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UNIVERSITI PUTRA MALAYSIA

IDENTIFICATION OF LIPASE-PRODUCING THERMOPHILIC BACTERIA ISOLATED FROM HOT SPRINGS IN MALAYSIA

AZURA LIANA BINTI KAMARUZAMAN

FSTM 2008 7

IDENTIFICATION OF LIPASE-PRODUCING THERMOPHILIC BACTERIA

ISOLATED FROM HOT SPRINGS IN MALAYSIA

By

AZURA LIANA BINTI KAMARUZAMAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirements for the Degree of Master of Science

January 2008

Specially for my loving parents, Kamaruzaman Mohd Yatim and Rashidah Daud,

and siblings, Azila Asfar and Azrul Azlan

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science

IDENTIFICATION OF LIPASE-PRODUCING THERMOPHILIC BACTERIA ISOLATED FROM HOT SPRINGS IN MALAYSIA

By

AZURA LIANA BINTI KAMARUZAMAN

January 2008

Chairman: Associate Professor Nazamid Saari, PhD Faculty: Food Science and Technology

Lipase-producing thermophilic microorganisms were successfully isolated from water

samples collected from six hot springs in the west coast of Peninsular Malaysia and were

identified up to species level. Five hundred and forty-four microbial colonies were

qualitatively screened using Rhodamine B-olive oil agar plate method and sixty-six were

found to be positive as bacterial lipase producers after they formed orange fluorescent

colour around the colonies when irradiated with UV light. Lipolytic activities were

assayed using titrimetric method with olive oil emulsion as substrate. Comparative

analysis among the positive bacterial lipase producers indicated that isolate ST 7 had the

highest lipolytic activity of 4.58 U/ml, followed by isolate ST 6 with an activity of 3.51

U/ml. Eighteen isolates that showed high lipolytic activity were further examined for

thermostability. Thermostability was determined by incubation of the crude lipase at

temperature ranging from 40 to 80°C for 30 minutes. It was found that isolates ST 7 and

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ST 8 produced the most thermostable lipase, which retained 86.70% and 87.52% of the

original activity after incubation at temperature 80°C for 30 minutes, respectively.

Eighteen lipase-producing thermophilic bacteria were further characterized and

identified using morphological characteristics, Biolog Microlog® Bacterial

Identification System and conventional biochemical tests. Strains KW 6, KW 7, ST 1,

ST 6 and ST 10 were identified as CDC Group IVc-2 (Alcaligenes-like) or Ralstonia

paucula. Strains KW 8, KW 10 and DT 17 were identified as Burkholderia

cocovenenans, B. glumae and Photobacterium logei, respectively. Biolog identified

strains ST 7, ST 8, SY 7, KW 1, KW 9, KW 12 and TBN 3 were belonging to genus

Bacilli and strains SY 9, DT 9 and DT 12 as Staphylococcus species. Four strains of the

Bacillus genus with the highest thermostability, ST 7, ST 8, SY 7 and KW 12 were

chosen for further identification using 16S rRNA gene sequence analysis.

The protocol of DNA extraction method applied in this study was Qiagen DNeasy

Tissue Kit. Based on the quantification of extracted DNA and estimation of the purity by

UV spectrophotometer, the Qiagen DNeasy Tissue Kit produced a high DNA yield of all

samples. The primer pair used for specific-PCR generated the same expected PCR

product for all the strains with molecular weight of 1500 bp. Sequences were determined

using an automated sequencer; which was aligned with reference sequences of the

closest related organisms in NCBI database. Based on 16S rRNA gene sequencing, the

highest sequence similarity was found between strain ST 8 and Bacillus subtilis gene for

16S rRNA partial sequence (AB110598 with 100% similarity between nucleotides 1-

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1532). Strains KW 12 and ST 7 were also showed a high sequence similarity at 99.0%;

comparable to those of Anoxybacillus kamchatkensis 16S rRNA gene partial (accession

number: AF510985) and Anoxybacillus flavithermus isolate AB05 16S rRNA gene

partial sequence (accession number: AF001964), respectively. Strain SY 7 displayed

98.0% sequence similarity to Bacillus cereus strain J-1 16S rRNA gene partial sequence

