SCREENING, CLONING AND EXPRESSION OF A XYLANASE FROM PALM OIL MILL

SCREENING, CLONING AND EXPRESSION OF A XYLANASE FROM PALM OIL MILL

EFFLUENT (POME) BY FUNCTIONAL METAGENOMICS APPROACH

BY

ADIBAH BINTI PARMAN

A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)

Kulliyyah of Engineering

International Islamic University Malaysia

SEPTEMBER 2020

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Metagenomics approach is an alternative method to study the novel enzyme. Therefore, a metagenomic fosmid library of approximately 50,000 clones was screened to identify the novel xylanase enzymes. The metagenomics deoxyribonucleic acid (DNA) used in this study is from the samples of palm oil mill effluent (POME) from Felda Palm Industry Sdn. Bhd. in Mempaga, Pahang. Clones of the metagenomics were screened using fluorescence substrate of chlorocoumarin xylobioside. The high score reads obtained from screening were sent for next-generation sequencing (NGS) of Illumina HiSeq2000. The sequences were further analysed to identify the predicted xylanase genes using automated and manual bioinformatic tools. A total of 34 predicted xylanases were identified, and five predicted xylanase genes #11, #15, #16, #17, and

#18 of various microbial origins were chosen to be further analysed. The translated sequences of these five genes later were analysed to determine the primary, secondary, and tertiary protein structures in predicting feature and function of predicted xylanases.

Next, based on the integrity checking in agarose gel electrophoresis (GE), Gene #15 with approximately 1.2 kb was chosen to be cloned into the pBAD-TOPO vector. Gene

#15 has the percentage identity of 99.3% to the Ochrobactrum intermedium with glycoside hydrolase (GH) 10 as the conserved domain. After cloning, pBAD-TOPO- Xyl was used to transform E. coli cells and expressed using the inducer, L-arabinose.

Protein #15 (P15) was later purified using immobilised metal affinity chromatography (IMAC), and the molecular mass of SDS-PAGE of approximately 46 kDa was confirmed. The P15 also fluoresced when checked with chlorocoumarin xylobioside substrate, which suggests the protein is a xylanase enzyme.

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ثحبلا ةصلاخ

برتعي تايمونيجاتيلما جنه

)ةيثارولا ةدالما ةسارد(

ةليدب ةقيرط

تايمزنلإا ةساردل ةركتبمو .

ت كلذل

صحف

ديمزوف ةبتكم ( ةينيلجا ةداملل

ةيمونيجاتيم )

لح لاو 50.000

خاسنتسا ديدحتل

تايمزنإ نلايزلا يز .ةديدلجا

تو صلاختسا

لحا ضم لا يوون لا نم يزوبير عوز

جسكلأا نم ين

تانيع نم لا عتجرلما لئاس رصاعلم تيز

( ليخنلا POME

نم ) ةكرش ف ادل ل ةيمنتل ةمادتسلما في

ةعانص ليخنلا ةنيدبم اجافمم ةيلاو في هبا

ا جن

يازيلابم تو . صحف خاسنتسا ( ةيثارولا تانيلجا

ةيمونيجاتيلما )

مادختسبا

ةعشلما نيرامويكورولكلا ةدام

( Chloro-coumarin xylo-bioside )

تو . لاسرإ لا تاءارق ةيلاعلا تلا ت لوصلحا اهيلع نم

صحفلا ا ةساردل لسلستل نييلجا ل ليجل لاتلا ( NGS ) نم Illumina HiSeq2000 تو .

ليلتح

تايلاوتلما

لكشب بركأ ديدحتل تانيج لا يمزنا نلايز يز ةعقوتلما مادختسبا

تاودأ ةيتامولعلما

ةيويلحا ةيللآا

ةيوديلاو . تو يدتح د ام هعوممج 34 ينج عقوتم لا يمزنلإ نلايز ي

،ز و كلذك ت رايتخا ةسخم تانيج نلايز ي ز

ةعقوتم رايتخا ت ًاضيأو . #

11 و # 15 و # 16 و # 17 و # 18 نم تانيج لوصلأا

ةيبوركيلما

ةفلتخلما ديزلم نم و صحفلا تو .ليلحتلا

ليلتح تايلاوتلما

ةجمترلما هذله تانيلجا ةسملخا في تقو قحلا

لت ديدح بيكارت ينتوبرلا ةيلولأا ةيوناثلاو ةيثلاثلاو في ؤبنتلا تامسب فئاظوو لا تايمزنإ نلايز

يز ةعقوتلما . دعبو

،كلذ اًدانتسا و لإ صحف ةملاسلا في نلاحرلا يئبارهكلا

ملاهلل ( GE ) ، ت رايتخا ينلجا مقر

# 15 عم

لاوح 1.2 تيباوليك

هخاسنتسلا

في لقنا pBAD-TOPO كلتيمو .

