SYNTHESIS AND CHARACTERIZATION OF ALCOHOL-FREE TYROSINASE

ENCAPSULATED SILICA AEROGEL

NOR SURIANI BINTI HJ. SANI

UNIVERSITI TEKNOLOGI MALAYSIA

SYNTHESIS AND CHARACTERIZATION OF ALCOHOL-FREE TYROSINASE ENCAPSULATED SILICA AEROGEL

NOR SURIANI BINTI HJ. SANI

A thesis submitted in fulfilment of the requirements for the award of the degree of

Master of Science (Chemistry)

Faculty of Science Universiti Teknologi Malaysia

AUGUST 2009

iii

YÉÜ Åç uxÄÉäxw ytÅ|Äç? a|~ T{Åtw a|étÅ u|Ça|~ `tÄx~ tÇw

a|~ T{Åtw mtÜxxy u|Ça|~ T{Åtw a|étÅ

iv

ACKNOWLEDGEMENT

In the name of Allah the Almighty Lord of the world. Thanks to Him for

giving me the opportunity and will to finish this thesis. Heartfelt thanks to my

project supervisor, Prof. Dr. Halimaton Hamdan, who introduced me to the field of

catalysis. Her patience, criticism and thoughtful guidance throughout this study are

greatly appreciated. Without her continued support and interest, this thesis would not

be the same as presented here. I would like to express my sincere appreciation and

gratitude to Dr. Lee Siew Ling for her knowledge, guidance, evaluation and

supporting me throughout the undertaking of this research. I am also very thankful to

my research team, especially to Mrs. Fitri Hayati, Mrs. Rozana Abu Bakar and Ms.

Nurul Hidayah Yunus for giving me the motivation, knowledge, assistance and

valuable advice in completing this project.

My appreciation also goes to all the staff at Faculty of Science, UTM and

Department of Chemistry, who in many ways contributes to the success of my study.

Not forgetting my beloved husband, Nik Ahmad Nizam Nik Malek for his endless

support, encouragement, friendship, advice and understanding. I greatly appreciate

it.

I want to extend my utmost gratitude and appreciation to my parents, family,

fellow friends and those who provide assistance in this research either intentionally

or unintentionally throughout the progress of this research. Finally, I would like to

acknowledge MOSTI, IRPA and NSF for financial support and scholarship.

v

ABSTRACT

Encapsulation of tyrosinase enzyme into nanoporous silica aerogel via an alcohol-free colloidal sol-gel route using rice husk ash (RHA) as silica source was studied. Tyrosinase encapsulated silica aerogel (TESA) was synthesized with and without solvent extraction process at room temperature and neutral pH in order to study their effect on the enzyme activity and to minimize enzyme denaturation. The physicochemical properties of TESA was characterized by X-ray diffraction (XRD) technique, fourier transformed-infrared (FTIR) spectroscopy, field emission-scanning electron microscopy (FESEM), energy dispersive X-ray (EDX) analysis, transmission electron microscopy (TEM) and thermogravimetric analysis (TGA). These characterizations confirmed tyrosinase in TESA was located inside the network of silica aerogel. Enzymatic activity of tyrosinase was assayed through the reduction of ascorbic acid using UV Visible spectrophotometer. Almost 98% of tyrosinase was successfully loaded into silica aerogel as determined by the leaching test of TESA. TESA without solvent extraction showed higher tyrosinase activity than TESA extracted by amyl acetate/acetone (v/v:1/1). The highest activities for both TESA were obtained with 10.00 mg/mL of enzyme loading that was aged for 2 days. The stability of tyrosinase in TESA was enhanced towards extreme temperature as well as acidic and basic conditions. Free tyrosinase was totally inactivated at pH < 4 and pH > 9 and at temperature exceeding 55 °C, while TESA showed a significant activity at these conditions. In the application of TESA, about 80% of phenol was removed after 3 hours contact with TESA. The reusability of tyrosinase in TESA was observed to be very high since TESA can be reused to remove phenol up to 10 times without significant loss. As a conclusion, nanoporous silica aerogel from RHA prepared with and without solvent extraction techniques can be used as suitable support for the improvement of the tyrosinase stability and it can be applied to remove phenol.

