CHAPTER 5

CHARACTERIZATION AND APPLICATION OF ENZYMES FROM THE CORM

63

CHARACTERIZATION AND APPLICATION OF ENZYMES FROM THE CORM

OVERVIEW OF THE CHAPTER

Amorphophallus skinned corms were screened for cellulase and polyphenol oxidase (PPO)

enzyme. The extract showed the presence of cellulase and PPO enzyme activity which was

further confirmed by Congo red plate diffusion and catechol agarose plate assay respectively.

The major useful factors in controlling enzyme activity i.e. temperature, pH, chemicals which

can inhibit enzyme action, substrates alteration were checked. Extracted PPO was purified by

acetone precipitation and ion exchange chromatography and characterized using

spectrophotometric methods. Crude enzyme fractions having cellulase activity could clarify

apple juices where as crude enzyme having polyphenol oxidase activity shows dough raising

application.

64

CHARACTERIZATION AND APPLICATION OF ENZYMES FROM CORM

5.1 INTRODUCTION

Plants usually contain 35–50% cellulose on dry weight basis making it the abundant organic

compound in nature. Cellulose is found in most abundance and represents 1.5x10 12 tons of

total annual biomass production through photosynthesis[149]. It is a highly stable polymer

consisting of β-1, 4-linked glucose units[150-151]. Lignin, hemicelluloses and other back-up

substances provide support against cellulolytic activities. Cellulases refer to cellulose-

degrading enzymes with potential to convert cellulosic material into its subunit-glucose [50].

Increasing demand for renewable energy sources has sparked growing interest in enzymes

capable of degrading cellulose to sugars that can then be used for the production of ethanol

[152] Enzymatic hydrolysis is an economic process in the conversion of cellulose to easily

fermentable low cost sugars. Cellulases have been commercially available for more than 30

years, and these enzymes have represented a target for both academic as well as industrial

research[70], [153], [154]. Higher plant cellulases like Lantana camara and Cuscuta reflexa

are mostly involved in fruit ripening and senescence. Cellulases are used in the textile

industry for cotton softening and denim finishing, in laundry, detergent market for

colour care, cleaning, in the food industry for mashing; Cellulase is used for commercial

food processing in coffee and it also performs hydrolysis of cellulose during drying of

beans[155]–[157]. In pulp and paper industries it has found application in deinking, drainage

improvement, and fibre modifications. High cost and low activity are the major impediments

to the commercial use of cellulases [158].

Unfavourable oxidative browning (mainly due to the oxidation of widely distributed enzyme

polyphenol oxidase) occurs in the damaged tissues during drying processes and other

technological operations. Polyphenol oxidase (PPO, a copper-containing enzyme) is one of

the most common browning agents in nature and enzyme catalyzes the hydroxylation of

monophenols to O-diphenols and oxidation of O-diphenols to O-diquinones. Browning is a

prominent problem for elephant foot yam derived products. This undesirable enzymatic

browning occurs in many plants and vegetables and is of a great concern to food

technologists and processors. In plants, research on PPO is mainly focused on its roles in

65

darkening of the damaged tissue along with browning [159]. Poly-phenolic compounds are

closely related to the sensory, aroma and nutritional qualities of plants. Storage, processing

and handling also leads to browning as demonstrated in the case of potato tubers [160],

artichoke [161][162], yacon roots [163], lettuce [164] and green beans[165] making them

less appealing to consumers and therefore results in the loss of their marketability.

This aroid Amorphophallus paeoniifolius has not been explored much; the identification of

cellulolytic enzymes and PPO enzyme from corm is unique work and can be commercially

harnessed by the fruit juice industry and bakery industry which is among the largest agro-

based industries worldwide. High juice yield is an important goal for juice production. Many

modern processes for fruit and vegetable juice production employ enzymes as important

processing aids to obtain higher yields and clarity[166].