لجا ين مقر

# 15 ةبسن ةيوئم

99.3 ٪ ( Ochrobactrum intermedium عم )

لها ديسوكيلج لوردي

يز 10 ( GH ) لاجمك

ظوفمح . دعبو ،خاسنتسلاا

ت مادختسا

pBAD-TOPO-Xyl

ليوحتل يالاخ E. coli

تو

يربعتلا هنع مادختسبا

،ضرلمحا L-arabinose

تو . ةيقنت ينتوبرلا مقر

# 15 ( P15 اًقحلا ) دختسبا ما

ايفارغوتامورك

براقت ندعلما تبث ُ لما ( IMAC )

، تو ديكتأ ةلتكلا ةيئيزلجا ـل SDS-PAGE

تلا

غلبت 46 وليك نوتلاد و .اًبيرقت ينتوبرلا نأ حضتا P15

اًضيأ عشم دنع هصحف عم نيرامويكورولكلا ةدام

( Chloro-coumarin xylo-bioside رك )

ةزيك لل ( لعافت

substrate

) امم ، يرشي يو دكؤ نأ

ينتوبرلا

وه

يمزنإ

نلايز

يز

.

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I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science (Biotechnology Engineering)

………

Hamzah Mohd. Salleh Supervisor

………

Ibrahim Ali Noorbatcha Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Science (Biotechnology Engineering)

………

Raha Ahmad Raus Internal Examiner

………

Farah Diba Abu Bakar External Examiner

This thesis was submitted to the Department of Biotechnology Engineering and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)

………

Nor Fadhillah Mohamed Azmin Head, Department of

Biotechnology Engineering

This thesis was submitted to the Kulliyyah of Engineering. and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)

………

Sany Izan Ihsan

Dean, Kulliyyah of Engineering

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DECLARATION

I hereby declare that this thesis is the result of my own investigations, except

where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Adibah Parman

Signature ... Date ...

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INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

SCREENING, CLONING AND EXPRESSION OF A XYLANASE FROM PALM OIL MILL EFFLUENT (POME) BY FUNCTIONAL

METAGENOMICS APPROACH

I declare that the copyright holders of this thesis are jointly owned by the student and IIUM.

Copyright © 2020 Adibah Parman and International Islamic University Malaysia. All rights reserved.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

1. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

2. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by Adibah Parman

……..……….. ………..

Signature Date

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ACKNOWLEDGEMENTS

ب م س ّاللَ

ّرلا م ح ن ّرلا ح مي

In the name of Allah, the Most Beneficent, the Most Merciful. All praises belong to Allah, the supreme power, who is the right guider of humankind. Firstly, a special thanks to both of my supervisors, Prof. Dr. Hamzah Mohd. Salleh and Prof. Dr. Ibrahim Ali Noorbatcha for their continuous guide, teaching, support, encouragement, and advice. I will be forever grateful. Jazakumullahu khairan kathiran. May Allah always repay both of you with goodness. I also would like to thank Prof. S.G. Withers (University of British Columbia, Canada) for kindly providing chlorocoumarin xylobioside needed for this project.

I wish to express my appreciation and thanks to those who provided their time, effort, and support for this project, especially my colleague, sister Farah. She’s my partner in research who has a lot of experience in molecular technique and the one that always shared and understood the hardship in finishing this vast research. Both of us always sit together, discussing and wondering when we will be able to finish this research. To the members under the same supervisory committee, brother Oualid and brother Aziz, thank you very much for guiding me in the skills of laboratory work. I am very grateful to have another three Ph.D. students who are in the supervisory committee that can always guide me in the upcoming problem of this research.

Finally, it is my highest pleasure to dedicate this work to my dear parents, Rohenah Binti Hasan and Parman Bin Sirad. My utmost appreciation to both, especially when I always in need of motivational advice and support in continuing my research.

