UNIVERSITI PUTRA MALAYSIA

ADSORPTION IMMOBILIZATION OF CANDIDA RUGOSA LIPASE ONTO ALUMINOSILICATE SUPPORTS AS BIOCATALYSTS FOR

FATTY ACID SUGAR ESTER SYNTHESES

USWATUN HASANAH BT. ZAIDAN

FS 2011 85

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ADSORPTION IMMOBILIZATION OF CANDIDA RUGOSA LIPASE ONTO

ALUMINOSILICATE SUPPORTS AS BIOCATALYSTS FOR FATTY ACID SUGAR ESTER SYNTHESES

By

USWATUN HASANAH BT. ZAIDAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra

Malaysia, in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

June 2011

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement of the degree of Doctor of Philosophy

ADSORPTION IMMOBILIZATION OF CANDIDA RUGOSA LIPASE ONTO ALUMINOSILICATE SUPPORTS AS BIOCATALYSTS FOR

FATTY ACID SUGAR ESTER SYNTHESES

By

USWATUN HASANAH BT. ZAIDAN

June 2011

Chairman : Professor Mohd Basyaruddin Abdul Rahman, PhD Faculty : Science

Nowadays, the industrial applications of the biocatalysts have not yet reached a

significant level of use due to the high cost, instability and the inconvenience of

separating, recycling and reusing of the enzymes. These deficiencies have

motivated researchers to improve their catalytic properties by immobilization of

enzymes onto selected supports. The high cost of popular supports such as

calcium carbonate, rice husks and rice straw or kaolin causes many to search

for cheaper substitutes from the natural environment. Instead of these

alternatives, feldspar and mica appear to be most attractive since they are

naturally available and have the potential to be used as low cost sorbents.

In this study, aluminosilicates of feldspar and mica originated from the quary

area of Tanah Putih, Gua Musang, Kelantan (Malaysia) were used as enzyme

supports. They were found to have high surface area, large porosity and good

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mesoporous properties. Mica was physically modified either by acid treatment,

grafting with Amino-, Octyl-, Vinyl-, Mercapto- and Glycidoxy-triethoxysilanes,

and activation of pre-treated support with glutaraldehyde (Glu). The modified

mica preparations were used for immobilization of lipase from Candida rugosa

(CRL) and exhibited improved specific activity as compared to the free enzyme

by 1.8 - 2.4 folds. Lipase immobilized on mica showed enhanced protein

loading (up to 8.2 mg protein/g support) and immobilization (up to 78%)

compared to the free lipase (Free-CRL) and those immobilized on unmodified

mica. However, another two new approaches of enzyme-aggregate-coating

(EAC-CRL) and nanoscale-enzyme-reactor (NER-CRL) were found to perform

higher specific activities of about 2.6 folds.

The selected immobilized lipases were then used in the esterification reaction to

optimize the reaction conditions factors. Optimal production conditions (83%)

for the synthesis of fatty acid sugar esters (FASEs) were achieved after 48

hours with the use of capric acid (C10) and lactose sugar as substrates at

temperature of 55 °C. In addition, substrates molar ratio of 1:2 (capric acid to

lactose) exhibited the highest ester conversion in acetone which was the best

solvent used. On the other hand, the operational stability with half lives of over

13, 10, 7 and 6 reaction cycles for NER-CRL, Amino-CRL, EAC-CRL and

GluAmino-CRL, respectively, indicated the efficiency of the immobilization

process. The best performance by the immobilized lipase, in terms of both

activity and stability was achieved by NER-CRL, which was prepared through

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immobilization using the combination of physical adsorption and cross-linking

approaches of lipase on unmodified mica support.

The formation of lactose caprate from lactose sugar (Lac) and capric acid (CA)

using biocatalysts were also evaluated through a kinetic study. Determination of

the apparent kinetic parameters of Km and Vmax by means of Michaelis-Menten

kinetic model was used. The Ping-Pong Bi-Bi mechanism with one substrate

inhibition was adopted as it best explained the experimental findings. The

kinetic results showed lower Km values (CA = 14.5 mmol/L.mg); Lac = 9308

mmol/L.mg) of the NER-CRL when compared to the Free-CRL, indicating a

higher affinity of the NER-CRL towards the both substrates (Km,app (CA, Lac) < Km

(CA, Lac)), thus yielding an increase in reaction rate. The kinetic parameters

deduced from this model were used to simulate the initial rate data, which was

in excellent agreement with the experimental values.