This study will not only provide some fundamental understanding of the PPO and cellulase

enzyme of Amorphophallus but also provide a new enzyme source for different industrial

applications.

5.2 MATERIALS AND METHODS

5.2.1 Plant material and Chemicals

A. paeoniifolius corms were purchased from local vegetable market at Noida, India. Peels

were separated and processed separately for enzyme and phytochemical extraction[40],[167].

In this study, the peeled corm was washed under running water for 30 min to remove surface

contamination and then chopped into small cubes of 4mm, rinsed with distilled water and

stored at 4°C. Pyrocatechol, ascorbic acid, EDTA, D and L Tyrosine was procured from

CDH, New Delhi. PVP (Polyvinylpyrrolidone), potassium mono-hydrogen phosphate and

potassium di-hydrogen phosphate, sodium dodecyl sulphate (SDS), dithiothreitol (DTT) were

procured from Sigma Chemical Co (St. Louis, USA). Macro-prep DEAE support were taken

from BIO RAD. All the other reagents used were of analytical grade.

66

5.2.2 Preparation of corm homogenate as enzyme source Stored peeled corm was cleaned with distilled water, the weighed corm was chopped into

small pieces and homogenized in ratio of 1:3 with 0.1 M Citrate buffer, pH 5.0 for cellulase

enzyme and for PPO A. paeoniifolius peeled corm was homogenized in chilled pH 7.0, 0.1 M

phosphate buffer in a ratio of 1:3 along with 1% of polyvinylpyrrolidone (PVP). All the steps

of enzyme extraction were carried out at 4°C. Crude extract was centrifuged at 10,000 rpm

for 30 min at 4°C in a refrigerated centrifuge to obtain a clear supernatant that was used as

the enzyme source. The pellet was discarded whereas the supernatant was filtered through

Whatman No.1 filter paper and stored at -20°C. Protein and enzyme activity was estimated

for this extract prior to storing at -20°C.

5.2.3 Purification of the PPO extract

The crude protein extract was precipitated out by slowly adding (−20°C) pre chilled acetone.

This solution was stored at 4°C for 2 h. The acetone precipitate was collected and left for 24

h at 4°C to remove acetone. The precipitate was re-suspended in 100 mM phosphate buffer

(pH 7.0), stirred for 10 min and the suspension was centrifuged at 5000 rpm for 10 min. The

supernatant was pooled and loaded on DEAE (Diethylaminoethyl) anion exchanger (Macro-

prep DEAE support) column that had been pre-equilibrated with 100 mM phosphate buffer

(pH 8.0). Unbound of DEAE was collected in fractions of 2 ml each. Elution was done with

buffered solution (pH 8.0) containing 10, 20, 50 mM NaCl. Eluate fractions (2ml) were

collected and dialysed to remove salt. Both unbound and dialysate were assayed for

polyphenol oxidase activity. Active enzyme fraction were pooled and stored at -20°C.

5.2.4 Measurement of the enzyme activity

5.2.4.1 Carboxymethyl-cellulase (CMCase) and Filter-paperase (FPase) enzyme

activity assay

CMCase and FPase activity was assayed using a modified method described by (Wood and

Bhat, 1988),[168] with some modifications. 0.1 ml of supernatant was added to 900 μl of 0.1

M Citrate buffer, pH 5.0 and 1.0 ml of 1% CMC (for CMCase) and Whatman no. 1 filter

paper strip (1 x 3 cm, 25 mg) (for FPase) was added in a test tube and incubated at 50°C for

60 min. The reaction was terminated by adding 3.0 ml of 3, 5-dinitrosalicylic acid (DNS)

reagent and by subsequently placing the reagent tubes in water bath at 100°C for 15 min

67

(Miller, 1959). One ml of Rochelle salt solution 40% was then added to stabilize the colour.

The absorbance was recorded at 540 nm against the blank (without enzyme filtrate). One

international unit of CMCase and FPase activity was expressed as 1 μ mole of glucose

liberated from per ml enzyme per min under assay condition.