vi

ABSTRAK

Kajian terhadap enzim tirosinase yang dikapsulkan ke dalam aerogel berasaskan silika melalui kaedah koloid sol-gel tanpa melibatkan alkohol telah dijalankan. Pengkapsulan tirosinase ke dalam aerogel berasaskan silika (TESA) yang mempunyai liang bersaiz nano telah disintesis menggunakan abu sekam padi (RHA) sebagai sumber silika. Di dalam kaedah ini, TESA disintesis pada suhu bilik dan pH neutral melalui proses pengekstrakan dan tanpa pengekstrakan bagi mengkaji kesannya terhadap aktiviti enzim di samping mengurangkan penyahaslian enzim. Sifat-sifat fiziko-kimia TESA telah dicirikan dengan kaedah XRD, FTIR, FESEM, EDX, TEM dan TGA. Pencirian mengesahkan bahawa tirosinase telah berjaya dikapsulkan ke dalam silika aerogel dan ianya terletak di dalam rangkaian jaringan silika aerogel. Aktiviti tirosinase telah ditentukan dengan kaedah spektofotometer UV Tampak melalui proses penurunan asid askorbik. Hasil Ujian Larut Lesap terhadap TESA menunjukkan hampir 98% tirosinase berjaya dimuatkan di dalam silika aerogel. TESA yang disintesis tanpa melibatkan pengekstrakan mempunyai aktiviti yang lebih tinggi berbanding TESA yang disintesis melalui pengekstrakan menggunakan pelarut amil asetat/aseton (v/v:1/1). Aktiviti yang tinggi bagi kedua-dua TESA diperolehi apabila sebanyak 10.00 mg/mL enzim dikapsulkan ke dalam silika aerogel pada suhu bilik selama 2 hari. Kestabilan tirosinase di dalam TESA terhadap asid dan alkali meningkat. Tirosinase tidak menunjukkan sebarang aktiviti pada pH < 4 dan pH > 9 juga pada suhu melebihi 55 °C tetapi tirosinase di dalam TESA menunjukkan aktiviti pada keadaan tersebut. Kajian penggunaan TESA terhadap penyingkiran fenol menunjukkan 80% fenol berjaya disingkirkan selepas bertindak balas dengan TESA selama 3 jam. Kebolehan tirosinase di dalam TESA untuk menyingkirkan fenol berulang-kali adalah tinggi kerana TESA boleh digunakan sehingga 10 kali tanpa menunjukkan sebarang penyusutan yang ketara. Kesimpulannya, silika aerogel daripada RHA, dengan liang bersaiz nano, yang disintesis sama ada melalui proses pengekstrakan atau tanpa proses pengekstrakan, mampu menjadi tapak kepada enzim bagi meningkatkan kestabilan tirosinase dan menyingkirkan fenol.

vii

TABLE OF CONTENTS

CHAPTER TITLE PAGE

TITLE i

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES xi

LIST OF FIGURES xii

LIST OF SYMBOLS xv

LIST OF ABBREVIATIONS xvi

LIST OF APPENDICES xvii

I INTRODUCTION 1

1.1 Background of Research 1

1.2 Problem Statement 4

1.3 Hypothesis 5

1.4 Objectives of Research 7

1.5 Scope of Research 7

1.6 Outline of Research 9

viii

II LITERATURE REVIEW 10

2.1 Rice Husk Ash as a Silica Source 10

2.2 Silica Aerogel 12

2.3 The Sol-Gel Process 14

2.3.1 Hydrolysis and Condensation 15

2.3.2 Gelation and Aging 19

2.3.3 Drying 19

2.4 Immobilization of Enzyme 22

2.4.1 Enzyme as Catalyst 23

2.4.2 Immobilization Techniques 24

2.4.3 Immobilization of Enzymes by

Encapsulation via Sol-Gel Method

25

2.5 Tyrosinase 27

2.5.1 Immobilized Tyrosinase in the

Removal of Phenol

30

III RESEARCH METHODOLOGY 33

3.1 Raw Materials and Chemical Reagents 33

3.2 Synthesis of Tyrosinase Encapsulated Silica

Aerogel (TESA)

