UNIVERSITI PUTRA MALAYSIA

L-PHENYLALANINE PRODUCTION BY STABILISED CALCIUM ALGINATE IMMOBILISED AMINOACYLASE

SlAW YEW SEONG

FSMB 1993 2

L-PHENYLALANINE PRODUCTION BY STABILISED CALCI UM ALGINATE I HHOBILISED AMINOACYLASE

By

SlAW Y EW S EONG

The s i s Sub.i tted i n Ful fi l.ent of t he Re quire.ent s for t he Degree of Mas ter of Science in

t he Faculty of Food Science and Biotechnology , Universiti Pertanian Malaysia

Marc h 1993

ACDOWLEDGEHENTS

First of all , I would l ike to express .y sincere gratitude

to .y for.er supervi sors Dr . Lee Kong Hung and Dr . Pat H . Lee

for t heir guidance and advice t hroughout t he proj ect . I a.

also very grateful to Dr . Shari fah Kharida h Syed Huha •• ad , who

a s su.es t he C ha i r.an o f .y Supervi sory Co •• i t te e after t he

r e s i gnation o f Dr . Lee Kong Hung i n January 199 2 , for her

countless co •• ents and kind assistance during t he preparation

of t hi s t he si s . Hany thanks also go to t he othe r .e.bers o f

t he Supervisory Co •• i ttee , Dr . Baharuddin Ghani and Associate

Professor Dr . Hoha.ed Is.ai l Abdul Kari •.

I would l ike to acknowledge t he financial support provided

by t he IRPA fund for t hi s researc h (awarded to Dr . K . H . Lee ,

Grant No . 503 7 5 ) and also t he JICA grant for t he develop.ent of

t he Depart.ent o f B i o te c hnology , U . P . H . . Ms . Junnaida h (a

staff .e.ber of t he Biotechnology Laboratory) , Mr . Ho and Ms .

Ha.idah ( Elec tron Microscopy Laboratory staff .e.ber s ) . and Mr .

Az.an Moha.ed eU. p. M . p hotographer) are also hig hly apprec iated

for t heir tec hnical help .

My s pe c i a l app r e c i a t i o n i s a l s o e x t e n d e d t o .y b e s t

friends Hr . Seow Teck Keong , Mr . Leong Wooi Chai t and Hr . Chan

i i

Tin Wan for t heir kind friends hip during .y graduate s tudy in

t hi s uni versi ty .

Finall y , I wi s h to express .y s incere appreciation to .y

parents , .y brothers and s i sters , .y grandaot her , .y aunts and

.y fiancee Hs . Wong Jien Hin for t heir .oral encourage.ent ,

patience and understanding t hroughout .y s tudies .

i i i

TABLE or CONTENTS

Page

ACKNOWLEDGEMENTS . . . . • . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i i

LIST OF TAB LES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x

LIST or FIGUR ES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

LIST OF PLA TES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvi i

ABSTRAK

CHAPTER

I

I I

xix

GEN ERAL INTRODUCTION 1

LI TERATURE REVI EW 6

Production of A.ino Acids . . . . . . . . . . . . . . . . . . . 6

Enzy.atic Prope rt i e s of A.inoacylase . . . . . . . . 9

I .. obi l i sation of A.inoacyla se . . . . . . . . . . . . . . 1 3

Calciu. Alginate as a n I •• obi l i sation Mat ri x . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

III CHARAC TERISATION OF FREE AHINOACYLASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Int roduct ion 20

Mat e rial s and Met hods . . . . . . . . . . . . . . . . . . . . . . . 20

Mate rial s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Met hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1

iv

Page

Resul ts and Discus s ion . . . . . . . . . . . . . . . . . . . . . . 26

Deteraination of t he Initial Vel ocity of Free Aainoacylase-Catalysed Reaction . . . . . . . . . . . . . . . . . . . . . . . 26

