UNIVERSITI PUTRA MALAYSIA

CHEMICAL MODIFICATION OP LIPASE AND ITS IMMOBILIZATION ON POLYMER BEADS FOR USE IN ORGANIC SYNTHESIS

MAHlRAN BASRI

FSAS 1993 2

CHEMICAL MODIPICATION OP LIPASE AND ITS ItK>BILIZATION ON POLYMER BEADS fOR USB IN ORGANIC SYNTHBSIS

By

MAHlRAN BASRI

Thesis Presented in Fulfil lment of the Requirements for the Degree of Doctor of Phi losophy in the Faculty of Science & Environmental Studies ,

Universiti Pertanian Mal aysia .

1993

DEDICATIONS

To my beloved grandmother . . . .

who has taught me al l there is about beauty,

love and human grace .

To abah and mak • . • •

who have made me feel very special.

To adik-adik • • • •

who have always given me the cha l l enge to work harder .

To Jamal • • • •

who has given me a l l the patience , love and support .

To Gaja and Jordie • • • •

who make l iving a joyous happening .

ACKNOWLBDGEMENTS

Special Acknowledgeaent

I would like to express my sincere appreciation to Dr .

Kamaruzaman Ampon for his compas s ion and opt imism, Dr . Abu

Bakar Salleh for his vision and persistence , Dr . Wan Zin Wan

Yunus for his unders tanding and patience and Dr . Che Nyonya

Abd . Razak for her constant support . Their contribution� have

been an inspiration in the completion of this thesis .

Acknowledge-.ent

Special thanks are also extended to my collegues , friends

and department staff whose help , advi ce , encouragement and

humour were invaluable during the course of the research:

Puan Raja Nor Zaliha Raja Ab(1. Rahman , En . Yusof Samad , Puan

Zainoha Zakari a , Puan Ros i ah Musani , Puan Normaya t i , Cik

Rohaidah .

En . Atan Shar i f , Dr . Sidik Silong , Puan Kamaliah S i rat , En .

Nazari Ahmad , En . Buharan Nordin , En . Kamal Margona .

Dr . Khati jah Yusof , Dr . Juzu Hayati .

Members of Enzyme and Microbial Technology Lab.

Members of Jabatan Biokimia & Mikrobiologi .

Members of Jabatan Kimia .

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The Pub l i c S e rvice De pa rtme nt Ma l ays ia , The Uni v e r s i t i

Pertanian Malaysia , The Southeast Asian Ministers of Education

Organization ( SEAMEO) and Southeast Asia Regional Center for

Graduate Studies and Research in Agriculture ( SEARCA ) and the

Ministry of Science , Technology and Environment , Malaysia ( IRPA

Project no . 1 07 05 086 ) for their continued supports .

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TABLE O' CONTENTS

ACKNOWLEDGEMENTS

LIST OP TABLES

LIST OF FIGURES

LIST OP PLATES

LIST OF ABBREVIATIONS

ABSTRACT

ABSTRAK

CHAPTER

1

2

IN'l'RO�ION' • • • • • • • • • • • • • • • • • •

LITBRA'.ruRB REVIEW . . . . . . . . . . . . . . .

Enzymes as Biocatalysts

Lipases

Reactions Catalyzed by Lipases .

Lipase Selectivity . .

Importance of Esters . •

Lipase Catalysis in Organi c Solvents •

Chemical Modification of Enzyme

Modification to Alter Properties

of Enzymes . . . . . . . . . . .

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ii

x

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xvi

xvii

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xxi

1

7

7

17

17

20

26

32

34

3S

3

Modification of Lipase with PEG

Amidination of Lipase with Imidoesters

Reductive Alkylation of Lipase

with Aldehydes . . .

Immobilization of Enzyme • • • •

Immobilization Techniques

Immobilization by Adsorption •

MATERIALS AND MB11IODS

Purification of Lipase •

Protein Determination . . .

Activity Determination

Synthetic Activity

Hydrolytic Activity

Chemical Modification Reagents

. . . .