34

3.2.1 Preparation of Sodium Silicate 34

3.2.2 Synthesis of Wet Gel 35

3.2.3 Drying of Wet Gel 35

3.3 Characterization of Tyrosinase Encapsulated

Silica Aerogel (TESA)

36

3.3.1 X-Ray Diffraction (XRD) Technique 37

3.3.2 Fourier Transformed-Infrared (FTIR)

Spectroscopy

40

3.3.3 Field Emission-Scanning Electron

Microscopy (FESEM)

43

3.3.4 Energy Dispersive X-ray Analysis

(EDX)

44

ix

3.3.5 Transmission Electron Microscopy

(TEM)

45

3.3.6 Thermogravimetry Analysis 46

3.4 Optimization of Synthesis Condition 47

3.4.1 Effect of Solvent Extraction 47

3.4.2 Effect of Aging Period 48

3.4.3 Effect of Enzyme Loading 48

3.5 Assays of Enzymatic Activity 49

3.5.1 Assay of Free Tyrosinase Activity 50

3.5.2 Assay of Encapsulated Tyrosinase

Activity

51

3.5.3 Leaching Study 53

3.5.4 Influence of Temperatures 53

3.5.5 Influence of pH 54

3.6 Application of Tyrosinase Encapsulated Silica

Aerogel (TESA)

54

3.6.1 The Removal of Phenol 54

3.6.2 Reusability 55

IV RESULTS AND DISCUSSION 56

4.1 Synthesis of Tyrosinase Encapsulated Silica

Aerogel (TESA)

56

4.2 Characterization of Tyrosinase Encapsulated

Silica Aerogel (TESA)

58

4.2.1 Physical Appearance 59

4.2.2 X-ray Diffraction (XRD) Analysis 59

4.2.3 FTIR Spectroscopy 60

4.2.4 Field Emission Scanning Electron

Microscopy (FESEM)

64

4.2.5 Energy Dispersive X-ray Analysis

(EDX)

68

4.2.6 Transmission Electron Microscopy

(TEM)

69

x

4.2.7 Thermal Analysis 73

4.3 Optimization of Synthesis Conditions 75

4.3.1 Effect of Solvent Extraction 75

4.3.2 Effect of Aging Period 78

4.3.3 Effect of Enzyme Loading 79

4.4 Enzymatic Activity 81

4.4.1 Assay of Free Tyrosinase and

Tyrosinase Encapsulated Silica

Aerogel (TESA) Activity

81

4.4.2 Leaching Study 83

4.4.3 Influence of Temperatures 83

4.4.4 Influence of pH 85

4.5 Application of Tyrosinase Encapsulated Silica

Aerogel (TESA)

87

4.5.1 The Removal of Phenol 87

4.5.2 Reusability 88

V CONCLUSIONS AND RECOMMENDATIONS 91

5.1 Conclusions 91

5.2 Recommendations 94

REFERENCES 96

APPENDICES 111

xi

LIST OF TABLES

TABLE NO. TITLE PAGE 3.1 The assignments of the main FTIR bands for silica

42

4.1 FTIR wavenumbers and assignment for the functional group present in the tyrosinase

62

4.2 FTIR assignments of silica aerogel

62

4.3 Major elemental analysis (mass% ± SD) of free tyrosinase, silica aerogel (with and without solvent extraction) and TESA (with and without solvent extraction)

68

xii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

1.1 The encapsulation of tyrosinase into silica aerogel

network

6

1.2 Flow diagram of research activities

9

2.1 (a) Hydrolysis, (b) condensation and (c) polycondensation reactions, during the sol-gel process in the synthesis of silica aerogel