Deteraination of t he Specific Activity of Free Aainoacylase

Effect of Cobalt Chloride on

28

Free Aainoacylase Activity . . . . . . . . . . . . . . . 29

Effec t of pH on Free Aainoacylase Acti vi ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Deteraination o f the Optiaua Teaperature for Free Aainoacylase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1

Deteraination of t he Activati on Energy of Free Aainoacylase . . . . . . . . . . . . . . 34

Deteraination of t he Michae l i s Constant ( Ka) and Maxiaal Velocity (Vaa ) for Free Aainoac Ylase-�atalysed React ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Theraal Stabi l i ty of Free Aai noacylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

Suaaary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

IV CHARACTERISATION OF AHINOACYLAS E I HHOBILISED IN CALCI UM ALGINATE B EADS (ACAG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Introduct ion 40

Materials and Met hods . . . . . . . . . . . . . . . . . . . . . . . 4 1

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1

Met hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1

v

V

Page

Result s and Di scussion . . . . . . . . . . . . . . . . . . . . . . 46

Det er.inat ion of t he Loading Capacity o f Calciu. Alginate Beads . . . . . . . . . . . . . . . . 46

Deter.inati on of t he Initial Velocity o f ACAG-Catalysed Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Deter.inati on of t he Specific Activity of ACAG . . . . . . . . . . . . . . . . . . . . . . . . . 48

Effect of pH on ACAG Activity

Deter.inati on of t he Opti.u. Teaperature for ACAG Activity

Deter.inat ion of t he Activation

50

50

Energy of ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

Deter.inati on of t he Mi c hae l i s Constant (K.) and Maxi.al Velocity (V.a ) for ACAG-Catalyse� Reaction . . . . . . . . . . . . . . . . . . 53

Ther.al Stability of ACAG . . . . . . . . . . . . . . . . 56

Bat c h Operat ional Stabi l ity o f ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Structural Exa.ination o f ACAG by Scanning Electron Microstopy . . . . . . . . . . . . . 58

Suaaary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

CHARACTERISATION OF AMINOACYLASE I HHOBILISED IN CALCI UM ALGINATE B EADS COATED WI TH POLYETHYLENEIMINE (PEl-Coated ACAG) AND GLUTARALDEHYDE (GLU-Coated ACAG) . . . . . . . . . . . . 63

Introduc tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

Material s and Met hods . . . . . . . . . . . . . . . . . . . . . . . 65

Material s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

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Met hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

Result s and Di s cus sion . . . . . . . . . . . . . . . . . . . . . . 70

Effec t of Polyethylenei.ine Coating Ti.e on t he Perfor.ance of P El-Coat ed ACAG 70

Effect of Glutaralde hyde Coating Ti.e on t he Perfor.ance of GLU-Coated ACAG 7 1

Batc h Operational Stabi l i ty o f P El-Coated ACAG , GLU-Coated ACAG and P EI -GLU-Coated ACAG . . . . . . . . . . . . . . . . . . 72

Characteri sation of P El-coated ACAG . . . . . . 73

Su •• ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

VI CHARACTERISATION OF AMINOACYLASE I HHOBILISED IN CALCI UM ALGINATE B EADS COATED WITH POLY-L-LYSINE ( PLL-Coated ACAG) . . . . . . . . . . . . . 96

Introduc tion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Materials and Met hods . . . . . . . . . . . . . . . . . . . . . . . 97

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Met hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Results and Discus s ion . . . . . . . . . . . . . . . . . . . . . . 101

Effect of Poly-L-Lysine Coat ing Ti.e on t he Perfor.ance of PLL-Coated ACAG 101

Effect of pH on Poly-L-Lys ine Coating Procedure . . . . . . . . . . . . . . . . . . . . . . . . 102