. . . .

Activation of Monomethoxypolyethylene

Glycol . • • • • • . •

Synthesis of Imidoesters

Chemical Modification of Lipase

Modification with Activated PEG •

Amidination with Imidoesters

Reductive Alkylation with Aldehydes

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38

40

41

42

44

52

56

56

57

58

58

60

60

6 1

63

63

64

65

Analysis of Modified Lipases using

Hydrophobic Chromatography •

Immobi l ization of Lipase

Adsorption of Native and

Modified Lipases • •

Bffect of Temperature on Bnzyme

Adsorption

Bffect of pH on Bnzyme Adsorption

Bffect of Bead Sizes on

Bnzyme Adsorption

Operational Stabi lity

Glutaraldehyde Cross1inking

Characteristics of Lipases •

Bffect of Temperature on the

Bsterification Activity

Catalytic Activity in the Presence of

Organi c Solvents •

Patty Acid Selectivity in the

Bsterification Reaction

Thermostabi1ity

Stabi lity in Organic Solvents

Storage Stability • • • •

Kineti c Studies . . . . . . . .

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4 RBSULTS . . . . . . . . . . . . . . . .

Modificat ion of Lipase with Activated PEG

Properties of Activated

Monomethoxypolyethylene Glycol . . . . •

Reaction of Lipase wi th Activated PEG

Amidination of Lipase wi th Imidoesters .

Propert ies of Imidoesters

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75

75

75

78

78

78

Amidination of Lipase . • • • • • 86

Reductive Alkylation of Lipase with Aldehydes . . 86

Reductive Alkylation of Lipase • . 86

Characteristics of Modified Lipases 87

Catalyt ic Activity of PEG-Lipases 87

Catalytic Activity of Amidinated Lipases 87

Catalytic Activity of Alkylated Lipases 90

The Effect of Temperature on

Esterification Activity

Catalytic Activity in the

Presence of Organic Solvents

Patty Acid Selectivity in the

Esterificat ion Reaction

Thermostabil i ty

Stabi lity in Organic Solvents

Storage Stabi l i ty • • • • . .

vii

94

100

104

108

115

120

5

Hydrophobicity of Modified Lipases •

Immobi lization of Modified Lipases

Adsorption of Native and

Modified Lipases . .

Adsorption Temperature •

Adsorption pH

Effect of Bead Sizes on Adsorption •

Characteristics of Immobilized Lipases •

Catalytic Activity of Immobi l ized

Lipases

The Effect of Temperature on

Esterification Activity

Catalyti c Activity in the Presence

Organic Solvents •

Patty Acid Sel ectivity in the

Esterification Reaction

Thermostability

Stabil ity in Organic Solvents

Storage Stability

Operational Stabi lity

Kinetic Studies . . . . . .

DISCUSSION • . . . . .

Modification of Lipase •

Characteri stics of Modified Lipases

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Immobi l i zation of Modified Lipases • •

Characteristics of Immobilized Lipases

Kinetic Studies

SUMMARY

Recommendations for Further Studies

BIBLIOGRAPHY

APPENDIX

VITA • • . . . . . . . .

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Tabl e

1

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15

15

LIS T or TABLES

Properties of the Different Polymer Beads .

Characteristics of Imidoesters

Catalytic Activity of PEG-l ipases •

Catalytic Activity of Amidinated Lipases

Catalytic Activity of Alkylated Lipases

Catalytic Activity of PEG-1 ipases in Various Organic Solvents • • • •

Catalytic Activity of Imidoester Lipases in Various Organic Solvents

Catalytic Activity of Alkylated Lipases in Various Organic Solvents

Storage Stability of PEG-lipase after 60 days • • • • • • • • .