(accession number: AY305275). The 16S rRNA gene sequences for ST 8, ST 7, KW 12

and SY 7 have been deposited into Genbank Data Library and assigned the accession

number DQ401073, DQ193516, DQ401072 and DQ401074, respectively.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains

PENGENALPASTIAN BAKTERIA TAHAN HABA PENGHASIL ENZIM LIPASE DIPENCILKAN DARI KOLAM AIR PANAS DI MALAYSIA

Oleh

AZURA LIANA BINTI KAMARUZAMAN

Januari 2008

Pengerusi: Profesor Madya Nazamid Saari, PhD Fakulti: Sains dan Teknologi Makanan

Mikroorganisma tahan haba penghasil enzim lipase telah berjaya diasingkan dari

sampel-sampel air yang diambil dari enam buah kolam air panas yang terletak di pantai

barat Semenanjung Malaysia dan dikenalpasti hingga ke paras spesies. Lima ratus empat

puluh empat koloni mikroorganisma telah disaring secara kualitatif menggunakan

kaedah piring agar Rhodamine B-minyak zaitun dan enam puluh enam didapati positif

sebagai penghasil enzim lipase setelah membentuk lingkaran cahaya berwarna jingga

mengelilingi koloni apabila disinari cahaya UV. Aktiviti lipolitik dicerakin

menggunakan kaedah titrimetrik dengan emulsi minyak zaitun sebagai substrat. Analisis

perbandingan di antara semua bakteria penghasil lipase yang positif menunjukkan

pencilan ST 7 mempunyai aktiviti lipolitik yang tertinggi iaitu 4.58 U/ml, diikuti oleh

pencilan ST 6 dengan aktiviti 3.51 U/ml. Lapan belas pencilan yang menunjukkan

aktiviti lipolitik yang tinggi kemudiannya diperiksa untuk ujian thermostabiliti.

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Thermostabiliti ditentukan dengan menginkubasi lipase mentah pada suhu 40 hingga

80°C selama 30 minit. Pencilan ST 7 dan ST 8 didapati menghasilkan enzim lipase

yang paling tahan haba, iaitu mengekalkan 86.70% dan 87.52% daripada aktiviti asal

setelah diinkubasi pada suhu 80°C selama 30 minit.

Pencirian dan pengenalpastian lapan belas bakteria tahan haba penghasil enzim lipase

telah dilakukan menggunakan kaedah pencirian morfologi, sistem ‘Biolog Microlog®’

pengenalpastian bakteria dan ujian-ujian biokimia. Strain KW 6, KW 7, ST 1, ST 6 dan

ST 10 telah dikenalpasti sebagai CDC Group IVc-2 (Alcaligenes-like) atau Ralstonia

paucula. Bagi strain KW 8, KW 10 dan DT 17 pula, mereka telah dikenalpasti sebagai

Burkholderia cocovenenans, B. glumae, dan Photobacterium logei. Hasil ujikaji Biolog

mendapati strain ST 7, ST 8, SY 7, KW 1, KW 9, KW 12 dan TBN 3 tergolong sebagai

genus Bacilli dan strain SY 9, DT 9 dan DT 12 sebagai spesies Staphylococcus. Empat

strain daripada genus Bacillus dengan thermostabiliti yang tertinggi, ST 7, ST 8, SY 7

dan KW 12 telah dipilih untuk pengenalpastian selanjutnya menggunakan kaedah ‘16S

rRNA Gene Sequence Analysis’.

Kaedah untuk pengekstrakan DNA yang digunakan di dalam kajian ini ialah Qiagen

DNeasy Tissue Kit. Berdasarkan kepada keputusan kuantifikasi DNA ekstrak dan

penganggaran ketulenannya menggunakan spektrofotometer UV, kaedah Qiagen

DNeasy Tissue Kit memberikan kualiti DNA tertinggi untuk untuk semua strain.