Characterisat ion of PLL-Coated ACAG . . . . . . 104

Su •• ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120

vii

VII CHARACTERISATION OF AMINOACYLASE I HHOBILISED IN CALCIUM ALGINATE BEADS BY CARBODI IMIDE

Pale

CROSS-LINKING METHOD (Cros s-Linked ACAG) . . . . 1 26

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

Material s and Met hods . . . . . . . . . . . . . . . . . . . . . . . 127

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

Met hods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

Resul t s and Discuss ion . . . . . . . . . . . . . . . . . . . . . . 132

Part ial Puri fication of A.inoacylase 132

Characteri sation of t he Part ially Purified A.inoacylase . . . . . . . . . . . . . . . . . . . . 134

Opt i.isation of t he Process for t he Pre paration of Cross-Linked ACAG . . . . . . . . . 146

Characterisation of Cros s-Linked ACAG . . . . 149

Su •• ary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160

VII I GENERAL DISCUSSION AND CONCLUSIONS . . . . . . . . . . 165

Kinetic Cons tant Study . . . . . . . . . . . . . . . . . . . . . . 169

Opt i.UII pH Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 70

Opti.UII Te.perature Study . . . . . . . . . . . . . . . . . . . 170

Activation Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1

Ther.al Stability Study . . . . . . . . . . . . . . . . . . . . . 1 7 1

Speci fic Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

Ope rational Stability Study . . . . . . . . . . . . . . . . . 1 73

BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

BIOGRAPHICAL SKETCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

viii

Table

1

2

3

4

5

6

7

8

9

10

11

12

LIST OF TABLES

P roduction o f A.i no Acids . . . . . . . . . . . . . . . . . .

Su •• a ry of t he Enzy.at i c P rope rties of F ree A.i noacylase . . . . . . . . . . . . . . . . . . . . . . .

Su •• a ry o f t he Enz�at i c P roperties of ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Polyet hylenei.i ne Coati ng Ti.e on t he Activity and Stabi l i ty of P El -Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ef fect of Glutaralde hyde Coati ng Ti.e on t he Activity and Stability of GLU-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Sn..ary o f the Enzy.at i c P roperties of P El -Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Poly-L-Lysi ne Coati ng Ti.e on t he Activity and Stab i lity of PLL-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of p H on Poly-L-Lysi ne Coati ng P rocedure . . . . . . . . . . . . . . . . . . . . . . . . . .

Sua.a ry of t he Enzy.at i c Properties of PLL-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . .

The A.i noacylase Activity and Protein Cont e nt of Co • • e rc ial A.i noacylase P re c ipitated Us i ng A •• oni n. Sulp hate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The A.i noacylase Activity and Protein Content of Co •• e rcial A.i noacylase Before and Afte r Puri f i cation . . . . . . . . . . . . . .

Effect of Carbodii.ide Reaction Ti.e on the Ac tivity and Stabi lity of Cros s-Li nked ACAG . . . . . . . . . . . . . . . . . . . . . . .

i x

Pale

9

39

62

71

72

95

101

103

125

133

134

147

13

14

15

16

Effect of Carbodi i.ide Concentration on t he Activity and Stabi lity of Cross-Linked ACAG . . .. . . . . . . . . . . . . . . . . . . .

The Specific Activity and Stabi l i ty of ACAG , Cross-Linked ACAG , and Cross­Linked ACAG wi t h Exce s s Carbodii.ide Re.oved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Su •• ary of t he Enzy.ati c Properties of Cross-Linked ACAG and Par t ially Puri fied A.i noacylase . . . . . . . . . . . . . . . . . . . . . .

The Enz �at ic Properties of Free and I •• obi l i sed A.inoacylase . . . . . . . . . . . . . . . . . . .

x

Page

148

149

164

168

Figure

1

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4

5

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7

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LIST OF FIGURES

Effect of Incubation Ti.e on the React ion Rate of Free AIlinoacylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Cobalt Chloride Concentrat ion on Free AIlinoacylase Activity . . . . . . . . . . . . . . .

Effect of pH on Free A.inoacylase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Te.perature on Free AIlinoacylase Acti vi ty . . . . . . . . . . . . . . . . . . . . . . .

Arrhenius Plot for the Free A.inoacylase Reaction . . . . . . . . . . . . . . . . . . . . . . .