Storage Stabi lity of Amidinated Lipase after 60 days • • . • • . • . •

Storage Stability of Alkylated Lipase after 60 days . . . . • . . • . • •

Hydrophobi city of Modified Lipases

Immobil ized Activity of Modified Lipases on Different Types of Polymer Beads

Effect of Increasing the Degree of Lipase Modification on the Immobi li zed Activity . . . . . . . . . . . . . . . . . . .

a : Specific Esterification Activities of XAD7 Immobil ized Lipases in Various Organi c Solvents • • . • • . .

b : Specific Esterification Activities of PL1900 Immobi l ized onto Several Types of Polymer Beads in Various Organic Solvents

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141

Table

16

17

Stabi lity of XAD7 Immobi li zed Lipase Preparations in Different Storage Conditions after 60 days • •

Kinetic Parameters of Lipases •

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Figure

1

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7

8

LIST OP PIGURES

Reactions Catalyzed by Lipases • . • . . • • •

A Gas Chromatogram Showing the Synthesis of Propyl Oleate from Oleic Acid and Propanol in the Presence of Lipase . . . . . . . . . . . . . . .

a : IR Spectrum of Acti vated PEGl900 •

b : IR Spectrum of Unactivated PEGl900 •

Modification of Lipase with Different Amounts of Activated PEGl900 (APEG) • • • • • . • . • .

a : IR Spectrum of Methyl 4-phenylbutyrimidate { Imidoester ( VI » • • • • • • • • . • • •

b : IR Spectrum of 4-phenyl-butyronitrile , the Corresponding Nitrile of Methyl 4-phenylbutyrimidate • • • • • • •

a: H-NMR Spectrum of Methyl 4-phenylbutyrimidate { Imidoester (VI » • • • • • • • • • • • • • • •

b : H-NMR Spectrum of 4-phenyl-butyronitrile , the Corresponding Nitrile of Methyl 4-phenylbutyrimidate • • • • .

a : The Effect of Increasing the Degree of Modification on the Esterification Activity of Lipase Modified with Methyl Acetimidate ( Imidoester 1) . • . • • • . • • • •

b : The Effect of Increasing the Degree of Modification on the Esterification Activity of Lipase Modified with Methyl 4-phenyl­butyrimidate ( Imidoester VI ) • . • • • • •

a : The Effect of Increasing the Degree of Modification on the Esterification Activity of Lipase Modified with Acetaldehyde

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b: The Bffect of Increasing the Degree of Modif ication on the Bsterif ication Act ivity of Lipase Modified with Dodecyldehyde

The Bffect of Temperature on the Esterif ication Reaction by PBG-1 ipases

The Bffect of Temperature on the Bsteri fication Reaction by Imidoester Lipases

The Bffect of Temperature on the Bsterification Reaction by Alkylated l ipases

The Bffect of Chainlength of Patty Acids as Acyl Donor in the Bsterification Reaction by PBG-lipase • • • • • • • • •

The Bffect of Chainlength of Patty Acids as Acyl Donor in the Bsterification Reaction by Imidoester Lipase • • • • • •

The Bffect of Chainlength of Patty Acids as Acyl Donor in the Bsterification React ion by Alkylated Lipase • • • • •

a : Thermostabil i ty of PBG-l ipases Incubated for One Hour in Benzene . • • • . • •

b : Stabil i ty PBG1900-l ipase with Respect to Time at 40 ·C and 50 ·C • . . . • • • •

a : Thermostabi1 ity of Imidoester Lipases Incubated for One Hour in Benzene

b : Stability of Imidoester Lipase (VI ) with Respect to Time at 40· C and 50· C

a : Thermostabi lity o f Alkylated Lipases Incubated for One Hour in Benzene •

b : Stabi lity of Dodecyl Lipase with

Page

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1 1 1

1 12

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Respect to Time at 40· C and 50 · C • • • • • • • 116

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18 Stability of PEG-lipases Incubated in Benzene for Ten Days at Room Temperature • • • • • • • • . • •

19 Stability of Imidoester Lipases Incubated in Benzene for Ten Days at Room Temperature • • • • • • • • • • . •

20 Stabil ity of Alkylated Lipases Incubated in Benzene for Ten Days at

21

22

23

24

25

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29

Room Temperature .

