UNIVERSITI PUTRA MALAYSIA

AHMAD AFIF ABDUL AZIZ

FBSB 2013 34

ISOLATION AND ENZYMATIC STUDIES OF DYE-DEGRADING WHITE-ROT FUNGUS

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ISOLATION AND ENZYMATIC STUDIES OF DYE-DEGRADING WHITE-ROT

FUNGUS

By

AHMAD AFIF ABDUL AZIZ

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

Fulfilment of the Requirements for the Degree of Master of Science

December 2013

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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

ISOLATION AND ENZYMATIC STUDIES OF DYE-DEGRADING WHITE

ROT FUNGUS

By

AHMAD AFIF ABDUL AZIZ

December 2013

Chairman: Assoc. Prof. Mohd Yunus Abd Shukor, PhD

Faculty : Biotechnology and Biomolecular Sciences

Dye is present continuously in the environment. They are designed to be permanent

and resistant to degradation by their physical and chemical properties. Therefore,

increasing discharge and improper management of solid and liquid industrial dye

wastes have generated a great concern among industrialists and the scientific

community due to their negative effects. To date, effective method to treat

recalcitrant dye is still lacking, to ensure safe disposal of dye effluents. In this

regards, this study had been designated to isolate and screen dye degrading white rot

fungi from local environments in Peninsular Malaysia; to identify the selected white

rot fungus that showed the best degrading ability; to evaluate the degradation of azo

dye by the selected white-rot fungus and determine its optimum conditions; and to

partially purify and partially characterize the ligninolytic enzyme. Thirty nine white

rot fungi (WRF) from soil and wood samples were isolated in Selangor, Kelantan,

Pahang and Terengganu and tested for their capability to degrade textile azo dyes

(Orange G (C.I. 16230), Ponceau 2R (C.I. 16450), Amaranth (C.I. 16185), Trypan

Blue (C.I. 23850) dan Direct Blue 71 (C.I. 34140). Thirty-three isolates showed

positive results with varying degrees of dye degradation. Two isolates (Isolate 4-

UPM and Isolate 17-UPM) from Universiti Putra Malaysia (UPM) campus in

Selangor were selected for further studies owing to their ability to completely

decolourize all the azo dyes within the shortest time. Qualitative study on defined

solid media showed Isolate 17-UPM and Isolate 4-UPM were capable of degrading

all five dyes under nitrogen-limiting conditions, with glucose as the source of energy.

When cultured in two-stage liquid medium for quantitative screening, Isolate 17-

UPM degradation rate was in the range of 96 to 99% of 0.2 g/L while Isolate 4-UPM

showed a range of 38 to 96 % of all the tested azo dyes. Both isolates degraded the

dyes within one to ten days at different rates. Isolate 17-UPM and Ponceau 2R were

used for further studies. Overall, the degradation rates of Isolate 17-UPM in agitated

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cultures were higher by nearly ten times compared to static cultures. Ponceau 2R was

degraded optimally when incubated between 35 to 40°C in agitated cultures at the

initial pH of 6.

The assays for lignin modifying enzymes involved in the azo dye degradation

showed the presence of laccase only, while lignin peroxidase and manganese

peroxidases were absent. There was a significant correlation between the laccase

activity profile in agitated liquid cultures and the azo dye degradation profile where

both optimum temperature and initial pH were 40°C and pH 6, respectively. The

laccase produced by Isolate 17-UPM during azo dye degradation was partially

purified using DEAE Cellulose anion exchanger and ZorbaxR GF-250 gel filtration

column. The partial purified enzyme showed a Km (app) value of 0.28 mM, Vmax (app)

value of 100 μmol/min.ml, optimum temperature activity at 40 to 50°C and pH 3.0 to

5.0 when 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) was used as the

substrate. It was also shown to be most stable at room temperature and pH 6.0 to

7.0.The Isolate 17-UPM was further characterized at molecular level through ITS

region gene sequencing. The internal transcribe spacer region of the isolated DNA

was amplified by PCR using the primers recognized as, primer ITS 1F and ITS 4.