Pasangan primer yang digunakan untuk PCR-spesifik menghasilkan ‘produk PCR’ yang

diharapkan untuk semua strain dengan berat molekul 1500 bp. Rangkaian jujukan

dikenalpasti dengan menggunakan program rangkaian jujukan automatik; di mana

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rangkaian jujukan akan dibandingkan dengan rujukan yang terhampir dengannya di

dalam pangkalan maklumat NCBI. Keputusan dari analisis ‘jujukan gen 16S rRNA’

mendapati rangkaian jujukan ST 8 telah mendapat nilai persamaan tertinggi dengan

Bacillus subtilis gene for 16S rRNA partial sequence (AB110598 dengan 100%

persamaan antara nukleotida 1-1532). Strain KW 12 dan ST 7 juga menunjukkan nilai

persamaan yang tinggi pada 99.0%; dibandingkan dengan Anoxybacillus kamchatkensis

16S rRNA gene partial (nombor kemasukan: AF510985) dan Anoxybacillus flavithermus

isolate AB05 16S rRNA gene partial sequence (nombor kemasukan: AF001964). Strain

SY 7 mempamerkan 98.0% persamaan rangkaian jujukan dengan Bacillus cereus strain

J-1 16S rRNA gene partial sequence (nombor kemasukan: AY305275). Hasil dari ujikaji

jujukan gen 16S rRNA untuk ST 8, ST 7, KW 12 dan SY 7 telah disimpan dalam

‘Genbank Data Library’ dan ditetapkan di bawah nombor kemasukan DQ401073,

DQ193516, DQ401072 dan DQ401074.

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ACKNOWLEDGEMENT

In the name of Allah, The Most Gracious and The Most Merciful. First and foremost, I

would like to express my heartfelt thanks to Almighty for giving me the will, strength

and the perseverance to pursue and complete my Master of Science. It would not have

been possible to complete this study without His grace.

I would like to express my sincere gratitude and appreciation to the chairman of my

supervisory committee, Assoc. Prof. Dr. Nazamid Saari for his invaluable guidance,

dedicated efforts, his tremendous help in teaching me to write proper scientific papers

and continuous support throughout the study. Because of his guidance, I have been able

to make my dream come true of combining two of my favourite fields, microbiology and

biotechnology together to write this thesis.

My special thanks and appreciation goes to my co-supervisor, Dr. Nazimah Sheikh Abd.

Hamid and Professor Dr. Son Radu for their advises, suggestions and consistent

encouragement. Their strong enthusiasm for research helps me a lot in maintaining my

confidence to achieve such high-level results in scientific work.

I am indebted to my dear friend, Halifah Mat Yasin for her willingness and constant

assistance and help in time especially when I was having difficulties with my molecular

works during my early years of research. Words cannot express my gratitude for her

patience and support.

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My sincere gratitude also goes to all the staff in the biochemistry lab and microbiology

lab of the Faculty of Food Science and Technology, Universiti Putra Malaysia, who has

contributed one way or another throughout the course of study.

Needless to say, that I am grateful to all of my friends, Yuli Haryani, Marlina, Tunung,

Rozila, Lesley, Belinda, Rasyila, Kqueen, Jurin, Tuan, Suwaibah, Yin Tze, Sia Yen,

Daniel, Henie, Tung and Maslihana from Microbiology Lab. Sincere thanks are also

extended to my friends, Ermina Sari, Wan Zunairah, Azimah, Ching, Reza, Raja Rohaya

and Rosnizam from Nutraceutical and Enzymology Lab. Thank you for being around

and sharing so much fun and several good times together during my research in the lab.

All of you have made my student life an interesting and a delightful one. I am thankful

to all of you and wish you the best of luck.

All of my hard work would have never been done properly without continuous support

and encouragement especially from my dearest parent and my loving brother and sister.

Because of their financial and spiritual support, I have been able to study at an advanced

academic level.

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I certify that an Examination Committee met on 22 January 2008 to conduct the final examination of Azura Liana Binti Kamaruzaman on her Master of Science thesis entitled “Identification of Lipase-Producing Thermophilic Bacteria Isolated from Hot Springs in Malaysia” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the student be awarded the Master of Science. Members of the Examination Committee are as follows:

Yaakob Che Man, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Farinazleen Mohd Ghazali, PhD Lecturer Faculty of Food Science and Technology Universiti Putra Malaysia (Internal Examiner) Shuhaimi Mustafa, PhD Associate Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner) Thong Kwai Lin, PhD Professor Faculty of Science University Malaya (External Examiner)

________________________________

HASANAH MOHD GHAZALI, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 10 July 2008

