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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
vi
Page
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
2
3
4
5
6
7
8
9
10
11
12
13
14
15
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 . . . . . . . . .
xi
Page
27
30
32
33
35
37
38
47
49
51
52
54
55
57
59
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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
Page
74
76
79
81
82
84
86
88
89
91
105
107
109
110
112
113
32
33
34
35
36
37
38
39
40
41
42
43
44
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
x i i i
Page
ll5
ll7
ll8
ll9
121
122
135
137
139
141
142
144
145
45
46
47
48
49
50
51
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 . . . . . . . . . . . . . . . . . . . . . . . . . . .
xiv
Page
151
153
154
156
158
159
161
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