5.2.4.2 Polyphenol oxidase enzyme assay

PPO activity was determined spectrophotometrically by measuring the initial rate of quinone

formation, which is indicated by an increase in absorbance at 420 nm. The reaction mixture

contained 1000 µl of 0.1 M phosphate buffer (pH 7), 50 mM catechol solution, and 100 µl of

the enzyme solution and 900 µl of distilled water. The reaction was carried out for 15 min at

35°C. One unit of PPO activity was defined as the amount of enzyme that caused an increase

of 0.001 unit of absorbance per minute under standard assay conditions.

5.2.5 Protein assay Protein concentrations were determined according to the dye binding method of (Bradford,

1976) [169], using bovine serum albumin as standard

5.2.6 Congo red plate diffusion assay The evaluation of cellulase enzyme was identified employing the Congo red plate assay as

described by (Teather and Wood, 1982) [170]. Agar plates were prepared containing 0.5%

carboxy methyl cellulose incorporated into 1% w/v agar in milli Q water. The experimental

steps were conducted in sterile condition in an incubator set at 50°C. The 50 μl enzyme

sample was applied onto each agar well. Following a 24–48 hours incubation period at 50°C,

the wells were washed off with distilled water and were stained with Congo red solution for

30 min. The gels were soaked in 1 M NaCl until clear yellow zones were detected.

5.2.7 Polyacrylamide gel electrophoresis and Activity staining of cellulase

SDS-PAGE was performed on a slab gel containing 10% (w/v) polyacrylamide by the

method of Laemmli (1970) [171]. The gel was stained with Coomassie brilliant blue (0.5%

w/v) for 30 min and de-stained in 10% methanol and 5% acetic acid for a limited period of

time.

68

The activity staining method was performed by modified method as reported by Trudel and

Asselin (1989) [172]. Carboxymethyl cellulose (0.5% and 1%) substrate was boiled and

incorporated into the analytical 10% (w/v) polyacrylamide gel. The isolated protein samples

were run in the substrate incorporated gel at 100V constant voltage for 2-3 hours. After

electrophoresis the gels were soaked in 0.1 M citrate buffer pH 5.0 for two 15 min changes

for 24 hours to renature the enzymes in gel. The polyacrylamide gels were stained for 30 min

in Congo red and de-stained in 1 M NaCl for at least 30 min. Bands with lytic activity

appeared as clear zones against bright red background.

5.2.8 Gel diffusion assay for polyphenol oxidase quantification

Agarose (1.5% (w/v); phosphate buffer pH 7.0) was heated in microwave oven for 5 min, the

solution was gradually cooled to 50°C and 50 mM catechol was added [173]. 25 ml of this

buffered solution was transferred into round petri dishes, cooled at 350 C and stored at 4°C in

dark conditions. Wells of 5 mm diameter were created using a cork borer and gel plugs were

removed. Crude enzyme extract (20-100 ul) was loaded into each 5 mm well and the effect of

altering concentrations on sensitivity and linearity of the gel diffusion assay was studied by

monitoring the dark ring diameters formed after 12 h of incubation. Plates were covered and

left for incubation at 35°C in incubator for 12 h. The diameters of the intensely stained zones

were also recorded.

5.2.9 Detection of PPO activity by SDS PAGE electrophoretic-blot technique

Preparation of Catechol paper

3mm chromatography (15x12 cm) paper was immersed in 50 mM catechol solution for 10

min. The catechol paper was dried at 350C for 15 min and stored in dark condition till further

use.

SDS PAGE and Electrophoretic-blot

20 µg of protein from crude, acetone and unbound fractions were loaded on 10% SDS PAGE

gel. For determining the molecular weight of A. paeoniifolius PPO enzyme, the gel was

checked for activity staining by the electrophoretic blot process. After electrophoresis, one

69

part of gel was stained with Coomassie Blue R-250 to determine the molecular weight

whereas the other side of the glass plate along with the gel was removed and immediately

blotted onto the top of the dried catechol paper. A dark activity brown band developing

within 5- 10 min validates the presence and molecular weight of PPO [174].