Isolate 17-UPM was identified as Coriolopsis sp. strain aff17. In this study, a white-

rot fungus capable of degrading azo dyes was isolated, identified and optimized for

dye degradation, and the enzyme involved was partially purified and characterized.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk ijazah Master Sains

PEMENCILAN DAN KAJIAN ENZIM KULAT REPUT-PUTIH YANG

MENGURAI PEWARNA

Oleh

AHMAD AFIF ABDUL AZIZ

Disember 2013

Pengerusi: Prof. Madya. Mohd Yunus Abd Shukor, PhD

Fakulti : Bioteknologi dan Sains Biomolekul

Pewarna wujud secara berterusan dalam alam sekitar. Ia direka untuk menjadi kekal

dan tahan penguraian kepada sifat-sifat fizikal dan kimia pewarna tersebut. Oleh itu,

peningkatan pelepasan dan salah pengurusan sisa pewarna industri pepejal dan cecair

telah menjadi satu kebimbangan besar di kalangan usahawan-usahawan dan komuniti

saintifik disebabkan oleh kesan negatif mereka. Setakat ini, kaedah yang berkesan

untuk merawat sisa pewarna masih kurang, untuk memastikan pelupusan selamat

efluen pewarna. Oleh itu, kajian ini telah direka untuk memencil dan menyaring

kulat reput putih dari persekitaran tempatan di Semenanjung Malaysia yang boleh

mengurai pewarna; untuk mengenal pasti kulat reput putih yang dipilih; untuk

menilai keupayaan kulat reput putih penguraian pewarna azo yang dipilih dan

menentukan syarat-syarat alam sekitar yang optimum; dan untuk penulenan separa

dan pencirian separa enzim pengubah lignin. Tiga puluh sembilan kulat reput putih

dari sampel tanah dan kayu di Selangor, Kelantan , Pahang dan Terengganu telah

dipencilkan dan diuji keupayaan untuk menguraikan pewarna tekstil azo Orange G

(CI 16230), Ponceau 2R (CI 16450), Amaranth (CI 16185), Trypan Blue (CI 23850)

dan Direct Blue 71 (CI 34140). Tiga puluh tiga pencilan menunjukkan hasil yang

positif dengan pelbagai peringkat penguraian pewarna. Dua pencilan (Isolat 4-UPM

dan Isolat 17-UPM) dari Universiti Putra Malaysia (UPM) kampus di Selangor telah

dipilih untuk kajian lanjut berdasarkan keupayaan untuk menguraikan keseluruhan

warna pewarna azo dalam masa paling singkat. Kajian kualitatif di media pepejal

menunjukkan Isolat 17-UPM dan Isolat 4-UPM mampu menguraikan kesemua lima

pewarna dalam keadaan nitrogen yang terhad, dengan glukosa sebagai sumber

tenaga. Apabila dikultur dalam medium cecair dua peringkat bagi penyaringan

kuantitatif, kadar penguraian Isolat 17-UPM adalah dalam lingkungan 96 hingga 99

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% daripada 0.2 g/L manakala Isolat 4- UPM menunjukkan lingkungan 38 hingga

96% bagi semua pewarna azo yang diuji. Kedua-dua pencilan menguraikan pewarna

dalam tempoh satu hingga sepuluh hari pada kadar yang berbeza. Isolat 17- UPM

dan Ponceau 2R telah digunakan untuk kajian lanjutan. Secara keseluruhan, kadar

penguraian Isolat 17-UPM dalam kultur goncang lebih tinggi hampir sepuluh kali

berbanding kultur statik. Ponceau 2R telah diuraikan secara optimum apabila dieram

di antara 35 hingga 40°C dalam kultur goncang pada pH awal 6.

Pencerakinan untuk enzim-enzim pengubah lignin yang terlibat dengan penguraian

pewarna azo hanya menunjukkan kehadiran lakase (E.C. 1.10.3.2) manakala lignin

peroksidase (E.C. 1.11.1.14) dan mangan peroksidase (E.C. 1.11.1.13) tidak dapat

dikesan. Terdapat hubungan yang signifikan antara profil aktiviti lakase dalam kultur

cecair goncang dan profil degradasi pewarna azo di mana suhu dan pH awal

optimum bagi kedua-dua adalah masing-masing 40°C dan pH 6. Penulenan separa

lakase yang dihasilkan oleh Isolat 17-UPM semasa degradasi pewarna azo melalui

penukar anion DEAE Cellulose dan kolum penurasan gel ZorbaxR GF -250. Enzim

separa tulen menunjukkan Km (app) bernilai 0.28 mM, Vmax (app) bernilai 100

μmol/min.ml, aktiviti suhu optimum pada 40 hingga 50°C dan pH 3.0 hingga 5.0

apabila 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) digunakan

sebagai substrat. Ia juga mempamerkan kestabilan tinggi pada suhu bilik dan pH 6.0

hingga 7.0. Isolat 17- UPM terus dicirikan pada peringkat molekul melalui

penjujukan rantai gen ITS. Rantai ruangan salinan dalaman DNA yang dipencilkan

telah dikuatkan melalui PCR menggunakan primer dikenali sebagai primer ITS-1F

dan ITS 4. Isolat 17- UPM telah dikenalpasti sebagai Coriolopsis sp. starin aff17.