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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 Master of Science. The members of the Supervisory Committee were as follows: Nazamid Saari, PhD Associate Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Chairman) Nazimah Sheikh Abd. Hamid, PhD Faculty of Food Science and Technology Universiti Putra Malaysia (Member) Son Radu, PhD Professor Faculty of Food Science and Technology Universiti Putra Malaysia (Member)

AINI IDERIS, PhD Professor and Dean School of Graduate Studies Universiti Putra Malaysia Date: 10 July 2008

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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 any other institution. AZURA LIANA BINTI KAMARUZAMAN Date:

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TABLE OF CONTENTS

Page DEDICATION ii ABSTRACT iii ABSTRAK vi ACKNOWLEDGEMENTS ix APPROVAL xi DECLARATION xiii LIST OF TABLES xvi LIST OF FIGURES xvii LIST OF ABBREVIATIONS xix CHAPTER 1 INTRODUCTION 1 1.1 General Objectives of the Study 4 2 LITERATURE REVIEW 5

2.1 Extremophilic Microorganisms 5 2.1.1 Thermophiles 6 2.1.2 Resistance of Thermophiles 7 2.2 Sources of Thermophilic Microorganisms 8 2.3 Lipase-producing Bacteria 10

2.3.1 Pseudomonas 12 2.3.2 Bacillus 14 2.4 Screening and Isolation of Lipase- Producing Thermophilic Bacteria 15 2.4.1 Assay of Lipase Activity 18

2.5 Identification of Lipase-Producing Thermophilic Bacteria 21 2.5.1 Traditional Phenotypic Identification 22 2.5.2 Modern Genotypic Identification 23

3 SCREENING AND ISOLATION OF LIPASE- PRODUCING THERMOPHILIC BACTERIA FROM MALAYSIAN HOT SPRINGS 30 3.1 Introduction 30

3.2 Materials and Methods 32 3.2.1 Materials 32 3.2.2 Methods 33 3.3 Results and Discussion 36 3.3.1 Screening and isolation of lipase-

producing bacteria 36 3.3.2 Comparative lipolytic activity of

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positive bacterial lipase producer 39 3.3.3. Thermostability of lipases 43 3.4 Conclusion 49 4 IDENTIFICATION OF LIPASE PRODUCING THERMOPHILIC BACTERIA ISOLATED FROM MALAYSIAN HOT SPRINGS 50 4.1 Introduction 50 4.2 Materials and Methods 52 4.2.1 Materials 52 4.2.2 Methods 53 4.3 Results and Discussion 57 4.3.1 Morphological characteristics 57 4.3.2 Identification of strains 59 4.4 Conclusion 87 5 CONFIRMATION OF BACILLUS GENUS LIPASE- PRODUCING THERMOPHILIC BACTERIA ISOLATED FROM MALAYSIAN HOT SPRINGS BY 16S RIBOSOMAL RNA GENE SEQUENCING 88 5.1 Introduction 88 5.2 Materials and Methods 90 5.2.1 Samples 90 5.2.2 DNA Extraction Method 90 5.2.3 Quantification Of DNA Quality 92 5.2.4 Polymerase Chain Reaction (PCR) 92 5.2.5 Sequencing of 16S rRNA Gene 94 5.3 Results and Discussion 96 5.3.1 DNA Extraction 96 5.3.1.1 DNA Yield and Purity 96 5.3.2 Specific PCR for Strains Identification 98 5.3.3 16s rRNA Sequence Analysis for Confirmation of PCR products 99 5.4 Conclusion 114 6 CONCLUSION 116 REFERENCES 118 APPENDICES 135 BIODATA OF STUDENT 158 LIST OF PUBLICATIONS 161

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LIST OF TABLES Table Page

2.1 Lipase-Producing Bacteria 11

3.1 The Occurrence of Lipase-Producing Bacteria from Six Hot Springs in West-Coast of Peninsular Malaysia 39 3.2 Lipases Thermostability of the 18 Lipase- Producing Bacteria Isolates 44 4.1 Morphological Characteristics of Lipase-