They are indeed the backbone and supporter of my work either in terms of financial or emotional advice. Not forget to my siblings, who always have a firm belief in my ability to accomplish this goal, thank you for all of your support and patience.

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Abstract ii

Abstract in Arabic iii

Approval Page iv

Declaration v

Copyright Page vi

Acknowledgements vii

Table of Contents viii

List of Tables xi

List of Figures xii

List of Abbreviations xiii

List of Symbols xv

CHAPTER ONE: INTRODUCTION 1

1.1 Background 1

1.2 Problem Statement 2

1.3 Research Objectives 3

1.4 Significance of Study 3

1.5 Research Methodology 4

1.6 Scope of Research 4

1.7 Dissertation Organisation 5

CHAPTER TWO: LITERATURE REVIEW 6

2.1 Introduction 6

2.2 Agro-Residue Biomass 7

2.2.1 Palm Oil Mill Effluent (POME) 7

2.2.2 POME as Microbial Fermentation Medium 8

2.3 Xylan From Hemicellulose 9

2.3.1 Xylan 10

2.3.2 Endo-β-1,4-xylanase 10

2.3.3 Glycoside Hydrolase (GH) 10 11

2.3.4 Glycoside Hydrolase (GH) 11 12

2.3.5 Microbes Producing Xylanase 12

2.4 Functional Metagenomics Screening Approach 13 2.5 Identifying Unique Sequence Through Metagenomics Analysis 15

2.5.1 MG-RAST 15

2.5.2 IMG/M 17

2.5.3 EBI Metagenomics 17

2.6 Cloning and Expression of Recombinant Xylanase 19

2.7 Xylanase in Industrial Applications 20

2.8.1 The Paper and Pulp Industries 20

2.8.2 Biofuel, Pharmaceutical, Food and Animal Feed Industries 21

2.8 Chapter Summary 21

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CHAPTER THREE: RESEARCH METHODOLOGY 23

3.1 Introduction 23

3.2 Materials 23

3.2.1 Sample 23

3.2.2 Chemicals and Reagents 24

3.2.3 Extraction, Fosmid Production and Cloning Kits 24

3.2.4 Sequencing Services 24

3.2.5 Purification Column 24

3.3 Research Methodology Flowchart 25

3.4 Metagenomics Library Screening 26

3.4.1 Plate Culturing and Screening 26

3.4.2 Selection of Hit Readings 27

3.4.3 Auto-induction 28

3.4.4 Fosmid DNA Purification 28

3.4.5 Gel Electrophoresis 30

3.5 Unique DNA Sequence Identification of Xylanase 30

3.5.1 Metagenomics Analysis of NGS data 30

3.5.2 Metagenomics Analysis by Auto-Bioinformatics Pipeline 31

3.5.2.1 MG-RAST 31

3.5.2.2 IMG/M 32

3.5.2.3 EBI Metagenomics 32

3.6 Modelling the Unique Sequence of Xylanase 33 3.6.1 Sequence’s Physical Parameter and Primary Structure

Analysis 33

3.6.2 Secondary Structure Analysis 34

3.6.3 Tertiary Structure Analysis 34

3.7 Cloning and Expression of Recombinant Xylanase 34

3.7.1 Designing PCR Primers 34

3.7.2 Producing PCR Products 35

3.7.3 TOPO Cloning Reaction 36

3.7.4 Transforming One Shot Top10 Competent Cell 36

3.7.5 Expressing the PCR product 37

3.8 Purification of Recombinant Xylanase 37

3.8.1 Purification of Recombinant Xylanase 38

3.8.2 SDS-PAGE Analysis 39

3.9 Chapter Summary 39

CHAPTER FOUR: RESULTS AND DISCUSSION 41

4.1 Introduction 41

4.2 Screening of Metagenomic Library 41

4.3 Identifying the Unique Sequence 43

4.3.1 Metagenomics Analysis by Automated Pipelines 44

4.3.2 Manual Pos-Processing of NGS 46

4.4 Protein Homology Modeling of Predicted Xylanase 48 4.4.1 Sequence Physical Parameter and Primary Structure Analysis 48

4.4.2 Secondary Structure Analysis 49

4.4.3 Tertiary Structure Analysis 50

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4.5 Cloning and Expression 55