Dalam kajian ini, kulat reput putih mampu menguraikan pewarna azo telah berjaya

dipencilkan, dikenalpasti dan dioptimumkan bagi penguraian pewarna, dan

penulenan dan pencirian separa enzim yang terlibat.

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ACKNOWLEDGEMENTS

Firstly I would like to express my gratitude to Allah for showering me His blessing

and consent to make all of this possible. My sincere thanks and wholehearted

appreciation to my supervisor Assoc. Prof. Dr. Mohd Yunus Abd Shukor for his

invaluable guidance, advice, expertise and motivation throughout my research. My

thanks also go to Prof. Dr Mohd Arif Syed for the advice and support on many

aspects of this work.

Special thanks to my laboratory mates especially those from lab 204 namely Mohd

Haris, Mohd Ezuan, Badrin and Khalizan for being the source of strength, constant

sharing of knowledge and advice that will be remembered and more importantly is

the friendship we treasure.

Last but not least, I would like to express my warm gratitude to my family especially

my mother and father for giving me the strength and unremitting love, not to forget

for my lovely wife Dania Aziz for being patient and giving constant encouragement

and support throughout the completion of this thesis. I certainly wouldn’t be able to

achieve what I have today without the unconditional love and support from family

and friends. Thank you again.

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I certify that a Thesis Examination Committee has met on (16th

December 2013) to

conduct the final examination of Ahmad Afif Abdul Aziz on his thesis entitled

Isolation And Enzymatic Studies Of Dye-Degrading White Rot Fungus 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 Masters of Science.

Members of the Thesis Examination Committee were as follows:

Nor’Aini bt. Abdul Rahman, PhD

Faculty of Biotechnology and Science Biomolecule

Universiti Putra Malaysia

(Chairman)

Norhani bt. Abdullah, PhD

Professor

Institute of Tropical Agriculture

Universiti Putra Malaysia

(Internal Examiner)

Phang Lai Yee, PhD

Faculty of Biotechnology and Science Biomolecule

Universiti Putra Malaysia

(Internal Examiner)

Mahmoud Abdel-Mongy Ahmed Ismail, PhD

Associate Professor

Genetic Engineering and Biotechnology Research Institute

Minufiya University

Egypt

(External Examiner)

________________________________

NORITAH OMAR, PhD

Associate Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 17 February 2014

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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:

Mohd Yunus Bin Abd Shukor, PhD Associate Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Chairman)

Mohd Arif Bin Syed, PhD

Professor

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

_______________________________

(BUJANG BIN KIM HUAT, PhD)

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

Declaration by graduate student

I hereby confirm that:

this thesis is my original work;

quotations, illustrations and citations have been duly referenced;

this thesis has not been submitted previously or concurrently for any other

degree at any other institutions;

intellectual property from the thesis and copyright of thesis are fully-owned

by Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Research and Innovation) before thesis is published (in the

form of written, printed or in electronic form) including books, journals,

modules, proceedings, popular writings, seminar papers, manuscripts,

posters, reports, lecture notes, learning modules or any other materials as

stated in the Universiti Putra Malaysia (Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and

scholarly integrity is upheld as according to the Universiti Putra Malaysia

(Graduate Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra

Malaysia (Research) Rules 2012. The thesis has undergone plagiarism

detection software.

Signature: _______________________ Date: ___16 December 2013____

Name and Matric No.: ___AHMAD AFIF ABDUL AZIZ (GS19573)___

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Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the writing of this thesis was under our

supervision;

supervision responsibilities as stated in the Universiti Putra Malaysia

(Graduate Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature: ____________________ Signature:_________________

Name of Name of

Chairman of Member of

Supervisory Supervisory

Committee: Mohd Yunus Bin Abd Shukor, Committee: Mohd Arif Bin Syed,

PhD PhD

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK iv

ACKNOWLEDGEMENTS vi

APPROVAL vii

DECLARATION ix

LIST OF TABLES xiv

LIST OF FIGURES xv

LIST OF ABBREVIATIONS xix

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW

2.1 Azo Dye

2.1.1 Structure and stability of azo dyes

2.2 Environmental effect of azo dye residual

2.2.1 Toxicology and persistence of azo dyes

2.3 Methods of azo dye removal

2.3.1 Physical and chemical method

2.3.2 Biological method

2.4 Bioremediation

2.5 Mycoremediation of azo dyes

2.6 White Rot Fungi

2.7 Laccase characteristics

2.7.1 Optimum pH

2.7.2 Optimum temperature

2.7.3 Laccase stability and substrate specificity

2.8 Application of laccase

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3 MATERIALS AND METHODS

3.1 General Overview

3.2 Chemicals and Equipments Used

3.3 Isolation and Screening of White-Rot Fungi

3.3.1 White-Rot Fungi Sampling

3.3.2 Cleaning and Disinfection of White-Rot Fungi

Samples

3.3.3 Growth and Isolation

3.3.4 Maintenance of White-Rot Fungi Isolates

3.3.5 Screening of White-Rot Cultures for Azo Dye

Degrading Ability

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3.3.5.1 Primary Qualitative Screening