The physicochemical properties of synthesized FASEs product of lactose

caprate have been studied in order to meet their potential as high value-added

biosurfactants. The present study had shown that Candida rugosa lipase

immobilized on natural and low cost mica support was successfully employed

as potential biocatalyst for FASEs syntheses. Furthermore, immobilized lipase

preparations were able to enhance the productivity and stability, suggesting that

the preparations are more versatile and adaptable for FASEs production as

potential biosurfactant in industrial applications.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah

PENYEKATGERAKAN LIPASE DARIPADA CANDIDA RUGOSA KE ATAS PENYOKONG ALUMINOSILIKAT SEBAGAI BIOPEMANGKIN DALAM

SINTESIS ESTER GULA ASID LEMAK

Oleh

USWATUN HASANAH BT. ZAIDAN

June 2011

Pengerusi : Profesor Dr. Mohd Basyaruddin Abdul Rahman, PhD Fakulti : Sains

Pada masa ini, aplikasi industri terhadap biokatalis belum mencapai tahap yang

signifikan disebabkan oleh kos enzim yang tinggi, ketidakstabilan serta

kesukaran dalam pemisahan dan penggunaan semula enzim. Semua

kekurangan ini telah menggalakkan penyelidik meningkatkan ciri-ciri

pemangkinan melalui penyekatgerakan enzim ke atas bahan penyokong

terpilih. Kos bahan penyokong yang tinggi seperti kalsium karbonat, sekam padi

dan jerami padi atau kaolin menyebabkan ramai mencari pilihan yang lebih

murah dari sumber semulajadi. Selain alternatif ini, felspar dan mika menonjol

dan menarik kerana wujud secara semulajadi dan berpotensi untuk digunakan

sebagai penyokong kos rendah.

Dalam kajian ini, aluminosilikat felspar dan mika yang berasal dari tapak kuari

di Tanah Putih, Gua Musang, Kelantan (Malaysia) telah digunakan sebagai

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penyokong enzim. Mereka didapati mempunyai luas permukaan dan porositi

yang tinggi serta memiliki ciri-ciri bahan mesoporos. Mika telah diubahsuai

melalui rawatan asid, penambahan kumpulan Amino-, Oktil-, Vinil-, Merkapto-

dan Glisidoksi-trietoksisilan, dan pengaktifan menggunakan glutaraldehid (Glu).

Mika terubahsuai telah digunakan untuk penyekatgerakan lipase daripada

Candida rugosa (CRL) dan menunjukkan peningkatan aktiviti spesifik

berbanding enzim bebas iaitu pada 1.8 - 2.4 kali ganda. Lipase tersekatgerak

pada mika menunjukkan penjerapan protein (sehingga 8.2 mg protein/g) dan

penyekatgerakan (sehingga 78%) berbanding dengan lipase bebas (Free-CRL)

dan lipase pada mika takterubahsuai. Namun, dua pendekatan baru enzim-

agregasi-lapisan (EAC-CRL) dan nano-enzim-reaktor (NER-CRL) masing-

masing didapati mempamerkan aktiviti lebih tinggi iaitu sehingga 2.6 kali ganda.

Lipase tersekatgerak terpilih telah digunakan dalam sintesis ester untuk

mengkaji faktor-faktor tindakbalas. Keadaan optimum (83%) sintesis ester gula

asid lemak (FASEs) telah dicapai pada 48 jam menggunakan asid kaprik (C10)

dan laktosa sebagai substrat pada suhu 55 °C. Selain itu, nisbah molar substrat

1:2 (asid kaprik kepada laktosa) telah mempamerkan penukaran ester tertinggi

dalam aseton sebagai pelarut terbaik. Malah, kestabilan operasi separuh hayat

melebihi 13, 10, 7 dan 6 kitaran tindakbalas bagi NER-CRL, Amino-CRL, EAC-

CRL dan GluAmino-CRL, masing-masing menunjukkan kecekapan proses

penyekatgerakan. Prestasi terbaik lipase tersekatgerak, baik dari segi aktiviti

dan kestabilan telah dicapai oleh NER-CRL, yang telah disediakan melalui

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penyekatgerakan menggunakan kombinasi penjerapan fizikal dan pendekatan

interaksi lipase pada sokongan mika takterubahsuai.

Sintesis laktosa kaprat daripada laktosa (Lac) dan asid kaprik (CA)

menggunakan biokatalis juga telah dinilai melalui kajian kinetik. Penentuan

parameter kinetik Km and Vmax berdasarkan model Michaelis-Menten telah

digunakan. Mekanisma Ping-Pong Bi-Bi dengan satu perencatan substrat

adalah yang terbaik menjelaskan penemuan eksperimen. Keputusan kinetik

menunjukkan nilai Km (CA = 14.5 mmol/L.mg); Lac = 9308 mmol/L.mg) bagi

NER-CRL lebih rendah berbanding dengan Free-CRL, dan menunjukkan

bahawa NER-CRL mempunyai afiniti yang lebih tinggi terhadap kedua-dua

substrat (Km,app (CA, Lac) < Km (CA, Lac)), seterusnya meningkatan kadar tindakbalas.