17

2.2 Polymeric structure of silica framework in acidic and basic conditions

18

2.3 Monooxygenation reaction catalyzed by tyrosinase

28

2.4 Oxidation of o-diphenol to o-quinone

29

2.5 Oxidation of phenol catalyzed by tyrosinase

31

3.1 Derivation of Bragg’s law for X-ray diffraction

38

3.2 The illustration of the X-ray powder diffraction method

39

3.3 Types of silanols exist in silica surface

41

4.1 Optical absorption spectra of (a) free tyrosinase, λmax = 289.14 nm, (b) TESA (with solvent extraction), λmax = 291.28 nm and (c) TESA (without solvent extraction), λmax = 292.91 nm

57

4.2 XRD diffractogram of (a) silica aerogel and (b) TESA

60

xiii

4.3 The FTIR spectra of (a) silica aerogel and (b) free tyrosinase

61

4.4 FTIR spectra of (a) free tyrosinase, (b) silica aerogel, (c) TESA with SE and (d) TESA without SE

64

4.5 FESEM micrographs showing the surface morphology of (a) silica aerogel with SE and (b) TESA with SE

65

4.6 FESEM micrographs showing the surface morphology of (a) silica aerogel without SE and (b) TESA without SE

66

4.7 FESEM micrograph showing the surface morphology of free tyrosinase

67

4.8 TEM micrographs showing the surface morphology of (a) silica aerogel with SE and (b) TESA with SE

70

4.9 TEM micrographs showing the surface morphology of (a) silica aerogel without SE and (b) TESA without SE

71

4.10 Thermogravimetry analysis (TGA) and Derivative thermogravimetry (DTG) curves of (a) silica aerogel without SE, weight loss = 21.102%, (b) silica aerogel with SE, weight loss = 16.284%, (c) TESA without SE, weight loss = 10.696%, (d) TESA with SE, weight loss = 6.632% and (e) free tyrosinase, weight loss = 100%

74

4.11 Effect of solvent extraction to the enzymatic activity of TESA with SE

76

4.12 Enzymatic activities of (a) TESA with SE and (b) TESA without SE; at different aging periods

78

4.13 Enzymatic activities of (a) TESA with SE and (b) TESA without SE; of different enzyme loadings

80

4.14 Enzymatic activity of (a) free tyrosinase, (b) TESA without SE, (c) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1) and (d) without tyrosinase; at pH 7

82

4.15 Enzymatic activities of (a) free tyrosinase, (b) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1) and (c) TESA without SE; at different temperatures

84

xiv

4.16 Enzymatic activities of (a) free tyrosinase, (b) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1) and (c) TESA without SE; at different pH

86

4.17 The effect of contact time on the percent removal of phenol by (a) free tyrosinase, (b) TESA without SE and (c) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1)

88

4.18 Reusability studies of (a) TESA without SE and (b) TESA with SE; extracted by amyl acetate/acetone (v/v:1/1), in the removal of phenol