Lineweaver-Burk Plot of Free A.inoacylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ther.al Stab i l ity of Free A.inoacylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of the A.ount o f A.inoacylase Loaded into Calciu. Alginate Beads on the Enz �e Activity . . . . . . . . . . . . . . . . . . . . . .

Effect of Incubat ion Ti.e on the Reac t ion Rate o f ACAG . . . . . . . . . . . . . . . . . . . . . . .

Effect o f pH on the Activity o f ACAG . . . . . . . .

Effect of Te.perature on the Act i vity of ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Arrhenius Plot for the ACAG . . . . . . . . . . • . . . . . .

Lineweaver-Burke Plot of ACAG . . . . . . . . . . . . . . .

Ther.al Stab i l ity of ACAG . . . . . . . . . . . . . . . . . . .

Batch Operat i onal Stabi l i ty of ACAG . . . . . . . . .

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Batch Operational Stab i l i ty of ACAG , PEl-Coated ACAG , Glu-Coated ACAG , and PEI-GLU-Coated ACAG . . . . . . . . . . . . . . . . . . . . .

Effect of Incubation Ti.e on the Activity of PEl-Coated ACAG . . . . . . . . . . . . . . . . .

Effect o f pH on the Activity o f PEl-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Te.perature on the Activity of PEl -Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . .

Arrhenius Plot for the PEl -Coated ACAG . . . . . .

Lineweaver-Burk Plot of PEl-Coated ACAG . . . . .

Ther.al Stab i l ity of PEl-Coated ACAG . . . . . . . .

Effect o f Flow-Rate on the Perfor.ance of Packed-Bed Bioreactor Containing PEl-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Esti.ation o f the Final Convers ion o f Subs t rate i n t h e Packed-Bed Bioreactor Containing PEl-Coated ACAG . . . . . . . . . . . . . . . . . .

Continuous Operat ional Study Using Packed-Bed Bioreactor Containing PEl-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Incubation Ti.e on the Reacti on Rate of PLL-Coated ACAG

Effect o f pH on the Activity of PLL-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of Te.perature on the Activity o f PLL-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . .

Arrhenius Plot of PLL-Coated ACAG . . . . . . . . . . .

Lineweaver-Burk Plot of PLL-Coated ACAG . . . . .

The r.al Stabi l i ty of PLL-Coated ACAG . . . . . . . .

xii

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Batch Operati onal Stabi lity of PLL-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Storage Stab i l ity of PLL-Coated ACAG . . . . . . . .

Effect of Flow-Rate on the Perfor.ance of Packed-Bed Bioreact or Containing PLL-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Continuous Operational Study Us ing Packed-Bed Bioreactor Containing PLL-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Est i.ation o f the Final Convers ion o f Substrate in the Packed-Bed Bi oreator Containing PLL-Coated ACAG . . . . . . . . . . . . . .. . . .

Esti.ation of the Final Convers ion of Subs t rate in the Packed-Bed Bioreactor Containing PLL-Coated ACAG . . . . . . . . . . . . . . . . . .

The Protein and A.inoacylase Activity Pro f i l e of Co • • ercial A.inoacylase Obtained Us ing Gel Chro.atography Wi th Sephadex G-IOO . . . . . . . . . . . . . . . . . . . . . . . . .

Effect o f Incubation Ti.e on the Reaction Rate of Partially Pur i fied A.inoacylae . . . . . . . . . . . . . . . . . . . . . . . .

Effect of pH on Partially Purified A.i noacylase Activity . . . . . . . . . . . . . . . . . . . . . . .

Effect o f Teaperature on Partially Pur i fied A.inoacylase Activity . . . . . . . . . . . . . .

Arrhenius Plot for the Partially Pur i fied A.inoacylase Reaction . . . . . . . . . . . . . .

Lineweaver-Burk Plot of Part ially Puri fied A.inoacylase . . . . . . . . . . . . . . . . . . . . . . .

