UNIVERSITI PUTRA MALAYSIA ISOLATION, PURIFICATION...

UNIVERSITI PUTRA MALAYSIA

ISOLATION, PURIFICATION AND CHARACTERISATION OF PAPAYA PECTINESTERASE

FAYYAZ ASHRAF

FSMB 1993 4


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.

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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


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


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


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

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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

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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.


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

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