4.5.1 Cloning into Vector 55

4.5.2 Expression 56

4.6 Protein #15 Analysis 58

4.7 Purification Recombinant Xylanase 62

4.7.1 Purification of Recombinant Protein #15 62

4.8 Chapter Summary 65

CHAPTER FIVE: CONCLUSION 66

5.1 Conclusion 66

5.2 Contribution of Study 66

5.2 Recommendations 67

REFERENCES 68

ACHIEVEMENTS 76

APPENDIX A: Picture of POME Sample 77 APPENDIX B: List of Chemicals and Reagents 78 APPENDIX C: PCR Parameters of Gene #11 and #15 79 APPENDIX D: Picture of Metagenomic Library 80 APPENDIX E: Example of Robust Z-score Calculation 81 APPENDIX F: GH enzyme by Automated Pipelines 82 APPENDIX G: List of Possible Xylanases by BLAST 83 APPENDIX H: List of All Nucleotides and Amino Acids 85 APPENDIX I: Conserved Domain of Gene #11 and #15 92 APPENDIX J: Pictures of Secondary Structure Prediction 93 APPENDIX K: Example Picture of Swiss-Model Report 98 APPENDIX L: Sequence Alignment and Percentage Identity of Cwp19 99

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Table 2.1 The approximate compositions (%) of major constituents in POME 7 Table 2.2 Microorganisms found in POME by previous research 8

Table 2.3 Recent functional metagenomics research 13

Table 2.4 The list of each automated bioinformatic tool with databases 17 Table 2.5 Previous research on cloning and expression of xylanase metagenome 18

Table 3.1 Parameters of primer design of Gene #15 32

Table 3.2 The mixture in fluorescence activity checking 36

Table 3.3 SDS-PAGE recipe 36

Table 4.1 List of five selected possible xylanases by BLAST 44 Table 4.2 Physical parameters of five predicted xylanases 46 Table 4.3 Secondary prediction of the five predicted xylanase genes 47 Table 4.4 Tertiary structure analysis of the five xylanase genes 48

Table 4.5 Final 3D-model based on the QMEAN Z-Score 49

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L

F

Figure 2.1 Function of endo-β-1,4-xylanase 10

Figure 2.2 The basic flowchart of metagenomics analysis after sequencing 15

Figure 3.1 Flowchart of study 23

Figure 3.2 Four days screening of a 384 well-plate metagenomic library 24 Figure 4.1 Chlorocoumarin xylobioside structure after being hydrolysed

with endo-β-1,4-xylanase

39

Figure 4.2 Fifty clones with the highest robust z-score reading 40 Figure 4.3 (a) Phyla distribution of POME computed by each tool 42 Figure 4.3 (b) Major genus distribution in POME analysed by MG-RAST and

IMG/M only.

42

Figure 4.4 Model-template alignment of Gene #11 based on QMEAN 50 Figure 4.5 Model-template alignment of Gene #15 based on QMEAN 50 Figure 4.6 Model-template alignment of Gene #16 based on QMEAN 51 Figure 4.7 Model-template alignment of Gene #17 based on QMEAN 51 Figure 4.8 Model-template alignment of Gene #18 based on QMEAN 52

Figure 4.9 Agarose gel electrophoresis 53

Figure 4.10 Regulation of the PBAD promoter by L-Arabinose 57

Figure 4.#11 Top 10 blast hits of Protein #15 54

Figure 4.12 The multiple sequence alignment of Protein #15 55

Figure 4.13 Phylogeny tree of Protein #15 query 56

Figure 4.14 Purification of Protein #15 using HisTrap HP column 58 Figure 4.#15 The tubes tested with chlorocoumarin xylobioside 58 Figure 4.#16 SDS-PAGE profile of Protein #15 after purification 59

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L

A

4MU 4-methylumbelliferone

APS Ammonium Persulfate

BAC Bacterial Artificial Chromosomes BLAST Basic Local Alignment Search Tool BLAT BLAST-like Alignment Tool BOD Biochemical Oxygen Demand CAZy Carbohydrate-Active enZYmes CCX Chlorocoumarin xylobioside CDS Coding Sequence