5.2.10 Characterization of cellulase 5.2.10.1 Effect of temperature, pH and various metal ions on enzyme activity The cellulolytic activity of the crude enzyme was measured at different temperatures (30 -

90°C), pH values (2 – 10) and with different metal ions (100 mM). The pH was adjusted

using the following buffers (0.1 M) of citrate buffer (pH 2.0- 6.0), Tris (pH 7-10). The

reaction mixture was pre-incubated for 15 min with all the mentioned temperature, pH and

metal ions. After pre-incubation the activity of the enzyme was measured as per standard

assay conditions.

5.2.11 Determination of the PPO enzyme properties

5.2.11.1 Effect of pH and pH stability on PPO activity

The effect of pH on PPO activity was determined under standard assay conditions using 0.1

M hydrochloric acid potassium chloride buffer (HCl-KCl) ( pH 2), 0.1 M citrate buffer (pH

3-6), 0.1 M potassium phosphate buffer (pH 7-8), 0.1 M Tris-HCl buffer (pH 9). The blank

contained only substrate solution and the reaction buffer. The optimum pH and the enzyme

activity were expressed as the percentage of maximum enzyme activity under standard assay

condition.

The pH stability of the enzyme was determined according to a modified method of (Onsa et

al. 2000) [175]. 50µl of enzyme solution was incubated with 1 ml of different pH as

described for optimum activity for 24 h at 4°C, and the residual activity was determined and

calculated as relative activity (%) to the initial activity.

5.2.11.2 Effect of temperature on PPO activity

The buffered enzyme solution was pre-incubated in a water bath at different temperatures

(35°C to 75°C) for 10 minutes prior to addition of substrate 5mM catechol. Relative enzyme

70

activity was calculated as the percentage of the highest activity left after temperature

treatment.

5.2.11.3 Effects of inhibitors

Sodium azide, sodium chloride, ascorbic acid, citric acid CaCl2, ZnSO4 and EDTA were

used as PPO inhibitors. The reaction mixture contained 1 ml of 0.1 M phosphate buffer, pH

(6.9), 950ul of 100 mM inhibitor, 0.50 ml of enzyme extract and 1 ml of 50 mM catechol.

Percentage inhibition was calculated using the following equation:

Inhibition (%) = (Ao - Ai/Ao) 100,

Where Ao: initial PPO activity (without inhibitor) Ai: PPO activity with inhibitor

5.2.12 Application of crude extract

5.2.12.1 Apple juice clarification having cellulase activity

Crude enzyme extract having cellulase activity (30units) was incubated with 5ml of freshly

extracted apple juice, filtered through three layers of cheese cloth for 1 hour at 50°C. The

contents of the tubes were stirred well in order to mix the enzyme with juice. The reaction

was performed in water bath. Clarification of juice was observed after 50 min. Test for starch

and pectin was carried out to test the effectiveness of the enzyme treatment (results not

shown).

5.2.12.2 Dough rising application of crude extract having PPO activity

All the ingredients (salt, sugar, yeast and wheat flour) required to make dough were weighed

and mixed thoroughly. Ingredients were kneaded in water along with 1 ml of crude enzyme

extract (having PPO activity) to form dough whereas control has enzyme blank. The dough

mixture was transferred to marked beaker and kept at room temperature. Rise in dough level

was constantly monitored.

