SCREENING AND ISOLATION OF CYCLODEXTRIN PRODUCING FUNGI

AND BACTERIA FROM SELECTED UNIMAS AAS COLLECTION

Kathleen Michelle Mikal (18680)

Bachelor of Science with Honours

(Resource Biotechnology)

2010

Faculty of Resource Science and Technology

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ACKNOWLEDGEMENT

Above all, I would like praise God the highest for His blessing and for granting me the

strength and patience to accomplish my final year project. A special thanks and sincere

appreciation I dedicate to my supervisor, Dr. Mohd. Hasnain bin Md. Hussain and my co-

supervisor, Dr. Awang Ahmad Sallehin Awang Husaini for their advices, guidance and

encouragement in this project. I am deeply indebted to the members of Proteomic Laboratory

and Molecular Genetic Laboratory, especially to postgraduate students, Kak NurHidayah and

Fraser for all their help and valuable guidance I am especially grateful and thankful to my

beloved family and my dearest best friends for their great help and support in times of

difficulties. And finally, thank you for those who helped me throughout this project, fellow

course mates, lecturers, lab assistants and all those who involve directly and indirectly in this

project.

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TABLE OF CONTENTS

Content Page Number

ACKNOLEDGEMENT i

TABLE OF CONTENTS ii

LIST OF ABBREVIATIONS vi

LISTS OF TABLES vii

LISTS OF FIGURES viii

ABSTRACT 1

ABSTRAK 1

CHAPTER 1: INTRODUCTION

1.1 Introduction 2

1.2 Objective 3

CHAPTER 2: LITERATURE REVIEW

2.1 Cyclodextrin 4

2.1.1 History of cyclodextrin 4

2.1.2 Structure and properties of cyclodextrin 4

2.1.3 Types of cyclodextrin 5

2.1.4 Production of cyclodextrin 7

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2.2 Cyclodextrin glucosyltransferase (CGTase) 8

2.3 Uses of cyclodextrin 8

2.3.1 Cyclodextrin in perfuming industry 9

2.3.2 Cyclodextrin in food industry 10

2.3.3 Cyclodextrin in waste treatment 10

2.3.4 Cyclodextrin in paper industry 11

CHAPTER 3: MATERIALS AND METHODS

3.1 Collection and sample preparation

3.1.1 Collection and preparation of fungi samples 12

3.1.2 Preparation of bacteria samples 12

3.2 Screening of CGTase producers

3.2.1 Screening of CGTase-producing fungi 13

3.2.2 Screening of CGTase-producing bacteria 13

3.3 Isolation of CGTase-producing fungi 14

3.4 Effect of incubation time towards cyclodextrin production 14

3.5 Effect of yeast extract in production of cyclodextrin 14

3.6 Preparation of crude enzyme 15

3.7 Enzyme assay - Phenolphthalein reduction test 15

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3.8 Reconfirmation of identified CGTase producing fungi 17

3.9 Identification of unidentified CGTase producing fungi 18

CHAPTER 4: RESULT AND DISCUSSIONS

4.1 Screening and isolation of CGTase producers 19

4.1.1 Screening and isolation of CGTase-producing fungi 19

4.1.2 Screening and isolation of CGTase-producing bacteria 20

4.2 Effect of yeast extract in production of cyclodextrin 25

4.3 Effect of incubation time towards CGTase production 29

4.4 Reconfirmation of identified CGTase producing fungi

4.4.1 Morphological characteristics of 35

Aspergillus nomius (SW004)

4.4.1 Morphological characteristics of 36

Ceratocystis paradoxa (OSP)

4.4.1 Morphological characteristics of 37

Aspergillus flavus (NSH9)

4.5 Identification of unidentified CGTase producing fungi

4.5.1 Morphological characteristics of NSH6 . 38

4.5.2 Morphological characteristics of the Contaminant. 41

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CHAPTER 5: CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion 42