Producing Thermophilic Bacteria 58

4.2 Physiological Characteristics of Lipase- Producing Thermophilic Bacteria 63

4.3 Results for Biochemical Tests of the Gram-Negative Short Rods Bacteria at 45°C 64

4.4 Results for Biochemical Tests of the Gram-Negative Short Rods Bacteria at 35, 37 or 42°C 66

4.5 Results for Biochemical Tests of the Gram-Positive Rods Bacteria at 45°C 72

4.6 Results for Biochemical Tests of the Gram-Positive Rods Bacteria at 35, 37 or 42°C 74

4.7 Results for Biochemical Tests of the Gram-Positive Cocci Bacteria at 45°C 81

4.8 Results for Biochemical Tests of the Gram-Positive Cocci Bacteria at 35, 37 or 42°C 83

5.1 Oligonucleotide Primers for PCR Amplification and Sequencing 96

5.2 Comparison of Identification Results of Bacillus Genus Lipase-Producing Thermophilic Bacteria Against Three Identification Methods 113

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LIST OF FIGURES

Figure Page

2.1 The Different Steps in PCR 25

2.2 The Exponential Amplification of the Gene in the PCR 26

3.1 Six Hot Springs Sampling Locations in West-Coast of Peninsular Malaysia 37

3.2 The Difference between a Positive Lipase Producer and a Negative Lipase Producer 38 3.3 Lipolytic Activities of Positive Lipase-Producing Thermophilic Bacteria Isolated from Malaysian Hot Springs (A-F) 40

3.4 High Thermostable Lipases Secreted by ST 6, ST 7, ST 8, ST 10, SY 7 and KW 6 46

3.5 Moderate Thermostable Lipases Secreted by KW 7, KW 10, KW 12 and TBN 3 47

3.6 Low Thermostable Lipases Secreted by SY 9, KW 1, KW 8, KW 9, ST 1, DT 9, DT 12 and DT 17 48

5.1 Agarose Gel Electrophoresis Shows the Quality of DNA

Isolated from Thermophilic Bacteria by Qiagen Kit 98

5.2 Agarose Gel Electrophoresis of PCR Amplification Products of Thermophilic Bacteria 99

5.3 The Alignment of Sequencing Results from Each Primer to Provide a Partial Sequence for Genus Bacillus Strains 101 5.4 16S rRNA Gene Sequence Alignment Comparison of ST 8 as Subject Compared to the Published Sequence of Bacillus subtilis Gene for 16S rRNA Partial Sequence (AB110598) with 1532 Nucleotide 102

5.5 16S rRNA Gene Sequence Alignment Comparison of ST 7 as Subject Compared to the Published Sequence of Anoxybacillus flavithermus isolate AB05

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16S rRNA Gene Partial Sequence (AF001964) with 1411 Nucleotide 104 5.6 16S rRNA Gene Sequence Alignment Comparison of KW 12 as Subject Compared to the Published Sequence of Anoxybacillus kamchatkensis 16S rRNA Gene Partial Sequence (AF510985) with 1548 Nucleotide 107 5.7 16S rRNA Gene Sequence Alignment Comparison of SY 7 as Subject Compared to the Published Sequence of Bacillus cereus Strain J-1 16S rRNA Gene Partial Sequence (AY305275) with 1479 Nucleotide 110

xviii

LIST OF ABBREVIATIONS

rpm revolution per minute

v/v volume per volume

min minute

h hour

g gram

ml millileter

mg milligram

μm micrometer

nm nano meter

mM milliMolar

µmol micromole

ng nanogram

μl microliter

M Molar

N Normality

Gly Glycine

Ser Serine

kDa kilo Dalton

ATCC American Type Culture Collection

pI isoelectric point

U/ml unit per millileter

U/g unit per gram

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w/v weight per volume

UV ultraviolet

DNA deoxyribonucleic acid

rRNA ribosomal ribonucleic acid

bp base pair

kb kilo basepair

C18 stearate/ stearic acid

C18:1 oleate/ oleic acid

BUG Biolog Universal Growth medium

GN Gram-negative

GP Gram-positive

IF Inoculation fluid

CHAPTER 1

INTRODUCTION

Nowadays, lipases arise amongst the most important biocatalysts carrying out novel

reactions in both aqueous and non-aqueous media. This is mainly due to their ability

to utilize a wide spectrum of substrates, high stability towards extremes of

temperature, pH and organic solvents, and show chemo-, regio- and

enantioselectivity. In recent times, the determination of their three-dimensional

structures has thrown light into their unique structure–function relationships.