71

5.3 RESULTS AND DISCUSSION

5.3.1 Cellulase from stored Amorphophallus paeoniifolius in clarification of

apple juice

Cellulase enzyme was isolated from corm of Amorphophallus and enzyme activity was

estimated by the method of (Wood and Bhat, 1988)[168]. Optimum pH, temperatures were

determined and investigation was carried out to determine the role of metal ions on enzyme

activity. Optimum temperature for CMCase and FPase both was found to be 60°C as shown

in Figure 5.1(a). Thermal stability studies suggested that cellulase extracted from corm

retained activity even at 90°C. CMCase activity was found to be optimum at pH 10

whereas FPase activity was found to be optimum at pH 5, as shown in Figure 5.1(b).

CMCase retained its activity at both acidic and basic pH but interestingly at pH 7 both

the CMCase and FPase activity decreased.

Figure 5.1: Effect of (a) temperature and (b) pH on enzyme activity CMCase and FPase. Maximum specific

activity obtained for the enzymes was taken as 100% for the calculation of relative activity.

Congo red plate diffusion assay confirmed the presence of cellulase activity in the isolated

enzyme fraction and the clear zone were visualized under white light illumination. Control

with distilled water shows no clear zone. Radial diffusion of enzyme into CMC incorporated

gel produces hydrolysis zones, visualized by staining with congo red dye (see Figure 5.2).

72

Figure 5.2: Congo red plate diffusion assay

1% Agar along with 0.5% carboxymethyl cellulose plates were prepared. 50 microliters of crude extract was

loaded in wells and plates were left for incubation for 24–48-h at 50 °C, the wells were washed with distilled

water and stained with Congo red solution for 30 min., followed by washing in 1 M NaCl. Presence of clear

yellow zones around the wells 1-4 confirmed the presence of CMCase activity. C- control with distilled water

Polyacrylamide gel electrophoresis and zymography studies where substrate CMC was

polymerised in gel yielded bands with lytic activity after staining with Congo red as dark

zones shown in Figure 5.3. The molecular weight of the enzyme is 66 kDa as determined by

the zymographic studies.

73

Figure 5.3: SDS PAGE (a), Native(b) and Zymography(c) of crude enzyme

M: Marker, lane A1 is SDS PAGE analysis of crude extract, lane 1 is the Native gel analysis of the crude

extract, in lane 2 (substrate CMC 1%) and lane 3 (substrate CMC 0.5%) was polymerised in the native gel.

Clear zones in lane 2 and 3 indicate that molecular weight of enzyme is around 66 kDa.

a)

b)

c)

74

Investigation was also carried out to determine the role of metal ions in the stabilization of

cellulase enzymes.

Figure 5.4: Effect of metal ions on cellulase specific enzyme activity

Enzyme was incubated with various metal ions and control without the metal ions was also

taken. CMCase and FPase specific enzyme activity was calculated as shown in Figure 5.4.

Control specific enzyme activity (without the addition of metal ions) was taken as 100%.

Mn2+ has strong inhibitory effect on both CMCase and FPase, whereas Mg2+ activated the

CMCase activity maximum and Fe3+ strongly activated FPase activity.

This crude fraction rich in Cellulase was also checked for apple juice clarification. The

enzyme extract, clarified freshly extracted apple juice. Freshly extracted and filtered apple

juice was incubated with the enzyme extract in the ratio of 5:1 for 1 hour at 50°C.Assay was

done in triplicate and the clear juice was obtained after 1 hour of incubation with the enzyme

as compared to the control tube which did not contain the extract (see Figure 5.5).

0

50

100

150

200

250

Control MgSO4 CuCl 2 FeCl3 MnSO4 NaCl KCl HgCl2

Spe

cifi

c e

nzy

me

act

ivit

y %

Metal ions

CMCase

FPase

75

Figure 5.5: Clarification of apple juice with crude enzyme extract of Amorphophallus corm

Test tube no 1-3 shows clearing of apple juice as compared to the control tube (no 4) which did not contain the

extract.

Carbohydrases level increase on storage and ageing of tuberous crop and we report that these

native enzymes of Amorphophallus corms can degrade polysaccharide material of apple juice

thus facilitating clarification.