90

xv

LIST OF SYMBOLS

°C - Degree Celsius

cm - Centi meter

g - Gram

K - Kelvin

kV - Kilo Volt

L - Liter

M - Molar

m - Meter

mg - Mili gram

min - Minute

mL - Mili Liter

mM - Mili Molar

mm - Mili meter

nm - Nano meter

ppm - Part per million

rpm - Rotation per minute

v - Volume

W - Watt

Å - Angstrom

μg - Micro gram

μL - Micro Liter

λ - Lambda

θ - Theta

xvi

LIST OF ABBREVIATIONS

APD - Ambient Pressure Drying

CaA - Calsium Aluminosilicate

DDW - Double Distilled Deionized Water

DTG - Derivative Thermogravimetric Analysis

EDTA - Ethylene Diamine Tetracetic Acid

EDX - Energy Dispersive X-ray Technique

FESEM - Field Emission Scanning Electron Microscopy

FTIR - Fourier Transform Infrared

HMDSO - Hexamethyldisiloxane

HPLC - High Performance Liquid Chromatography

IR - Infrared

NaA - Sodium Aluminosilicate

NADH - Nicotinamide Adenine Dinucleotide

PFC - Plug Flow Combustor

RHA - Rice Husk Ash

SE - Solvent Extraction

SD - Standards Deviation

TEM - Transmission Electron Microscopy

TESA - Tyrosinase Encapsulated Silica Aerogel

TEOS - Tetraethyl Orthosilicate

TMOS - Tetramethyl Orthosilicate

TGA - Thermogravimetric Analysis

UV-Vis - Ultra Violet-Visible

XRD - X-Ray Diffraction

xvii

LIST OF APPENDICES

APPENDIX TITLE PAGE

A Determination of Ascorbic Acid using UV-Vis Spectrophotometer

111

B EDX Elemental Analysis of Tyrosinase, Silica Aerogel and TESA

114

C Determination of Phenol using UV-Vis Spectrophotometer

119

D Paper for R&D Nanotechnology Symposium 2007, Malaysia

121

E Paper for 26th International Symposium on Space Technology and Science (ISTS), Japan

128

CHAPTER 1

INTRODUCTION

1.1 Research Background

Nanotechnology, the process to generate, manipulate and employ

nanomaterials, represents an area holding significant promise for health care and

biotechnology in many years to come [1-4]. Nanotechnology is now poised to

revolutionize the biomedical field ranging from basic studies to disease diagnosis

and treatment. Understanding the principle of nanotechnology may provide insight

into critical biological system related to disease control, correction of genetic

disorder and longevity [3].

Biosensing is one of the most emerging sectors of nanotechnology. Such

growth is mainly derived from the expansion of R&D where nano dimension

research is introduced to analyze living cell constituents and for efficient drug

screening [5]. Biosensors are devices that incorporate biologically active element in

intimate contact with a physico-chemical signal; utilize the high sensitivity and

selectivity of biological sensing for analytical purposes in various fields of research

and technology [6]. Many proteins are expected to play the role as biocomponents

used in biosensors. Enzymes are large protein molecules that significantly increase

2

rates of reaction by lowering the activation energy. Enzymes specifically accelerate a

huge number of chemical reactions at room temperature and normal pressure [7].

Enzymes offer three major advantages [8]:

• Higher reaction rates: Up to a factor of 1012 times greater than the

uncatalyzed reactions

• Reaction conditions: Enzymes operate at room temperatures less than

100 oC, atmospheric pressures and near neutral pH

• Reaction specificity: Enzyme specificity for both substrate and product

produces fewer side reactions

Immobilizations of biological compounds into inorganic support are usually

applied in various fields such as biosensing [9-11], affinity chromatography [12-14]

and enzyme reactors [15-17]. One of the most challenging aspects in the

development of these matrices is the integration of biological molecules in the host

matrix and retaining the functionality of the biomolecules [10]. In biosensing, it is

advantageous to use immobilized enzymes rather than free enzymes [18-19].

Following are the reasons for immobilization of enzyme:

• To improve the stability of enzyme in adverse reaction conditions

• To improve the stability of enzyme in the presence of organic solvents

• To separate the enzyme from product stream

• To allow a continuous flow operations and repetitive usage

The application of immobilized enzymes in analytical chemistry is not a new

concept. The importance of immobilized enzymes as analytical reagents in clinical

chemistry [20-21], food analysis [22-23] and the pharmaceutical industry [24-25] has

3

been steadily increasing. To simplify the enzymatic measurement of glucose, the

principle of the litmus paper used for pH measurement has been implemented [6].

The first ‘enzyme test strip’ has been obtained by the impregnation of filter paper

with the glucose-converting enzymes. It can be regarded as the predecessor of

optoelectronic biosensors which initiated the development and application of ‘dry

chemistry’ [26]. Apart from the application of complex biocatalytic system, intense

effort are being made to broaden the spectrum of measurable substances and to

improve the analytical parameters of biosensors by the immobilization of several

enzymes in a silica host matrixes.

