SCREENING AND ISOLATION OF CYCLODEXTRIN and Isolation of Cyclodextrin...PDF file3.7 Enzyme assay -...

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  • 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

  • i

    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.

  • ii

    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

  • iii

    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

  • iv

    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

  • v

    CHAPTER 5: CONCLUSION AND RECOMMENDATIONS

    5.1 Conclusion 42

    5.2 Recommendation 43

    CHAPTER 6: REFFERENCES 44

    CHAPTER 7: APPENDIX 48

  • vi

    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

  • vii

    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

  • viii

    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

  • ix

    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

  • x

    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