Protein Adsorption Prof ile of Native and PEG-l ipase onto Several Types of Polymer Beads • • • • • • • • • • . •

Act ivity Adsorpti on Profi l e of Native and PEG-lipase onto Several Types of Polymer Beads ( Intact Beads ) • • • • •

Activity Adsorption Profile of Nat ive and PEG-lipase onto Several Types of Polymer Beads (Crushed Beads ) . • • •

The Effect of Different Temperature on the Immpbi lization of Lipase onto Several Types of Polymer Beads • • . •

The Effect of pH on the Immobi lization of Lipase onto Several Types of Polymer Beads . . • . . .

The Effect of XAD7 Bead Si zes on the Immobi lization of PEG-l ipase .

The Effect of Temperature on the Esteri fication Reaction by Immobi l ized Lipase . . . • • •

The Effect of Chainlength of Patty Acids as Acyl Donor in the Esteri fication Reaction by Immobilized Lipase • • . . •

Thermostabil i ty PEG-lipase Immobili zed onto Several Types of Polymer Beads Incubated for One Hour in Benzene

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30 Thermostabi l ity of XAD2 Immobi l ized , Modified Lipases Incubated for One Hour in Benzene . • • . . • . • . .

31 Stabi l ity of Immobi l ized PEG-lipases Incubated in Benzene for Ten Days

32

at Room Temperature

Stabil ity of XAD2 Immobil ized , Modified Lipases Incubated in Benzene for Ten Days at Room Temperature • • • • . •

33 Operational Stability of Immobil ized Lipases Before and After Crosslinking with Different Glutaraldehyde

34

Concentrations

A Lineweaver-Burk Plot Showing a Typical Graph of 1/V aga inst l/[S ] • • • • • . .

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Plate

1

LIS T 0' PLATES

A Thin-layer Chromatogram Showing the Synthesi s of Propyl Oleate from Oleic Acid and Propanol in the Presence of Native and Modified Lipases . . . • •

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PEG PEG 1900 PEG5000 APEG TNBS RCOOH RCN DVB PPLC IR NMR NL Imidoester I Imidoester I I Imidoester I I I Imidoester IV Imidoester V Imidoester VI PLl900 PL5000 IL( I ) IL( I I ) IL( I I I ) IL( IV) IL(V) IL(VI ) AL

DL XAD2NL XAD2PL XAD7NL XAD7PL XAD7IL XAD7AL RCOOHNL RCOOHPL

LIST or ABBRBVIAT IONS

monomethoxypolyethylene glycol monomethoxypo1yethy1ene glycol ( MW=1900) monomethoxypolyethylene glycol ( MW=5000) activated PEG trinitrobenzene sulfonate polycarboxylic acid polyacrylonitrile divinyl benzene Past performance liquid chromatography infra red nucl ear magnetic resonance nati ve l ipase methyl acetimidate methyl benzimidate methyl 4-biphenyl benzimidate methyl n-dodecanimidate methyl 3-phenylpropionimidate methyl 4-phenylbutyrimidate lipase modified with PEG1900 l ipase modified with PEG5000 lipase modi fied with imidoester I l ipase modified with imidoester II lipase modified with imidoester I I I l ipase modi fied with imidoester IV lipase modified with imidoester V lipase modi fied with imidoester VI lipase modified with acetaldehyde ( ethyl lipas e ) l ipase modified with dodecyldehyde native lipase immobil ized on XAD2 PEG-lipase immobil ized on XAD2 native l ipase immobilized on XAD7 PEG-lipase immobi l ized on XAD7 imidoester lipase immobil ized on XAD7 ethyl l ipase immobil i zed on XAD7 native l ipase immobilized on RCOOH PEG-lipase immobi lized on RCOOH

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Abs t r a c t of the t he s i s p r e s e n t e d t o t he S e n a t e of Univers i t i Pertanian Malaysia in fulfi lment of the requirements for the degree of Doctor of Phi losophy .