CMC Carboxymethyl cellulose COGs Clusters of Orthologous Groups

CUI Command User Interface

DH2O Distilled water

DNA Deoxyribonucleic Acid

DOE-JGI Department of Energy Joint Genome Institute

DTT Dithiothreitol

EBI European Bioinformatics Institute

EC Enzyme Commission Number

EFB Empty Fruit Brunches

EMBL European Molecular Biology Laboratory ENA European Nucleotide Archi

FFB Fresh Fruit Bunches

FPLC Fast Protein Liquid Chromatography

FR Forward

GE Gel Electrophoresis

GH Glycoside Hydrolase

GMQE Global Model Quality Estimation

GO Gene Ontology

GOLD Genomes Online Database

GRAVY Grand Average of Hydropathicity

GUI Graphical User Interface

HTS High-Throughput Screening

IMAC Immobilized Metal Affinity Chromatography IMG/M Integrated Microbial Genomes with Microbiomes KEGG Kyoto Encyclopedia of Genes and Genomes

KO KEGG Orthology

LB Luria-Bertani medium

M Marker

MAD Median Absolute Deviation MAP Metagenome Annotation Pipeline

Med Median

MGI Malaysia Genome Institute

MG-RAST Metagenome Rapid Annotation using Subsystem Technology NGS Next-Generation Sequencing

OD Optical Density

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OPF Oil Palm Fronds

OPT Oil Palm Trunks

PCR Polymerase Chain Reaction Pfam Protein Family Database

PHA Polyhydroxyalkanoates

PKC Palm Kernel Cake

PKS Palm Kernel Shell

POME Palm Oil Mill Effluent

PPF Palm Pressed Fibres

PRODIGAL PROkaryotic DynamIc programming Gene finding Algorithm QMEAN Z-Score Qualitative Model Energy Analysis of Z-Score

RBB Remazol Brilliant Blue RFU Relative Fluorescence Unit

RNA Ribonucleic Acid

RV Reverse

Rz Robust z-score

S.O.B Super Optimal Broth

S.O.C Super Optimal Broth with Catabolite repression

SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SOPMA Self-Optimized Prediction Method with Alignment

SSPACE SSAKE-based Scaffolding of Pre-Assembled Contigs after Extension

TE Tris-EDTA buffer

TEMED Tetramethylethylenediamine

X-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside

XOs Xylooligosaccharides

xv

L

S

kb kilo basepairs

ºC celcius

μg microgram

ml millilitre

µl microlitre

rpm revolutions per minute

g gram

g (RCF) relative centrifugal force

bp base pairs

s second

mM millimolar

kDa kilo dalton

1

CHAPTER ONE INTRODUCTION

1.1 BACKGROUND

The advancement in the genomics and metagenomics field has widened the view on microbial diversity and benefits future potential biotechnological applications, including the novel enzyme mining process. Metagenomics is an advanced alternative approach to genomics in understanding the microorganism’s diversity in a given sample without necessarily culturing any microbes. Cultivable microorganisms constitute only a tiny fraction of microbial diversity, which limits the mining process of novel enzymes (Ferrer et al., 2005). A lot of microorganisms cannot be cultured normally in the laboratory set-up. The metagenomics approach is the key to getting access to the uncultivable microbes that potentially possess unique enzymes.

The metagenomics approach will be used in this research to study potential xylan-degrading enzymes from the entire microorganisms living in palm oil mill effluent (POME) samples. POME is a by-product of processed fresh fruit bunches (FFB) from the palm oil industry, which contains high nutrient concentrations like carbohydrate, nitrogen, protein, phosphorus, potassium, magnesium, and calcium (Madaki & Seng, 2013).

Xylanase is one of the xylan degrading enzymes contained in hemicellulose rich POME. Xylanase is currently accessible in different industrial applications because of its catalytic ability in bio-bleaching of paper pulp, improvement of animal feed, bread making, application in solid waste treatment, preparation of juice from fruits or

2

vegetables, improve retting of flax fibers, production of biofuels, and others (Asish, 2015). Throughout this research, genes encoding xylanase enzymes are expected to be found from the POME sample through the metagenomics method.

The methodologies to be employed will be through the construction of the metagenomics library, fluorescence high throughput screening (HTS), next-generation sequencing (NGS), homology modeling, cloning, expression, and purification of the recombinant xylanase enzyme.

1.2 PROBLEM STATEMENT

To date, most of the available enzymes used in industrial applications originated from microbes. Enzymes are recognized as a favorable catalyst which can accelerate the rate of reaction of a process. The traditional method used in getting microbial enzymes of interest is by cultivating a microorganism through standard laboratory techniques.