Ther.al Stabi l ity of Partially Puri fied A.inoacylase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Effect o f Incubat ion Ti.e on the Reac t ion Rate of Partially Pur i fied A.inoacylase Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effect of pH on Cro s s-Linked ACAG Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . • . . .

Effect o f Te.perature on Cros s-Linked ACAG Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Arrhenius Plot of Cros s-Linked ACAG . . . . . . . . .

Lineweaver-Burk Plot of Cros s-Linked ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ther.al Stability of Cros s -Linked ACAG

Batch Ope rational Stab i l ity of Cros s-Linked ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . .

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LIST OF PLATES

Plate Page

1 Electron Micrograph of ACAG . . . . . . . . . . . . . . . . . • . . . . . . . . . • . . . . . . . . . . . 60

2 Electron Micrograph o f PEl-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

3 Electron Micrograph of PLL-Coated ACAG . . . . . . . . . . . . . . . . . . . . . . . • . . . . 123

4 Electron Micrograph of Cros s-Linked ACAG . . . . . . . . . . . . . . . . . . . . . . . . . . 162

xv

ACAG

GLU

Hepes

PEr

PLL

Tri s

LIST or ABBREVIATIONS

Calc iua Alginate r •• obi l i sed A.inoacylase

Glutaraldehyde

N- ( 2-hydroxyethyl ) piperaz ine-N'- ( 2-ethanesul fonic acid)

Hichae l i s -Henton Cons tant

Polyethylenei.ine

Poly-L-Lys ine

Tri s (hydroxy.etyl ) -a.ino.ethane

Haxi.al Velocity

xvi

Abstract of the s i s sub.itted to the Senate o f Univers i t i Pertanian Mal aysia i n ful f i l.ent of the require.ents for the degree of Master of Science .

L-PHENYLALANlNE PRODUCTION BY STABILISED CALCIUM ALGINATE IMNOBILISED AHlNOACYLASE

By

SlAW YEW SEONG

March 1993

Shari fah Khari dah Syed Muha .. ad , Ph . D .

Faculty Food Science and Biotechnology

Enz �atic resolut ion o f DL-a.ino acids using a.inoacylase

ia.obi l i sed on DEAE-s ephadex has been e.ployed in the indust ry

for the produc t i on o f the opt i c a l l y ac t i ve , L-a. ino a c i d.

There are , however , so.e l i.i tat ions to this technique . The

a i a s o f t h i s s t u d y w e r e t o e s t a b l i s h a n e f f i c i e n t

i •• obi l i s at i on .ethod for a.i noacyl a s e and t o report t h e

preparation , properties , and app l icabi l i ty of the resulting

i •• obi l i s e d allinoacyl ase . A.i noacyl a s e was i •• obi l i s ed i n

calciu. alginate beads (ACAG ) . The ACAG was further stab i l ised

by c o a t i n g w i t h p o l y e t h y l e n e i . i n e ( P EI ) , a m i x t u r e o f

polyethylenei.ine and glutaraldehyde (PEI-GLU) , poly-L-lys ine

(PLL) , or by cross-linking with carbodi i.ide . Each type o f the

i •• obi l i sed aminoacylase was then used to perfor. the optical

r e s o lution o f N-ac e t y l -DL-pheny l a l an i ne . PLL- c oated ACAG

xvi i

was found to be the bes t a.ong the i •• obi l i sed a.inoacylase

s tudied. It has a h i gher activity and s tabi l ity than that of

the other syste.s . In the batch operati onal s tabi l i ty s tudy ,

half of the ACAG act i vi ty was lost after the first cycle o f

reac t i on . The c r o s s - l i nked ACAG h a s a hal f-l i fe o f four

react ion cycles whi l e the PEl-coated , PEI-GLU-coated ACAG and

PLL- c oated ACAG were s t able up to ten c y c l e s o f reac t i on .