CHEMICAl. MODIPICATION OP LIPASE AND ITS IMMOBILIZATION ON POLYMER BEADS POR USE IN ORGANIC SYNTHESIS

By

Mahi ran Basri

April 1993

Chairman : Assoc iate Professor Dr . Kamaruzaman Ampon

Facul ty: Science & Environmental Studies

A s imple and effective method to produce a more active ,

stabl e and practical l i pase preparat ion was identified . Soluble

l ipase from Candida rugosa was modified with different types

of hydrophobi c chemi cal modifying reagents .

The es t e r i f i c a t ion a c t i v i t ies of the mod ified l ipases

were enhanced fol lowing the i r modifi cat i on . The degree of

activi ty enhancement depends on the type and molecular weight

of the modifiers used and the degree of modification of the

enzyme . A l ower degree of enzyme deriva t i zation was requi red

for modifi cation with the high molecular wei ght modifiers to

at t a i n ma x i ma l a c t i v i t i e s. In t h e c a s e of

monomethoxypolyethylene glycol ( PEG), however , maximal activity

was attained only after exhaust ive modificat ion .

xvi i i

The optimum esterificat ion temperature and preference of

fatty acids as acyl donors of the modified l ipases were very

simi lar to those of the native enzyme . Both were more active

in non-pol a r solvents than in pol a r sol vent s . The mod i f i ed

lipases showed higher thermostability , solvent stabi lity and

storage s tabi li ty compared to the nat i ve lipase . The lipase

modi f i ed with PEG 1900 was the mos t thermostabl e , and that

modified with methyl 4-phenylbutyrimidate ( imidoester vI) was

the most stable when incubated in benzene for ten days . The

bes t sto rage cond i t i on was at l ow temperature and in the

lyophi li zed form.

When porous polymer beads were added to a solution of the

modified lipase at room temperature and stirred gently for

0 . 5 to 2 hours , the enzyme was strongl y adsorbed onto them .

Native l ipase was only weakly adsorbed onto the supports .

The immobi li zed activity of modified l ipases were higher

compa re d w i t h t h e n a t i v e e n z yme . The magn i t ude o f t h e

difference depended o n the type of mod i f i e rs , the degree of

modification of the enzyme and the type of polymers used for

immob i 1 i z a t i on . L i p a s e that was mod i f i ed wi th the more

hydrophobic modifiers showed higher immobi lized activity

compared to those modified with the less hydrophobi c modifiers

( e xcept for PEG5000 ) . The highest immobi l i zed activity was

exhibited by lipase modified with PEGl900 ( 95% modification ) .

xix

Amberlite XAD7 , XAD8 and Polycarboxylic acid beads (ROOOH) were

good supports for enzyme immobi li zation by the above methods .

The o p t i mum e s t e r i f i c a t i o n t e mpe r a t u r e o f l i p a s e

immobi l i zed i n thi s manner was not changed . However , i t s

preference for certain fatty acids a s acyl donors was altered .

Medium chain length fatty acids were favoured over longer chain

fatty acids when the enzymes were immobi lized . Their activities

were increased even in the presence of polar sol vents . The

immobilized lipases also showed higher thermostabi lity , solvent

stabi l ity and s torage stabi l i ty compared with nat ive and

modified l ipases .

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Abs trak tes is yang d ikemukakan kepada Senat Universiti Pertanian Ma lays ia bagi memenuhi keperluan I jazah Doktor Palsafah .

PENGUBAHSUAIAN LIPASE SECARA KIMIA DAN SEKATGERAKANYA KEPADA BEBERAPA POLYMER UNTUK KEGUNAAN DALAM SINTESIS ORGANIK .

