ilnik, 1 989) .
Rombouts and P ilnik ( 1 978) also reviewed th e importance of th ese
enzymes in the fruit and vegetable juice processing technology and the
literature on the industrial production and application of these enzymes
was discussed by the same auth ors (Rombouts and P ilnik, 1 980) . The
s ignificance of the pectic enzymes in fruit and vegetable process ing
(Table 1 ) was reviewed by P ilnik ( 1982) , Voragen and P ilnik ( 1989) and
P ilnik and Voragen ( 199 1 ) . Th is ch apter discusses current information
available in th e l i terature on various aspects of pectinesterase s ,
specifically, and the other pectic enzymes in general .
P ectic enzymes are present in h igher plants and are produced by
some microorganisms (Pilnik and Voragen, 1991) . They act on a variety
of pectic substances wh ich occur as structural polysacch arides in th e
middle lamella and th e primary cell wall of h igh er plants (P ilnik and
Rombouts, 1978b) .
P ectic substances are complex heteropolysacch arides composed
principally of a-1 ,4 linked galacturonic acid or its methyl ester (Figure 1) .
4
Table 1 .
Technological Roles and Applications of Pectic Enzymes (Adopted from Voragen and Pilnik, 1989)
Enzyme (s) Technological roles/applications
Endogenous
PE - Cloud stability in citrus juices - Pectin manufacture from citrus pomace - Distillates from fermented fruit pulps - Enzymatic maceration of fruits and vegetables - Self-clarification of lemon/lime and apple juices - Pressing characteristics of citrus pomace - Ca2+ -firming of fruits and vegetables
PG - Softening during ripening (pears, peaches, avocado) - Texture loss of canned or bottled fruit products
PE + - High viscosity tomato juices and pastes (hot break) PG - Low viscosity tomato juices and pastes (cold break)
- Vegetable juices in combination with tomatoes
Cont.
Exogenous
- Manufacture low-ester pectins with reduced Ca2+ sensitivity
- Apple juice clarification
- Citrus juice stabilisation - Maceration (concentrated nectar bases, baby
food, vegetable juices) - V iscosity reduction of citrus concentrates - Manufacture of low viscosity pectins
- Clarification (apple juice, pear juice, grape musts or wines)
- Enzymic juice extraction (apples, soft fruit, stone fruit)
Cont. Table 1
Enzyme (s)
PE + ,PG
PL
PAL
Endogenous
PG ( + PE and/or PL) +(hemi)-cellulases
PE = Pectinesterase PL = Pectinlyases
Technological roles/applications
Exogenous
- Enzymic oil extraction (olives, palm fruit, coconut flesh
- Depectinizing citrus pulp wash - Clouding agent from citrus peel - Recovery of oil from citrus peel
- Maceration - Clarification; apple juice - Enzymic juice extraction
- Maceration
- Liquefaction; clear and cloudy juices (fruits + berries, tropical fruit, vegetables)
- Cell wall destruction for isolation of interesting cell constituents
- Liquefaction and saccarification of biomass
PG = Polygalacturonase PAL = Pectatelyases
0'\
,-0'
pectinesterase:
COOH
,,0
/pc(tillcstc:rase
COOCH, COIOCH, COOl-!
0/00 OH OH OH 01-1
CO 01-\ COOCH,
OH OH 0°0,°
�O�ygalacturollasc I OH _ r �_� ___ �� __ J pt'Cratc lyase:
COOH
Figure 1. Fragment of a Pectin Molecule and Points of Attack of Pectic Enzymes.
(Adopted from Rombouts and Pilnik, 1980)
01-1 o
-.)
8
P artially methyl-esterified compound is know n as pectin and the
demethylated compound is known as pectic acid or polygalacturonic acid.
The degree of esterification of a particular pectin depends upon its source
and extraction procedure (Macmillan and Sheiman, 1974) . Pectin also
contains varying amounts of neutral carbohydrates such as L-rhamnose ,
L-arabinose and D-galactose along w ith traces of other neutral sugars
(McCready and Gee, 1 960). Var ious numbers of L-rhamnosylpyranose
units are known to be linked through their carbon atoms C-2 and C- 1 in
the main galacturonan chain. More or less of the galacturonate residues
may be acetylated at C-2 and C-3 positions depending on the plant source
of the pectin (Rombouts and P ilnik, 1980) .
Pectic enzymes are classified on the basis of their attack on the
galacturonan part of the pectin molecule as shown in Figure 1 . They are
d iv ided into two main groups , namely deester ify ing enzymes
(pectinesterase) and chain- splitting enzymes (depolymerases) (Pilnik and
Voragen, 1 99 1 ; Voragen and P ilnik, 1 989; P ilnik, 1 990) . Pectinesterases
(PE, EC.3 . 1 . 1 . 1 1) removes methoxyl groups from pectin transforming it
into low methoxyl pectins or pectic acids (Figure 2). The depolymerases
(Fi gure 3, Table 2) spl it the gl yco s id ic l inka ges of the ir preferred
substrates either by hydrolysis (hydrolases) or by 13 -elimination (lyases) .
Polygalacturonases (PG; EC.3 .2 . 1 . 15 and 3 .2 . 1 .67) split the glycosidic
linkages next to free carboxyl groups by hydrolys is while pectate lyases
(pectic acid lyases, PAL; ECA .2.2.2 and 4.2 .2 .9) split glycosidic linkages
by 13-elimination next to free carboxyl groups (Figure 3). They have an
absolute requirement for calcium ions . Both endo- and exo-types of P Gs
and PALs are known. The endo-type (EC.3 .2. 1 . 15 , ECA .2.2.2) split the