5.3.2 Screening, purification and characterization of Polyphenol oxidase

from A. paeoniifolius corm

5.3.2.1 Extraction and partial purification of PPO

Poly phenol oxidase enzyme from corm of A. paeoniifolius was extracted in 0.1 M phosphate

buffer pH 7 and 1% PVP followed by acetone precipitation and ion exchange

chromatography by using DEAE. Undesirable reaction between the natural phenols and PPO

was prevented by adding 1 % polyvinylpolypyrrolidone (PVP). PVP is a phenol adsorbing

agent and added to the extraction buffer to remove phenolic substrates due to its binding

ability to the phenolics in order to prevent phenol-protein interactions. After acetone

precipitation the pellet was suspended and loaded onto a DEAE-cellulose ion-exchange

76

column. Activity rich DEAE unbound fractions were pooled and dialysed prior to studying

its kinetics. DEAE eluates of NaCl 10, 20 and 50 mM did not show any enzyme activity.

We report yield of 18.92 and 1.24 fold purification after acetone precipitation and unbound of

DEAE gave 5.54 fold purification (see Table 5.1).

Table 5.1: Purification of PPO from Amorphophallus paeoniifolius

Purification step Volume

Protein (mg/ml)

Total protein

Activity (U/ml)

Specific Activity(U/mg protein)

Total activity Yield

Purification fold

Crude extract 50 0.074 3.675 833 11333.33 41650 100 1 Acetone precipitation 4 0.14 0.56 1970 14071.43 7880 18.92 1.24 Unbound DEAE Anion Exchange chromatography 2.25 0.010 0.0212 590 62765.96 1327.5 3.19 5.54

Catechol agarose gel diffusion assay for quantification of PPO, Crude enzyme (20, 40, 50,

60, 100 ul) was added in the wells and kept for 12 hours along with control i.e. enzyme blank

well. Diameters of the oxidized zones (area turning black in colour) were measured. Graph

was plotted between the log units of the enzymes and the diameter of the oxidized circles. On

generation of standard curve linear relationship found between the diameters of dark oxidized

circles formed in the gel and the logarithm of the enzyme activity units added to the well,

with correlation coefficient of 0.8921. (see Figure 5.6 a & b).

77

Figure 5.6: Identification and quantitation of PPO activity. (a) catechol agarose plate assay: 5 mm diameter wells was loaded with crude enzyme (20-100 ul) incubated at

35°C in for 12 h in dark.

(b) Diameter (cm) of the dark circles formed was measured and the standard curve between the logarithm of

PPO activity and the diameter of stained zones formed on catechol agarose plates (y = 0.6478x + 0.1475

R² = 0.8921

5.3.2.2 pH optimum and stability

The effect of pH on PPO activity was determined by estimating enzyme activity at different

pH 2.0- 9.0 using catechol as substrate. We report that Amorphophallus PPO shows optimum

activity at pH 6.0 (see Figure. 5.7). The activity was found to increase upto pH 6.0 after

which it dropped considerably giving a bell-shaped curve. Our results were comparable to

PPO activity from other plant sources for example -the pH optimum of 6.5 for apple, pH 7.0

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 0.5 1 1.5 2 2.5

Dia

met

er(c

m)

Log enzyme activity (U) b)

a)

78

for artichoke and Barbados cherry respectively.[162], [176], [177] .The enzyme activity

gradually decreased as the pH was shifted either to extreme of alkaline or acidic region. The

enzyme was inactivated at pH 2 but maintained its activity at pH 8; showing relative activity

of 30.57%. Generally, vegetables and fruits show maximum activity at or near neutral pH

values, but these values may vary with the source of enzyme and substrate within a relatively

wide range of pH.