Silica host matrixes, made by the sol-gel process have emerged as a

promising platform for immobilization of enzymes [19, 26]. The advantages of their

usage in enzyme immobilization include:

• The high surface area of silica matrix provide the possibility of high

enzyme loadings in the matrix

• The silica matrix consists of surface hydroxyl groups that can be readily

attached by enzyme

• The open pore morphology of silica matrix allows substrates to quickly

move into the interior regions of the particle

• Solvents used in the processing of the silica materials are

environmentally benign thus avoid the denaturation of enzyme

Numerous techniques such as physical adsorption, covalent attachment,

entrapment and encapsulation in polymer and inorganic matrixes have been explored

over the years to achieve a high-yield, reproducible and robust immobilization

technique that preserves the activity of the biological molecules [10, 27-29].

Enzymes find a more stable environment upon encapsulation in a silica host, because

the polymeric framework grows around the biomolecules, creating a cage, thus

4

protecting the enzyme either from aggregation and unfolding or from microbial

attack. Encapsulations also offer a protection for the enzyme against deterioration by

the hydrophilic solvent, if the proper gel is selected [28]. Therefore, the encapsulated

biomolecules often retain a sufficient level of activity and functionality presumably

because of sufficient retention of their native state conformations. Moreover, the

matrix pores allow the diffusion of reactant molecules and their reaction with the

encapsulated biomolecules. Eventually, encapsulated enzyme can even improve the

activity and storage stability of the enzymes and will be easier to be used because

they can easily be recovered and washed [30].

In this research, tyrosinase was used as model enzyme because of its wide

application in medicine, environmental and industrial systems [8, 24]. Tyrosinase is

also suitable for the treatment of phenolic wastes [9, 11, 17]. Tyrosinase like most

other enzymes, is expensive and thus the use of the soluble enzyme is not practical

[2]. Therefore, the encapsulation of tyrosinase is very attractive in order to exploit its

catalytic properties and improve the cost effectiveness [5]. The improved stability of

the encapsulated enzyme allows it to be highly reusable. Moreover, since enzyme

does not dissolve in the solution, further purified process is not required and hence

the encapsulated tyrosinase is economical to be used repetitively [8].

1.2 Problem Statement

In most of the reported applications, an orthosilicate such as tetramethyl

orthosilicate (TMOS) or tetraethyl orthosilicate (TEOS) has been used as silica

source in the synthesis of proteins encapsulated silica monoliths [30-33]. These silica

sources present the advantages of relatively high purity sources of silica but lowering

the enzymatic activity. 70% reduction of enzymatic activity was reported when

lipase was encapsulated into TMOS-based silica matrix due to the presence of 5%

volume of methanol in the reaction [27]. The usage of TMOS or TEOS as starting

5

materials lead to the generation of alcohol as a by-product and the presence of

alcohol has been known to be detrimental to the activity of proteins by causing chain

unfolding, aggregation, destruction of secondary and tertiary protein structures to a

significant extent [28]. Moreover, such organic silicon precursors are usually too

expensive. So the production of silica aerogel in an industrial scale is not

economically practical.

Owing to their mesoporous structure, high specific surface area and

extremely low thermal conductivity, aerogels are considered as an efficient candidate

as a support for protein immobilization. However, the high level of sophistication

and the risks involved in the supercritical drying of the gels prohibit the commercial

production of the aerogels and their wide exploitation in various potential

applications [34-36]. Supercritical drying process is too energy intensive and

dangerous that real practice and commercialization are difficult. Therefore, it is

necessary to synthesize silica aerogels by an ambient pressure drying (APD)

technique at a reasonable cost [37].

1.3 Hypothesis

An alcohol-free aqueous colloidal sol-gel for the synthesis of silica monoliths

with encapsulated biological entities that uses rice husk ash as the cheap silicon

source for production of pure silicate solution has been developed [28, 38]. This

approach completely avoids the generation of alcohol and it allows encapsulation to

be carried out at neutral pH and ambient pressure in order to preserve biological

activity of proteins. Thus, denaturation of the biomolecule caused by the undesirable

interaction with alcohol molecules can be avoided and the degree of conformational

change can be reduced.