Although the stabi l it y of ACAG was enhanced by coating with PEl

and PEI-GLU, the speci fi c ac tivity of PEl & PEI-GLU-coated ACAG

were great l y reduc e d when c o.pared t o that o f ACAG . The

a c t i v i t y of ACAG , h owever , was not decreased s i g n i f i can t l y

after coating wi th PLL . Electron .icrographs of the uncoated

ACAG and coated ACAG revealed so.e di fference s in thei r surface

structure s . The surface of ACAG was s.ooth whi le that of PEl

and PLL-c oated ACAG had a cross-l inked network . The cros s-

l inked ACAG , however , had the s a.e surface structure as ACAG .

In the continuous operational stab i l i ty s tudy , the packed-bed

bioreactors containing PEl and PLL-coated ACAG were s table for

25 days .

xvi i i

Ab s t r a k t e s i s y a n g t e l ah d i kemukakan kepada Se n a t Uni versi t i Pertanian Halaysia sebagai me.enuh i syarat untuk i j azah Haster Sains .

PENGHASlLAN L-FENlLALANlNA OLEn AKlNOAS lLASE TERS EICAT-GERAK DALAK KALS lUM ALGlNAT YANG STABlL

Oleh

S lAW YEW SEONG

Hac 1 993

Pengeru s i Sharifah Kharidah Syed Huhammad , Ph . D .

Fakulti Sains Hakanan dan Bioteknologi

Re s o l u s i menggunakan e n z i m am inoa s i l a s e t e r s e kat-ge rak

p a d a DEAE- s e p h a d e x t e l ah d i gunaka n d a l am i n du s t r i u n tuk

mengha s i l kan a s i d amino yang akt i f s e c ara opt i ka l . Walau

bagaimanapun , terdapat beberapa kelemahan dala. teknik ini .

Tuj uan penye lidikan i n i adalah untuk mendapatkan s i s tem enzim

tersekat-gerak yang unggul untuk aminoa s i l ase dan lIe laporkan

cara-c ara penyedi aan , c i r i - c i r i , dan kegunaan s i s t em enzim

tersekat-gerak yang terhas i l . Aminoasilase telah di sekat-gerak

d a l am m a n i k ka l s i u ll a l g i n a t ( A CAG ) . ACAG i t u kemu d i a n

d i s t ab i l kan dengan penyalutan o l eh pol i e t i l e nai.ina (PEl ) ,

campuran pol ietilenaimina dan glutaraldehi da ( PEI-GLU) , poli-L-

l i s i n a ( PLL) atau i katan s i lang dengan karbodi i . i da ( ACAG

terikat s i lang ) . A.inoas i lase tersekat -gerak yang terhas i l

digunakan untuk me lakukan tindakbalas resolus i secara optikal

ke atas N-aset i l -DL- fenilalanina . ACAG yang di saluti dengan

xix

PLL .e rupakan s i s t e. e n z i . t e r s ekat-ge rak yang terbaik di

antara yang disel idiki . Ia lebih akt i f dan stabi l daripada

s i s t e ll y a n g l a i n . D i d a l a. ka j i a n ke s t a b i I a n ope r a s i

s e s eke l o llpok , separuh daripada akt i v i t i ACAG te lah h i l ang

s e l e p a s t i n d a kb a l a s yang p e r t a.a . ACAG t e r i ka t s i l a n g

lIe .pun y a i s e p a r a h a y a t s e l a.a e llp a t k i t a r a n t i ndakb a l a s

s ementara ACAG yang di s a lut i PEl , PEI -GLU , dan PLL .as i n g­

lIasing stab i l s eh ingga sepuluh ki taran t indakbalas . Walaupun

ke stabilan ACAG telah meningkat lIel a1ui penyalutan dengan PEl

dan PEI -GLU , t e tapi akt i v i t i s pe s i f i k mer eka t e l ah menurun

dengan ban yak berbanding dengan akt i vi ti ACAG . Sebal iknya ,

aktiviti ACAG tidak banyak berubah se lepas disaluti dengan PLL .