However, research has proven that only less than 1% of environmental microorganisms can be cultivated (Uchiyama & Miyazaki, 2009). This situation indicates that more than 99% of bacteria from the environment cannot be cultured using conventional approaches.

As a counter method, the metagenomics approach appeared to be an alternative to conventional microbial screening in studying the diversity of another 99% of non- cultured microorganisms (Kennedy et al., 2011). Therefore, this research will focus on the functional metagenomics approach to search for xylanases from the entire microorganisms in palm oil mill effluent (POME) without culturing the microorganisms from the POME sample. There are already microorganisms reported producing xylanases in POME, like Bacillus, Micrococcus, and Staphylococcus (Soleimaninanadegani & Manshad, 2014). However, by investigating through the

3

metagenomics approach, xylanase enzymes will be screened from the total microorganisms in POME, which involve 99% of still undiscovered microorganisms and including from 1% of cultivable microorganisms. The bacterial species of Bacillus, Micrococcus, and Staphylococcus can be considered as microorganisms from the 1%

that can be investigated through standard laboratory methods. On the contrary, the expected xylanase to be found in this research will be more diverse because of direct mining from the POME sample.

1.3 RESEARCH OBJECTIVES

1. To screen for xylanase from a metagenomics library of palm oil mill effluent (POME) sample.

2. To identify unique DNA sequences encoding xylanases and model the structure of xylanase from POME metagenome.

3. To clone, express, and purify a xylanase obtained from POME metagenomic library.

1.4 SIGNIFICANCE OF STUDY

Xylanase is a major xylan degrading enzyme for lignocellulosic materials. It plays a huge role in the paper and pulp bleaching industry during the past several years besides having potential applications in bioconversion of lignocellulosic biomass and agro- wastes into a fermentative product, the digestibility of animal feedstocks, and the clarification of juice (Motta et al., 2013).

4 1.5 RESEARCH METHODOLOGY

This functional metagenomics approach begins with metagenomic DNA extraction from palm oil mill effluent (POME) samples. Next, the metagenomic DNA was cloned into fosmid with a size of approximately 40 kb to construct a metagenomic library. In mining the xylanases enzyme, high throughput screening using chlorocoumarin xylobioside substrate was used in this study. Next, the gene of interest was further identified through Illumina HiSeq2000 next-generation sequencing (NGS) method and was analysed using metagenomics bioinformatics analysis. Several potential xylanases that have been found were further cloned and expressed using the pBAD-TOPO expression system. The recombinant enzyme was then finally purified using immobilised metal affinity chromatography (IMAC)

1.6 SCOPE OF RESEARCH

This study was conducted to study the possible xylanase enzymes in the microorganisms of palm oil mill effluent using the metagenomics approach. It was only limited to the lab-scale production of recombinant xylanases using molecular cloning method and a specific substrate. The library was constructed by inserting the fragmented metagenomic DNA of a specific size into the fosmid vector. Besides that, the fluorescence high-throughput screening (HTS), next-generation sequencing (NGS), and bioinformatics analysis method are also used to find the genes of interest. Later, the molecular method of cloning, expression, and purification were also done to further investigate on this enzyme.

5 1.7 DISSERTATION ORGANISATION

The thesis is systematically organised in 5 chapters with chapter 1 discussing an overview, background, significance, and objective of the study. Next, Chapter 2 is focusing on literature review and information related to the functional metagenomics approach, xylanases, and palm oil mill effluent, especially in the five recent years.

Following this chapter, chapter 3 elaborates on the materials and methodology being used in this study. Accordingly, chapter 4 details the results obtained in this study with a clear and concise discussion. Finally, chapter 5 is the concluding part of the thesis.

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CHAPTER TWO LITERATURE REVIEW

2.1 INTRODUCTION

Screening for microbial biocatalysts directly from environmental samples is more applicable and convenient compared to the conventional plate cultivation approach (Hu et al., 2008). The metagenomic technique has an advantage over conventional plate cultivation approach and provides an alternative approach in understanding 99%

missing biodiversity of unculturable or difficult to culture microbes (Ferrés et al., 2015).

In this study, the xylanase gene obtained from palm oil mill effluent (POME) through a functional metagenomic approach was screened, identified, analysed, cloned, expressed, and finally purified.