6

In this research, after the synthesis is complete, tyrosinase molecule is

expected to be encapsulated in the silica aerogel network since the tyrosinase was

added into the silica sol before the gelation stage. As shown in Figure 1.1, after the

addition of tyrosinase, the silica sol begins to link together in three-dimensional (3D)

network and subsequently, creates a cage around the tyrosinase molecule. It is also

known that when enzyme molecules are mixed with colloidal particles, the

interaction between them may result in enzyme adsorption on the particle surface.

Furthermore, the preparation of silica aerogel using water glass precursor followed

by an ambient pressure drying is the cheapest and safest method.

6

EnzymeSupport network

Figure 1.1 The encapsulation of tyrosinase into silica aerogel network

This research is proposed to develop an effective method for the

immobilization of biomolecule into silica aerogel matrix. Unrevealing the

interactions which take place in encapsulation of enzyme in the silica aerogel matrix

will contribute to better understanding of various chemical and biochemical

processes that occur when different synthesis conditions are applied. The

fundamental understanding of the enzyme-silica support interactions can help in

improving the fabrication of enzyme encapsulated silica aerogel as a potential

7

biosensor with enhanced thermal stability and enzymatic performance. Hence, this

research enables new materials for biosensors to be developed.

1.4 Objectives of Research

The objectives of this research are to:

i) Synthesize and characterize tyrosinase encapsulated silica aerogel

ii) Investigate the influence of synthesis conditions on biocatalytic activities of

tyrosinase encapsulated silica aerogel

iii) Investigate the enzymatic activity of tyrosinase encapsulated silica aerogel

iv) Study the application of tyrosinase encapsulated silica aerogel in the removal

of phenol

1.5 Scope of Research

The project is conducted following various phases as outlined in the flow

diagram in Figure 1.2. The tyrosinase encapsulated silica aerogel (TESA) was

synthesized via alcohol-free aqueous colloidal sol-gel process according to the

established method [28] but with some modification. TESA, which is synthesized

from rice husk ash as a silica source, was synthesized with and without solvent

extraction (SE) process in order to study their relationship with the enzyme activity.

The products were characterized for their textural properties by using X-ray

diffraction technique (XRD) and Fourier transformed-infrared spectroscopy (FTIR).

The interactions of tyrosinase-silica aerogel and its surface morphology were studied

8

by using Field emission-scanning electron microscopy (FESEM), Energy dispersive

X-ray spectroscopy (EDX), Transmission electron microscopy (TEM) and

Thermogravimetric analysis (TGA).

Some of the synthesis parameters in a sol-gel process were investigated for

their influences on biocatalytic activities of encapsulated enzyme namely effect of

solvent extraction and enzyme loading. In order to obtain information regarding the

tyrosinase-silica aerogel interaction, wet gels containing encapsulated tyrosinase was

submitted to different aging periods before drying phase.

Meanwhile, biocatalytic activities of the free tyrosinase and TESA were

assayed by examining the catecholase activity using UV-Vis spectrophotometer. The

properties of TESA were evaluated by studying the activity of tyrosinase at different

temperatures and pH ranges, as well as leaching test. TESA was used to remove

phenol in aqueous solution and their efficiency in removing phenol and the stability

of tyrosinase in TESA was determined through reusability study.

9

1.6 Outline of Research

Synthesis of tyrosinase encapsulated silica aerogel (TESA)

TESA without solvent extraction (SE) TESA with solvent extraction (SE)

Characterizations of TESA

X-ray diffraction technique (XRD) Fourier transformed-infrared spectroscopy (FTIR) Field emission-scanning electron microscopy (FESEM) Energy dispersive X-ray spectroscopy (EDX) Transmission electron microscopy (TEM) Thermogravimetry analysis (TGA)

Optimization of synthesis conditions Effect of solvent extraction Aging period Enzyme loading

Enzyme assay: UV-Vis Spectrophotometer Measurement of tyrosinase activity Leaching test Influence of temperatures Influence of pH

Application of TESA Removal of phenol Reusability

Figure 1.2 Flow diagram of research activities