Figure 5.7: Optimal pH requirement as well as effect on stability of enzyme on incubation for 24h at different

pH

Variation in optimum pH of PPO from different sources affected by factors such as cultivar,

nature of phenolic substrate, type of buffer and ionic strength of buffer, purity of enzyme,

extraction method and isoenzyme form [178], [179]. The stability of PPO was measured by

incubating the enzyme in different pH conditions at 4°C for 24 h. The pH stability of an

enzyme is considered as an important parameter for the determination of the conditions that

should be available through isolation, purification, handling and storage of the enzyme.

Protein structure of an enzyme molecule is influenced by the acidity or alkalinity of the

solution due to pH change as its various amino acid residues are in different states of

ionization. As shown in (Figure 5.7) enzyme from A. paeoniifolius when incubated at pH 5.0

retained more than 80% of its original activity. Maximum activity was found at pH 6.

However, the activity reduced to less than 45% of its original activity when incubated at pH

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0 1 2 3 4 5 6 7 8 9 10

Re

lati

ve e

nzy

me

act

ivit

y %

pH

pH optimum

pH stability

79

below 5.0 and retained 20% above 7.0. Therefore, acidic and alkaline conditions can be used

as a potential measure to control browning reaction in Amorphophallus corm.

5.3.2.3 Temperature optimum for PPO

An optimum temperature is necessary for the enzyme to achieve maximum activity. In

practical production, an efficient way to inhibit enzyme activity is avoiding proper

temperature of reaction [180]. Figure 5.8 shows the effect of various temperatures on PPO

activity. Result shows the optimum temperature of PPO was 35°C and its activity decreased

gradually as temperature increased. Optimum temperatures requirement for PPO of butter

lettuce is reported to be at 35oC[164], 40°C for Barbados cherry [177] and 25°C for artichoke

[161].

Figure 5.8: Optimal temperature requirement for A. paeoniifolius PPO

5.3.2.4 Effect of inhibitors on PPO activity

There are a number of inhibitors, such as sodium metabisulphite, L-cysteine, sodium azide,

tannic acid, benzoic acid [181], ascorbic acid [166], used by the researchers to prevent

enzymatic browning. The effect of seven inhibitors - L-ascorbic acid, sodium azide, NaCl,

CaCl2, ZnSO4, EDTA, Citric acid are shown in Figure 5.9. L-ascorbic is the most potent

inhibitor of A. paeoniifolius PPO. These inhibitors are known for their ability in modifying

the pH; monovalent cations in association with divalent cations are known convert insoluble

80

pectins to soluble pectins in the middle lamella of cells[182]. Ascorbate acts as an antioxidant

also by reducing the initial quinine formed by the enzyme to the original diphenol before it

undergoes secondary reactions which lead to browning.

Figure 5.9: Effect of inhibitors (100mM) on enzyme activity respectively

5.3.2.5 Molecular weight determination of Amorphophallus corm PPO

SDS PAGE and the semi native SDS PAGE ( protein sample was diluted with sample buffer

devoid of β-mercaptoethanol and loaded without heat treatment) revealed that the

approximate molecular weight of PPO to be 40 kDa. Figure 5.10(a) depicts the SDS PAGE of

the samples. Figure 5.10(b) shows the electrophorectic blot in which the native gel was kept

on dry catechol paper along with the glassplate for 5 minutes. Single dark brown band was

clearly visible in all the 3 lanes instantly. These bands confirm the presence of PPO in all the

three fractions (C; crude enzyme extract, A; acetone precipitated fraction, U; unbound

fraction of DEAE). Molecular weight of the enzyme can also be estimated very accurately by

this blot zymography studies. Polyphenol oxidase molecular weight ranges between 45 to 67

kDa as reported by many authors [183], [184]

81

a) b)

Figure 5.10: Detection of PPO activity by SDS PAGE(a) and electrophoretic-blot technique.b).

b)Single dark brown band was clearly visible in all the 3 lanes (C, Crude enzyme extract; A, Acetone

precipiated fraction; U, unbound fraction obtained after DEAE ion exchanger) depicts that the molecular weight

of A. paeoniifolius polyphenol oxidase is 40 kDa .The native gel was kept on dry catechol paper along with the

glassplate for 5 minutes.