3.3.5.2 Secondary Quantitative Screening

3.4 Azo Dye Degradation Studies

3.4.1 Effects of Glucose and Ammonium Chloride on Azo

Dye Degradation by Isolate 17-UPM

3.4.2 Effects of Aeration in Azo Dyes Degradation Profiles

(Static and Agitated Liquid Cultures)

3.4.2.1 Preparing the Degradation Media for Agitated

Cultures

3.4.3 Effects of Different Growth Conditions on Azo Dye

Degradation in Agitated Liquid Cultures

3.4.3.1 Effects of Incubation Temperatures on Azo

Dye Degradation

3.4.3.2 Effects of Different Degradation Medium pH

on Azo Dye Degradation

3.5 Enzymatic Studies on Azo Dye Degradation

3.5.1 Detection of LMEs Produced by Isolate 17-UPM in

Degradation Cultures and the Effects of Different

Growth Conditions on LMEs Activity

3.5.1.1 Assay for Laccase Activity

3.5.1.2 Assay for Lignin Peroxidase Activity

3.5.1.3 Assay for Manganese Peroxidase Activity

3.5.2 Azo Dye Degradation and LMEs Profile in 1 L

Agitated Degradation Cultures

3.5.3 Fungal Growth and Harvesting of Laccase from 1 L

Agitated Cultures for Enzymatic Studies

3.5.4 Partial Purification of Laccase

3.5.4.1 Protein determination

3.5.4.2 Concentration of crude laccase

3.5.4.3 Ion exchange chromatography using DEAE

cellulose

3.5.4.4Gel filtration chromatograhy using Zorbax®

GF 250

3.5.5 Laccase Characterization Studies

3.5.5.1 Determination of Laccase Km and Vmax with

ABTS as the Substrate

3.5.5.2 Effects of Different Temperatures on Laccase

Activity

3.5.5.3 Effects of Different pH on Laccase Activity

3.5.5.4 Determination of Laccase Temperature

Stability

3.5.5.5 Determination of Laccase pH Stability

3.6 Identification of Azo Dye-Degrading Fungus

3.6.1 ITS region analysis

3.6.1.1 Genomic Extraction

3.6.1.2 Polymerase Chain Reaction (PCR)

3.6.1.3 Purification of the amplified PCR products

3.6.1.4 Phylogenetic analysis

3.6.2 Statistical analysis

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4 RESULTS AND DISCUSSION

4.0 Isolation and screening of white-rot fungi

4.1 White-rot fungi isolated from sites in Peninsular Malaysia

4.2 Screening of white-rot cultures for azo dye degrading ability

4.2.1 Primary Qualitative Screening

4.2.2 Secondary Quantitative Screening

4.3 Optimization of Azo Dye Degradation

4.3.1 Effects of Glucose and Ammonium Chloride on Azo

Dye Degradation by Isolate 17-UPM

4.3.2 Effect Of Aeration on Azo Dyes Degradation Profiles

(Static and Agitated Liquid Cultures)

4.3.3 Effects Of Different Growth Conditions On Azo Dye

Degradation

4.3.3.1 Effects of incubation temperatures on azo dye

degradation

4.3.3.2 Effects of different medium initial pH on azo

dye degradation

4.4 Enzymatic Studies on Azo Dye Degradation

4.4.1 Detection and studies on the effects of growth

conditions on LMEs activity by Isolate 17-UPM in

degradation cultures

4.4.1.1 Effects of different incubation temperatures on

laccase activity in degradation medium

4.4.1.2 Effects of different degradation medium pH

on laccase activity

4.4.2 Azo dye degradation and LMEs profile in 1 L agitated

degradation cultures

4.4.3 Partial purification of laccase

4.4.3.1 Concentration of crude enzyme

4.4.3.2 Ion exchange chromatography

4.4.3.3 Size exclusion chromatography

4.4.4 Laccase characterization studies

4.4.4.1 Determination of Km and Vmax

4.4.4.2 Effects of temperatures on laccase activity

4.4.4.3 Effects of pH on laccase activity

4.4.4.4 Determination of Laccase Temperature

Stability

4.4.4.5 Determination of Laccase pH Stability

4.4.5 ITS Region Analysis

4.4.5.1 Genomic Extraction

4.4.5.2 Polymerase Chain Reaction (PCR)

4.4.5.3 ITS rRNA Gene Sequencing

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5 CONCLUSION 75

REFERENCES 76

APPENDICES 89

BIODATA OF STUDENT 93