Lipase or glycerol ester hydrolases (EC 3.1.1.3) is defined by the Enzyme

Commission of International Union of Biochemistry and Molecular Biology as the

enzyme that catalyzes hydrolysis of ester bonds at the interface between an insoluble

substrate phase and the aqueous phase in which the enzyme is dissolved, under a

natural conditions. Under certain experimental conditions, such as in the absence of

water, they are capable of reversing the reaction. The reverse reaction leads to

esterification and formation of glycerides from fatty acids and glycerol. The catalytic

action that occurs is as follows:

TRIACYLGLYCERIDE + H2O GLYCEROL + FREE FATTY ACIDS

(Ester) (Alcohol) (Acid)

In nature, lipases are ubiquitous and produced from various sources including

animals, plants and microorganisms. However, lipases of microbial origin are the

most versatile enzymes and are known to bring about a range of bioconversion

reaction including alcoholysis, aminolysis, hydrolysis, esterification and

interesterification (Pandey et al., 1999). Besides, microbes can be easily cultivated

and the lipases produced are mostly extracellular. Several Bacillus sp. have been

reported to be the main source of lipolytic enzymes (Schmidt et al., 1994).

The isolation of a large number of beneficial thermophilic microorganisms from

different exotic ecological zones of the earth and followed by the extraction of

thermostable lipases shows that the advances of this area have been possible

(Gowland et al., 1987; Dong-Woo et al., 1999; Kambourova et al., 2003).

Thermostable lipases, which are commercially available, are mostly produced from

mesophilic bacteria and fungi. Although many lipases from mesophilic group are

stable at elevated temperatures, lipases from thermophilic have become the subject of

special interest for structural investigation and biotechnological applications

(Herbert, 1992). Thermostable lipases, which have been isolated from thermophilic

organisms, play an important role in commercial applications because of their overall

inherent stability (Demirjian et al., 2001). They are also stable and active at

temperatures which are higher than optimum temperatures for the growth of the

microorganisms. This reduces the risk of contamination by common mesophiles.

Allowing a higher operation temperature has also a significant influence on the

bioavailability and solubility of organic compounds and thereby provides efficient

bioremediation (Becker et al., 1997).

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With the rapid development of enzyme technology, many novel biotechnology

applications for lipases have been identified. The esters produced play a relevant role

in the food industry as flavour and aroma constituents (Gandhi, 1997). Other

applications include the removal of the pitch from pulp in the paper manufacturing

industry, drug formulations in the pharmaceuticals industry and the removal of

subcutaneous fat in the leather industry (Pandey et al., 1999). Lipases are extensively

used in dairy industry for flavour development in cheese, butter and margarine and

hydrolysis of milk fat (Paiva et al., 2000). Other applications are in the fats and oil

industries including production of modified triacylglycerides and upgrading of low-

value oils and fats.

As industrial application increase, the requirements for large amounts of lipases

become a limiting factor. Therefore, several workers have tried to obtain

thermostable lipases from thermophilic or extreme thermophilic strains such as

Pseudomonas fluorescens SIK W1 (minimal activity at 60ºC) (Andersson et al.,

1979) or Bacillus sp. (maximal activity at 60ºC) (Emanuilova et al., 1993). The

alkalophilic and thermophilic Bacillus sp. has been the focus of a number of

investigations as the source of lipases that are stable and function optimally at

alkaline pH values and high temperature

This research was done with the ultimate goal of isolating lipase-producing

thermophilic bacteria from hot springs in Malaysia. The strain that produced a high

thermostable lipase was chosen for further studies. Identification and characterization

of the thermophilic bacteria producing lipase was carried out by using three methods;

3

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Biolog Microlog® Bacterial Identification System, conventional biochemical test and

16S rRNA gene sequencing.

1.1 General Objectives of the Study

The study was undertaken with the following general objectives:

a) To isolate lipase-producing thermophilic bacteria from hot springs in

Malaysia

b) To identify and characterize the thermophilic bacteria producing

lipase using three different methods

i) Biolog Microlog® Bacterial Identification System

ii) Conventional biochemical tests

iii) 16S rRNA gene sequencing