Oleh

Mahi ran Basri

Apri l 1 993

Pengerus i Prof. Madya Dr . Kamaruzaman Ampon

Pakulti Sains & Pengaj ian Alam Seki tar

Satu kaedah yang mudah dan berkesan telah dikenalpasti

un tuk mengha s i l ka n l i p a s e yang l eb i h akt i f , s t ab i l dan

prakt i ka l . L i pase dad Candida rugosa yang terlarut te lah

diubahsuai dengan beberapa bahan pengubahsuai hidrofobik .

Lipase terubahsuai telah menunjukkan kelebihan aktiviti

es terifika s i . Darjah pertambahan akt i v i t i bergantung kapada

jenis dam berat molekul bahan pengubahsuai yang digunakan dan

darjah ubahsuai enz im itu sendi ri . Dar jah yang lebih rendah

ada lah d iper lukan bagi mengubahsua i enz im dengan bahan yang

mempunya1 berat molekul yang tinggi bagi mendapatkan aktiviti

maksimum. Untuk monomethoxypolyethylena glycol (PEG) , aktiviti

maks imum di dapati dengan mengubahsuai l ipase habi s-habi san .

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Suhu optimum esterifikasi dan pemil ihan terhadap asid

1ernak sebagai penderma kumpu1an asi1 untuk l ipase terubahsuai

ada lah sama dengan l i pase nat i f . S e l a i n dari itu , kedua -dua

jenis l ipase didapati lebih akt i f di dalam pelarut organik yang

tidak berkutub berbanding dengan di dalam pelarut organik yang

lebih berkutub . Lipase terubahsua i menun jukkan kestabi l an

te rma , ke s t abi l a n da l am p e l a rut o rga n i k dan k e s t a b i l a n

penyimpanan yang l ebih t i nggi . Lipase yang diubahsuai

dengan PEG1900 menunjukkan kestabilan terms yang tertinggi dan

l ipase yang dimod i f ikas i dengan me t i l 4-fen i l buti rimidat

( imidoester VI ) adalah yang pal ing stabi l apabi la dieram dalam

benzena selama sepuluh hari . Keadaan penyimpanan terbaik adalah

pada suhu rendah dan keadaan beku-kering .

Apa b i l a bu t i r-but i r p o l i m e r y a n g b e r l i ang t e 1ah

dimasukkan ke dalam larutan l ipase terubahsuai pada suhu

bilik dan digoncang selama 0 . 5-2 jam , enzim didapati terjerap

lebih kuat . Lipase na t i f hanya terjerap dengan l emah kapada

butir-butir pol imer.

Lipase terubahsuai menunjukkan aktiviti tersekatgerak

yang lebih tinggi berbanding dengan l ipase nat1£ . Penambahan

akt i v i t i bergantung kepada jenis bahan pengubahsuai , darjah

uba hsua i dan j eni s pol imer yang d i gunakan . L i pa s e yang

diubahsua i dengan bahan yang l ebih hidro fobik menunjukkan

aktiviti tersekatgerak yang lebih tinggi berbanding dengan

xxii

l i pa s e d i ub a h s ua i d e nga n b a h a n y a n g kurang h i d ro f o b i k

( me la i nkan PBG5000 ) . Lipas e diubahsuai dengan PBG1 900 ( 95%

ubahsua i ) memberikan aktiv i t i ters ekatgerak pal ing t i nggi .

Amberlite XAD7. XADS dan pol ikarboks ilik asid (RCOOH ) adalah

polimer yang baik untuk menyekatgerakan enzim dalam kaj ian ini .

Suhu optimum l ipas e disekatgerak sepert i ini didapati

tidak bertuka r . Wa lau baga imanapun , pil i han terhadap a s id

l emak sebagai penderma kumpulan asil didapati bertukar. Asid

lemak rantai s ederhana l ebih di sukai berbanding dengan asid

l emak rantai panjang apabila enzim disekatgerak. Aktivitinya

juga tinggi di dalam pelarut organik yang l ebih polar. Lipase

tersekatgerak menunjukkan kestabilan terma , kestabilan dalam

pelarut organik dan kestabilan penyimpanan yang lebih tinggi

lagi daripada l ipase natif dan lipase terubahsuai .

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