Xylanase is one of the xylan degrading enzymes for hemicellulosic materials metabolism which is currently popular in different industries because of its catalytic ability in bio-bleaching of paper pulp, improvement of animal feed, bread making, application in solid waste treatment, preparation of juice from fruits or vegetables, improve retting of flax fibres, production of biofuels, and others (Asish, 2015).

However, this research will be limited to screening, cloning, and expression of a xylanase enzyme found from the POME sample only.

7 2.2 AGRO-RESIDUE BIOMASS

Malaysia is endowed with massive biomass supply from the agricultural and plantation residues. One of the major plantation residue in Malaysia besides rubber, cocoa, and pepper is the palm oil (Shafie et al., 2012). Malaysia was the largest producer and exporter of palm oil in the world from until 2007, when Indonesia replaced Malaysia as the largest palm oil producer. Nevertheless, Malaysia is still among the top palm oil producers and is only second to Indonesia (Otieno et al., 2016).

Agro-industrial wastes in the form of lignocellulosic biomass are accumulated every year in huge quantities (Mussatto & Teixeira, 2010). The wastes of palm oil industry include empty fruit bunches (EFB), palm pressed fibres (PPF), palm kernel cake (PKC) and palm kernel shell (PKS). Other wastes from the palm oil industry which contain lignocellulosic materials are oil palm trunks (OPT), oil palm fronds (OPF) and palm oil mill effluent (POME) (Abdullah & Sulaiman, 2013).

2.2.1 Palm Oil Mill Effluent (POME)

POME is the final stage effluent of palm oil mill production. Fresh POME is a hot (80- 90 °C) and thick brownish colloidal mixture of oil, water, and fine suspended solids. In this condition, POME is acidic at a pH of 4.5 with very high non-toxic biochemical oxygen demand (BOD) (Madaki & Seng, 2013). Raw POME contains high concentrations of carbohydrate (lignocellulosic materials), protein, nitrogenous compound, lipids and minerals which are summarised in Table 2.1 (Habib et al., 1997).

The nutrient rich POME makes it as an ideal habitat for microorganisms.

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Table 2.1: The approximate compositions (%) of major constituents in POME (Habib et al., 1997)

Major Constituents Composition (%)

Moisture 6.99

Crude Protein 12.75

Crude lipid 10.21

Ash 14.88

Carbohydrate 29.55

Nitrogen-free extract 26.39

Total Carotene 0.019

Total 100.789

2.2.2 POME as Microbial Fermentation Medium

POME has been used as microbial fermentation medium to produce several value added products like antibiotics, bio insecticides, solvents, polyhydroxyalkanoates (PHA), organic acids as well as enzymes (Wu et al., 2009). Microorganisms are known as a good source of useful enzymes as they could produce in extremely high rates and able to synthesise active biological product (Motta et al., 2013). Some of the bacteria found in POME is highlighted in Table 2.2. The indigenous microorganisms in POME or any habitat can be isolated and identified in laboratory conditions but the great majority (~99%) of the microbial population cannot be cultured, isolated and identified in laboratory conditions (Ferrés et al., 2015). As a counter method, metagenomics approach (also known as non-cultivable method) is a solution in making accessible of 99% biodiversity of microorganisms.

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Table 2.2: Microorganisms found in POME by previous research

References Microorganisms Genera

(Soleimaninanadegani & Manshad, 2014) Bacteria Bacillus sp.

Micrococcus sp.

Pseudomonas sp.

Staphylococcus sp.

Fungi Aspergillus sp.

Candida sp.

Fusarium sp.

Mucor sp.

Penicillium sp.

(Nwuche et al., 2014) Bacteria Flavobacterium sp.

Fungi Trichoderma sp.

(Bala et al., 2018) Bacteria Stenotrophomonas sp.

Providencia sp.

Klebsiella sp.

Fungi Meyerozyma sp.

2.3 XYLAN FROM HEMICELLULOSE

The agricultural biomass residue rich in lignocellulosic biomass materials mentioned previously in section 2.1 basically consists of 10 to 25% of lignin, 35 to 50% of cellulose and 20 to 35% of hemicellulose (Saha, 2003).

Hemicellulose consists of linear and branched polymer which basically made up of five and six different sugars of xylose, mannose, glucose, galactose and arabinose with other components of acetic, ferulic and glucuronic acid. It is being classified into xylans, glucans, mannans, glucuronoxylans, glucomannans, arabinoxylans,