Parameter kinetik dari model ini telah digunakan untuk mensimulasikan data

kadar awal tindakbalas, yang sangat menepati dengan nilai-nilai eksperimental.

Sifat-sifat fizik-kimia produk laktosa kaprat telah dikaji dalam rangka memenuhi

potensi produk sebagai biosurfaktan dengan nilai tambah yang tinggi.

Penyelidikan ini telah menunjukkan bahawa lipase tersekatgerak dari bahan

semulajadi dan berkos rendah telah berjaya digunakan sebagai suatu biokatalis

yang berpotensi untuk sintesis FASEs. Lebih-lebih lagi, lipase tersekatgerak

berupaya meningkatkan produktiviti dan kestabilan, seterusnya membuktikan

bahawa penyediaannya adalah lebih fleksibel dan sesuai untuk penghasilan

FASEs sebagai biosurfaktan yang berpotensi dalam aplikasi industri.

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ACKNOWLEDGEMENTS

In The Name of ALLAH, The Most Merciful and Most Beneficent

Alhamdulillah, all praises to Allah S.W.T., Lord of the universe, for giving me the

strength to endure all problems and only by His grace and mercy this thesis can

be completed.

First and foremost, I wish to express my sincere appreciation and gratitude

especially to Prof. Dr. Mohd. Basyaruddin Abdul Rahman and all co-

supervisors, Prof. Dr. Mahiran Basri, Prof. Dr. Abu Bakar Salleh and Dr. Siti

Salhah Othman of the Enzyme and Microbial Technology Research group for

their great concern, constant guidance, advices, patience, persistent

encouragement and precious assistance from the beginning till the end of this

study.

Special thanks are extended to the staff members of the Department of

Chemistry (Faculty of Science, UPM), Microscopy and Microanalysis Unit

(Institute of Bioscience, UPM), Institute of Advance Technology (ITMA, UPM),

Faculty of Science and Technology (USIM) and Malaysia Palm Oil Board

(MPOB, Bangi), who were so helpful and cooperative in many ways during the

course of the study.

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My deepest love and gratitude to my parents, Umi and Abah, for their never-

ending prayers and supports during my education. Never forgetting, I wish

especially to acknowledge my beloved husband, Mohd. Nasir Othman for his

continuous prayers, high encouragement, patience and understanding which

are truly essential and our dearest children, Muhammad Auza’ie and Afif Wahdi,

for simply being there and loving me with all their hearts.

My dearest friends, I wish to thank to Salwa, Ijah, Sook Han, Atena, Emmy,

Nora, Kak Shila and other members of Lab 401, for the bond of friendship, the

great work environment, the funny conversations, helpfulness in the daily life in

the lab and for making my stay in UPM a bearable one with many sweet

memories and experiences. Thank you for being friends in need.

Finally, thank you to the Universiti Putra Malaysia and Ministry of Higher

Education for their financial support and facilities.

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I certify that a Thesis Examination Committee has met on 24 June 2011 to conduct the final examination of Uswatun Hasanah bt. Zaidan on her thesis entitled “Adsorption immobilization of Candida rugosa lipase onto aluminosilicate supports as biocatalysts for fatty acid sugar ester syntheses” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U. (A) 106] 15 March 1998. The Committee recommends that the student be awarded the degree of Doctor of Philosophy. Members of the Examination Committee were as follows: Gwendoline Ee Cheng Lian, PhD Professor Faculty of Science Universiti Putra Malaysia (Chairman) Mohd. Zobir Hussein, PhD Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Mansor Hj. Ahmad @ Ayob, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Hossein Noureddini, PhD Professor Department of Chemical Engineering University of Nebraska-Lincoln United States (External Examiner) ________________________ NORITAH OMAR, PhD Associate Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows: Mohd. Basyaruddin Abdul Rahman, PhD Professor Faculty of Science Universiti Putra Malaysia (Chairman) Mahiran Basri, PhD Professor Faculty of Science Universiti Putra Malaysia (Member) Abu Bakar Salleh, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member) Siti Salhah Othman, PhD Faculty of Science and Technology Universiti Sains Islam Malaysia (Member) _____________________________ HASANAH MOHD. GHAZALI, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date:

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DECLARATION I declare that the thesis is my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously, and is not concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other institution.