5.2 Recommendation 43

CHAPTER 6: REFFERENCES 44

CHAPTER 7: APPENDIX 48

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LIST OF ABBREVIATION

AAS : Awang Ahmad Sallehin

ATCC : American Type Culture Collection

CD : Cyclodextrin

CGTase: Cyclodextrin glycosyltransferase

dH2O : Distilled water

EtOH : Ethanol

HII : Horikoshi II

HPLC : High Performance Liquid Chromatography

Km : Kinetic value

LB : Luria Bertani

mM : miliMolar

OD : Optical Density

PDA : Potato Dextrose Agar

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LIST OF TABLES

TABLES PAGE

Table 2. 1 Comparison between Alpha-, Beta- and Gamma-cyclodextrin 6

Table 4.1 Mean of the OD reading (at 550nm) on 2nd, 4th, 6th, 8th and 29

10th days of incubation

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LIST OF FIGURES

FIGURES PAGE

Figure 2.1 Molecular models of Alpha-,Beta- and Gamma-cyclodextrin 6

Figure 2.1 Chemical structure of Cyclodextrin 6

Figure 2.2 Schematic diagram of the four different transferase 9

catalyzed by CGTase

Figure 4.1 The original color of the screening plate before incubation. 20

Figure 4.2 SW004 after 4 days of incubation. 20

Figure 4.3 NSH9 after 8 days of incubation. 21

Figure 4.4 Contaminant (KCC01) from B. amyloliquefaciens screening plate 22

after 10 days.

Figure 4.5 KCC01 screening plate (HII with yeast) after 7 days. 23

Figure 4.6 OSP (HII with yeast extract) after 12 days. 25

Figure 4.7 OSP (HII without yeast extract) after 15 days. 25

Figure 4.8 SW004 (in a HII with yeast extract) after 4 days. 26

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Figure 4.9 SW004 (in a HII without yeast extract) after 6 days. 26

Figure 4.10 NSH9 (HII with yeast extract) after 8 days. 26

Figure 4.11 NSH6 with yeast extract on the 17th day of incubation. 27

Figure 4.12 NSH6 without yeast extract on the 17th day. 27

Figure 4.13 KCC01 screening plate (HII with yeast extract) after 7 days. 28

Figure 4.14 KCC01 screening plate (HII without yeast extract) after 8 days. 28

Figure 4.16 Enzyme Activity (U/ml) of 5 fungi samples in different period 30

of incubation (days).

Figure 4.17 SW004 (HII with yeast) after 14 days. 32

Figure 4.18 NSH9 (HII with yeast) after 7 days (early reduction). 34

Figure 4.19 Morphological characteristics of Aspergillus nomius (SW004) 35

Figure 4.20 Morphological characteristics of Ceratocystis paradoxa (OSP). 36

Figure 4.21 Morphological characteristics of Aspergillus flavus (NSH9) 37

Figure 4.22 Morphological characteristics of NSH6 38

Figure 4.22 Penicillium sp. conidiophores. 39

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Figure 4.23 Penicillium sp. conidiophores 39

Figure 4.24 The colony of one of the fungi in the Penicillium genera 39

Figure 4.25 Three types of conidiophores of Penicillium 40

Figure 4.26 Morphological characteristics of the Contaminant 41

1

Screening and Isolation of Cyclodextrin Producing Fungi and Bacteria from Selected

UNIMAS AAS Collection

Kathleen Michelle Mikal

Resource Biotechnology Programme

Faculty of Science and technology

Universiti Malaysia Sarawak

ABSTRACT

Cyclodextrin (CD) is cyclic oligosaccharides consisting of α-1,4-glucosidic linkage produced

by the reaction of cyclodextrin glycosyltransferase (CGTase EC 2.4.1.19) and starch. CGTase

are usually produced by bacteria mostly from the Bacillus genera. There is no research done

on fungi for production of CD. In this study, screening was done on fungi and bacteria to

identify which fungi and bacteria could produce CGTase that will later form CD. The

screening was done on the Horikoshi II media containing phenolphthalein and methyl orange

as CGTase indicator. Five fungi and three bacteria from the AAS UNIMAS collection were

screened. Screening and enzyme assay showed that two fungi, Aspergillus nomius and

Aspergillus flavus produce CGTase. Both fungi have high enzyme activity with 12.97U/ml for

A.nomius and 10.66U/ml for A.flavus based on CGTase assay.

Key words: cyclodextrin (CD), cyclodextrin glucosyltransferase (CGTase), screening, fungi

ABSTRAK

Siklodekstrin (CD) merupakan oligosakarida siklik yang terdiri daripada rantaian α-1,4-

glucosidik, hasil tindak balas siklodekstrin glucosiltransferase (CGTase, EC 2.4.1.19) dengan

kanji. Enzim ini kebanyakannya dihasilkan oleh bakteria dari genus Bacillus. Belum ada

penyelidikan dijalankan ke atas fungi untuk menghasilan CD. Dalam kajian ini, penyaringan

dilakukan ke atas fungi dan bakteria untuk mengenal pasti fungi dan bakteria yang boleh

mehasilkan CGTase yang akan membentuk CD. Penyaringan dilakukan pada media Horikoshi

II yang mempunyai fenolftalein dan metil oren sebagai penunjuk (indikator) penghasilan

CGTase. Lima fungi dan tiga bakteria yang diambil dari pada koleks