Hikrograf e l ekt ron untuk ACAG yang t i dak di s a lut i dan yang

telah disaluti .enunj ukkan perbezaan dala. struktur permukaan

mereka . Perllukaan ACAG adalah l i c i n s emen tara ACAG yang

di s aluti dengan PEl dan PLL mempunyai struktur rangkaian ikatan

s i lang . Walau bagaimanapun , ACAG t e r i kat s i l ang mellpunyai

struktur yang sama seperti ACAG. Di dal am ka j ian ke stabil an

o p e r a s i s e c a r a b e r t e r u s a n , b i o r e akt o r "p a c ke d - b e d" y a n g

lIe n g andun g i A C A G y a n g d i s a l u t i d e n g a n PEl dan P L L t e l ah

didapat i s tabi l selalla 25 har i .

xx

CHAPTER I

GENERAL IN'l'ROOOCl'IOIf

The wor l d de.and for a.i n o a c i ds i s vas t ; 6 7 5 tons o f

a.ino acids consu.ed i n 1985 . It represents an increase o f

3 4 I i n volu.e fro. 198 1 t o 1 98 5 ( K i n o s h i ta, 1 98 7 ) . The

turnover was esti.ated at 1 . 7 b i l l ion u.s. dol lars worldwide in

1 980 ( Crueger and Crueger , 1 98 4) . Al l the twenty a.ino ac i ds

that are requi red for the s ynthe s i s o f prot e i n have been

produced i n du s t r i a l l y w i t h L-gl u t a . i c acid being the .o s t ,

f o l l o w e d b y D , L - . e t h i o n i n e a n d L - l y s i n e . T o d a y , t h e

.anufacture o f a.ino acids i s largely do.inated by the Japane se

and there i s hardly any such indus t ry i n Halays i a except for

the production of .onosodiu. g luta.ate (HSG) by Aj ino.oto Co . .

A.ino acids are used in .edicine to strengthen the defence

.echanis. , to increase blood coagulat ion , for gluconeogene s i s ,

and for s ynthes i s of new protein in hospital i zed patients . In

food industry , a.ino acids are added to foods to enhance the

t a s t e , f l a v o u r a s we l l a s q ua l i t y o f t h e f o o d s . A n e w

app l i cat ion o f a.i n o a c i d s i n foods rec ent l y i s a s a l ow

calorie sweetener , asparta.e ( L-aspartyl-L-phenylalanine .ethyl

ester ) , which con s i s t s of L-phenylalanine and L-aspartic acid .

1

2

A.ino acids such as lys ine and .ethionine have been added to

feedstuff to upgrade i t .

I ndus t r i a l produc t ion o f a.i no a c i ds i s per for.ed by

fer.entati ve and che.ical synthetic .ethods . The latter .ethod

i s .ore s i.pl e , faster , and cheaper to perfor. than the for.er

.ethod . Nevertheless , the a.ino ac i ds produced che.ically are

the optically inac tive race.ic .ixture of D- and L-i so.ers .

The L-i s o.er i s a phys iologically active natural for. , the D-

i so.er i s generally inactive for. for hu.an nutrition , and i s

an unnatural for •. In order to obt a i n the L-a.ino ac i d ,

optical resolut ion i s neces sary . Opt i cal resolution of race.ic

a.ino acids has been carried out by phys ico-che.ical , che.i cal ,

biological , and enzy.atic .ethods . The enzy.atic resolut ion

us ing a.inoacylase i s the pre ferred .ethod as it has versatile

s ub s t ra t e s p e c i f i c i t y and ab solute s t e r eo spec i f i c i t y . The

reac tion catalysed by a.inoacylase is as fol lows :

N-acyl-DL-a.ino acid +

H20 ------------->

a.inoacylase

N-ac yl -D-a.ino acid +

L-a.ino acid

A.inoacylase selectively hydrolyses the N-acyl-L-i so.er .

T h e l i b e r a t e d L - a . i n o a c i d c a n b e s e p a r a t e d f r o . t h e

unhydrolysed N-acyl-D-a.ino acid based on the di fference in

their solub i l i ties . The N-acyl-D-a.ino acid can be race.ised

and the proc e s s be repeated .