______________________________ USWATUN HASANAH BT. ZAIDAN Date: 24 June 2011

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TABLE OF CONTENTS

Page

CHAPTER

1

2

INTRODUCTION Background of Research Problem Statements Objectives Research Objectives LITERATURE REVIEW Enzymes as Nature’s Catalysts Lipases Lipase-Catalyzed Reactions Structure and Properties of Candida rugosa Lipase Mechanisms of Lipase-Catalyzed Reactions Kinetics of Enzyme Immobilization Michaelis-Menten Equation Transformation of Michaelis-Menten Equation Immobilization of Enzymes Advantages of Immobilized Enzymes Effect of Immobilization Environment Supports for Enzyme Immobilization Aluminosilicate Minerals Structure and Properties of Mesoporous Silicates Functionalization of Mesoporous Aluminosilicates Enzyme Immobilization onto Mesoporous Aluminosilicates Enzyme Immobilization onto Functionalized Mesoporous Materials Fatty Acid Sugar Esters Applications of FASEs Enzymatic Synthesis of FASEs

1 2 3 4

5 9

11 14

17 22 24 25 27 29

30 32 34 37 38 40

41

42 43

46

ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPROVAL DECLARATION LIST OF TABLES LIST OF FIGURES LIST OF SCHEMES LIST OF ABBREVIATIONS

ii v

viii x xii xvi xvii xxii xxiii

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3

4

Immobilized Lipase-Catalyzed Synthesis of FASEs MATERIALS AND METHODS Materials Preparation of Natural Aluminosilicates Characterization of Aluminosilicates Fourier Transform Infra-red Spectroscopy Accelerated Surface Area and Porosimetry Analysis Scanning and Transmission Electron Microscopy Energy Dispersive X-ray Analysis Particle Size Analysis Functionalization of Aluminosilicates Hydroxylation of Natural Auminosilicates Post-synthesis Grafting via Silanization Glutaraldehyde-Activation of Support Preparation of Lipase Solution Immobilization of Lipase Protein Assay Bradford Method (Bradford, 1976) Characterization of Immobilized Lipases Enzymatic Esterification of Lipase Assay Optimization Study of FASEs Syntheses Screening of Immobilized Lipases Effect of Saccharides Structures Effect of Time Courses Effect of Fatty Acid Chain Lengths Effect of Various Organic Solvents Effect of Reaction Temperatures Effect of Molar Ratio of Substrates Operational Stability Study Kinetic Behaviours of Free and Immobilized Lipases Identification of Lactose Ester Product Thin Layer Chromatography Fourier Transform Infra-red Spectroscopy Ultra High Pressure Liquid Chromatography Gas Chromatography Mass Spectroscopy Physicochemical Characterizations of Synthesized FASEs RESULTS AND DISCUSSION Characterization of Natural Aluminosilicates X-ray Diffraction Fourier Transform Infra-red Spectroscopy Surface Area and Porosimetry Analysis

48

52 53 53

53 54 54 54

55 55 55

55 56 57 57 59 59 62 62 63 63 64 64 64 65 66 66 67 67 68 68 69 69

70 71

72 72

75 77

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Scanning Electron Microscopy coupled with Energy Dispersive X-ray Transmission Electron Microscopy (TEM) Immobilization of Lipase Preliminary Screening of Lipase Immobilization on Natural Supports Effect of Support Modification on Immobilization Lipase Activity in Esterification Characterization of Immobilized Lipases Lipase Immobilized on Natural Supports Lipase Immobilized on Modified Supports Optimization of the FASEs Syntheses Screening of Immobilized Lipases Effect of Saccharides Structures Effect of Time Courses Effect of Fatty Acid Chain Lengths Effect of Various Organic Solvents Effect of Reaction Temperatures Effect of Molar Ratio of Substrates Operational Stability Study SEM Analyses of Reused Immobilized Lipases Kinetic Performances Study of Free and Immobilized Lipase Initial Rates for the Free and Immobilized Lipase System Kinetic Constants Identification of Sugar Ester Thin Layer Chromatography Fourier Transform Infra-red Spectroscopy Ultra High Pressure Liquid Chromatography Gas Chromatography-Mass Spectrometry Physicochemical Properties of FASEs of Lactose Caprates as Potential Surfactants Hydrophilic-Lipophilic Balance Value Physical State and Solubility Refractive Index and Odor Potential Applications

CONCLUSION

REFERENCES

APPENDICES

BIODATA OF STUDENT

82

82 86 86

89 97

100 100 109 116 116 119 122 125 128 131 134 138 144 148

148

156 162 162 164 167 170 174

177 178 178 179

182

185

206

235