5.3.2.6 Application in dough rising

Previous studies have reported that polyphenoloxidase affects the dough matrices by

affixing dough enhancement additives to the bread dough, exerting an oxidizing effect to the

dough, strengthening gluten bonds, increasing volume, reducing stickiness which results in

improved freshness of the bread texture, softness of the baked product, improved crumb

structure, flavour and the improved machinability[128]. Role of crosslinking of esterified

ferulic acid on the arabinoxylan portion of the dough also brings about softening

phenomenon as there is breakdown of the cross-linked arabinoxylan network [184].

82

Figure 5.11 Application of crude PPO for dough rising in bakery. ‘A’ indicates control (without addition of

PPO) ‘B’ and ‘C’ shows the PPO treated dough samples with 1ml in duplicate. Bars indicate the dough-raising

in control and PPO treated dough samples. (0.8 cm more rise in height of dough)

Dough raising capability of the crude extract of A. paeoniifolius was monitored at regular

intervals. Maximum dough rising takes about 8 hrs after which there was no increase in

height of the risen dough. Crude enzyme treated dough showed better dough rising property

with a difference of 0.8 cm between control, and enzyme treated dough (Figure 5.11). A.

paeoniifolius PPO may be affecting the different phenolics existing in dough and its

association with biomolecules in flour can be accounted for the observed changes which need

to be investigated further.

5.4 CONCLUSIONS

Researches on cellulases and Polyphenol oxidase have progressed over some time. The

availability of enzymes from plant species for use in industrial processing needs remains a

feasible option as most of the industrial enzyme processes are run at high temperatures.

Crude enzyme fraction from stored corm of Amorphophallus paeoniifolius having cellulase

activity could clarify apple juices and the clear juice obtained indicated the potential for use

of crude extract of Amorphophallus in fruit juice processing. The use of cellulase from

locally produced corms having good shelf life value can be favourable and economical for

83

juice production. It is well known that for clarification of juices by cellulase, xylanases,

pectinases are utilized [185]. The availability of enzymes from plant species remains a best

viable option which needs to be further explored. The stored corm extract showed the

existence of cellulolytic activity with enhanced thermo stability and was stable under both

acidic as well as alkaline conditions. In both substrates (Filter Paper and CMC), the enzyme

extract showed two activity peaks because of dip in activity at pH 7.0. This is due to substrate

heterogeneity, such as the substrate portions degraded easily are hydrolysed first, end product

inhibition, thermal inactivation and irreversible adsorption of part of enzyme by the substrate.

This enzyme has potential to be exploited in the clarification of juices [186].

Enzymatic browning is a biochemical process which occurs in fruits and vegetables by the

enzyme PPO, resulting in the darkening and discoloration of vegetables. PPO activity causes

considerable economic and nutritional loss in the commercial production of fruits and

vegetables. To our knowledge no report is available on the A. paeoniifolius, browning

enzyme PPO. The present study deals with partial purification, characterization, inhibition

and dough rising application of PPO for the first time from A. paeoniifolius. It was shown

that Amorphophallus corm PPO has maximum substrate specificity for catechol. PPO activity

was quite sensitive to some of the common PPO inhibitors, especially to L ascorbic acid,

Citric acid, ZnSO4. This study led to the conclusion that enzymatic browning of A.

paeoniifolius corm might be minimized by addition of these inhibitor solutions. Acidic and

alkaline above pH 7 conditions can be used as a potential measure to control browning

reaction. A. paeoniifolius PPO enzyme will find application in dough raising and baking

industry where stability of enzyme at high temperature would be economical. Also it is to be

noted that further purification steps for the enzyme may be required to make this a good

candidate for some industrial or biotechnical applications.

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