3

The afore.entioned enzy.ati c resolution has been e.ployed

extensively in the i ndustrial production of L-a.ino acid . The

enzy.e reaction was initially per for.ed in batch process us ing

soluble enz�e . The drawbacks of thi s procedure are ( i ) the

enzy.e is i.pos s ible to recover after use , and ( i i ) the enzy.e

. i gh t n o t b e s u f f i c i e n t ly s t ab l e un d e r t h e o p e r a t i o n a l

condi tions . To overco.e thes e di sadvantage s , extensive work

has been c arried out to produce L-a.ino acids us ing i •• obi l i s ed

a.inoacylase (Ch ibata et a1., 1972 , Morikawa et a1., 1976 , Sato

et a1., 1 97 1 , Tosa et a1., 1967 , Weetal and Detar , 1974, Yokote

et a1., 197 5 ) . Chibata and his associates , in their evaluation

o f 43 different i •• obi l i sation .ethods for a.inoacylase , chose

DEAE-sephadex bonded enzyae for indus trial app l ication because

it allowed the. to regenerate the packed-bed enzy.e reac tor ' s

activity every so o ften ( wi thout unloading the reactor ) , by

flushing the sys te. with high salt concentrat ion and low pH to

re.ove the denatured enzy.e , subsequently adding fresh enz �e

directly to the reactor to allow i •• obi l i sation to take place

in s itu . The original DEAE-sephadex was left in the reac tor

for at least 10 year s . The l i.itations to this technique are

( i ) the e nz y.e .us t be a c t i ve w i t h i n the narrow range o f

environ.ental condi t ions (particularly pH) required t o keep the

enzy.e bonded t o the p o l y.er .atr i x , ( i i ) s o.e release o f

a.inoacylase .ay occur i f high concentration o f substrate i s

used , ( i i i ) the a.inoacylase used .ay contain other enzy.es

such a s c e l l u l a s e and dext rana s e wh i c h can hydr o l yse DEAE-

4

s ephadex , and ( i v ) DEAE-s ephadex i s an expens ive i on-exchange

r e s i n . F ur t h e r e f f o r t s s h o u l d t h u s be .ade t o p r e p a r e

i • • ob i l i s e d a . i n oa c y l a s e wh i c h h a s t h e a f o r e .e n t i o n e d

l i.itations re.oved .

The ob j e c t i ve o f thi s s t udy i n general i s t o obtain a

s table i •• obi l i sed a.inoacylase syste. for the production o f L­

phenyl a l a n i n e via the op t i c a l r e s o lu t i o n o f N-ac etyl-DL­

phenylalanine . The .ethod o f encapsulation of a.inoacylase in

calciua alginate beads was chosen due to the fact that ( i ) thi s

i •• obi l i s ation proces s i s s impl e and .i ld , thus the enzyme .ay

be .aintained in i t s nat ive for. after i •• obi l i sat ion , and ( i i )

the .atrix i s cheap and non-toxic . However , i t i s known that

the above.entioned .ethod is not sui table for i •• obi l i sat ion of

enzyae s as the pores on the surface of calc ium alginate beads

are large enough to al low enzy.es to diffuse out of the beads .

Thus , the spec i f i c ai. of this s tudy i s to prevent a.inoacylase

fro. leaking out of the calc ium alginate beads . To achieve

th i s , vari ous methods for the s t ab i l i s a t i on o f the c a l c i um

a l g i na t e i •• ob i l i sed a. i noac y l a s e have t o be de s i gned and

evaluated .

Th e f i r s t . e t h o d wou l d i n v o l v e c o a t i n g t h e c a l c i u.

alginate i •• obi l i sed a.inoacylase with ( i ) polyethylenei.ine ,

( i i ) a mixture of polyethyleneiaine and glutaraldehyde , and

( i i i ) poly-L-Iys ine . It i s hoped that by coat ing the calc iua

alginate beads wi th another l ayer of polymer , a coverage of the