UNIVERSITI PUTRA MALAYSIA

ISOLATION AND PURIFICATION OF RAW STARCH DEGRADING ENZYME FROM ENDOPHYTIC FUNGI AND ITS APPLICATION FOR

GLUCOSE PRODUCTION

YETTI MARLIDA

FSMB 2001 23

ISOLATION AND PURIFICATION OF RAW STARCH DEGRADING ENZYME FROM ENDOPHYTIC

FUNGI AND ITS APPLICATION FOR GLUCOSE PRODUCTION

YETTI MARL IDA

DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

2001

ISOLATION AND PURIFICATION OF RAW STARCH DEGRADING ENZYME FROM ENDOPHYTIC FUNGI AND ITS

APPLICATION FOR GLUCOSE PRODUCTION

By

YETII MARLIDA

Thesis Submitted in Fulfilment of the Requirement for the Degree of Doctor of Philosophy in the Faculty of Food Science and Biotechnology

Universiti Putra Malaysia

May 2001

Dedicated to my husband,

Mr. Syahril Amiruddin

And my love children,

Pramana Yuda Sayeti & Ameliora Reski Sayeti

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy

ISOLATION AND PURIFICATION OF RAW STARCH DEGRADING ENZYME FROM ENDOPHYTIC FUNGI AND ITS APPLICATION FOR

GLUCOSE PRODUCTION

By

YEITI MARLIDA

May 2001

Chairman: Dr. Nazamid bin Saari

Faculty Food Science and Biotechnology

Raw starch degrading enzymes are enzymes that degrade polymers of raw starches to

smaller molecules of oligo saccharides. Fifty-two strains of endophytic fungi were

isolated from tropical plant trees in Malaysia and screened for raw starch degrading

enzyme activity. Twelve endophytic strains with the ability to degrade raw starches

as indicated by size of clear zone formed on Czapek-Dox medium were obtained.

Three strains were found to be good raw starch degrading enzyme producers based

on the ability to degrade raw starches. Raw sago, potato and rice starches were

found to be appropriate substrate for enzyme production. The three strains of

endophytic fungi were identified as Gibbere/la pulicaris using I8S rRNA sequences

and, Acremonium sp. and Synnematous sp. taxonomically, respectively. Comparison

of their degradative activity on raw and gelatinized starches, substrate specificity,

optimum pH and product of hydrolysis showed that Acremonium sp. had a broad

activity towards both small and large sized granules of raw starches

while G. pulicaris and Synnematous sp. were dependent on the granule size and

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structures of starches. The enzyme from Acremonium sp. is unique since it does not

depend on the granule size and structures of starches. The results showed that the

enzyme from G. pulicaris and Acremonium sp were raw starch degrading

glucoamylase, an exo-acting enzyme which solely yielded glucose while enzyme

from Synnematous sp. was raw starch degrading a-amylase, an endo-acting enzyme

which yielded a mixture of glucose and maltose. Consequently, raw starch degrading

glucoamylase was more efficient than raw starch degrading a-amylase. Both

glucoamylase and a-amylase showed the highest degradation rate on raw rice starch.

Based on the substrate specificity, degradative pattern and product produced, the

Acremonium sp. was selected and pursued to produce raw starch degrading enzyme.

The optimum conditions for enzyme production based on carbon and nitrogen

sources were studied. Results showed that the highest raw starch degrading enzyme

activity was observed when the Acremonium sp. were grown in shake flask culture

of broth medium at 120 rpm, 30°C for 5 days and yielded glucoamylase activity of

67 U/ml. The enzyme was induced in broth medium by the addition of raw sago

starch as its carbon source and peptone and sodium nitrate as nitrogen sources.

Acremonium sp. showed maximum enzyme activity when grown at pH 5.0, 30°C for

5 days. The raw starch degrading enzyme was found to be inducible enzymes

depending on the amount of raw sago starch added to the culture medium. The raw

starch degrading enzyme from Acremonium sp. was purified to homogeneity by a

combination of ultrafiltration, DEAE-Toyopearl 650 S ion exchange and Sephadex

G-150 gel filtration. Pure raw starch degrading enzyme was shown to consist of two

sub-units of 22 and 39 kDa by sodium dodecyl sulphate-polyacrylamide gel

electrophoresis and a single band on native-polyacrylamide gel electrophoresis. The

optimum pH and temperature for hydrolysis of raw starches were 5.5 and 55°C,

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respectively. The enzyme was stable at pH range of 3.0 to 7.0 and at temperature up

to 60°C. The purified enzyme had Kmvalues of 10.0 and 3.8 mglml and Vmaxof 195

Ilmoles/ml/min and 391 J..lmoles/ml/min for amylose and amylopectin, respectively.

Pretreatment of sago starch at low pH and heating below gelatinization temperature

was found to increase the enzymatic hydrolysis.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Doktor Falsafah.

PEMENCILAN DAN PENULINAN ENZIM PENGURAI KANJI MENTAH DARIPADA FUNGI ENDOFITIK DAN KEGUNAANY A BAGI

PENGHASILAN GLUKOSA

Oleh

YETTI MARLIDA

Mei 2001

Pengerusi Nazamid Saari, Ph.D.

Fakulti Sains Makanan dan Bioteknologi

Enzim pengurai kanji mentah adalah enzim yang dapat menguraikan polimer kanji

mentah menjadi molekul oligosakarida yang lebih kecil. Lima puluh dua strain fungi

endofitik telah dipencilkan dari tumbuhan hutan tropika di Malaysia dan telah

disaring bagi mendapatkan aktiviti pengurai kanji mentah. Dua belas strain fungi

endofitik dengan keupayaan menguraikan kanji mentah ditentukan berdasarkan saiz

zon terang (clear zone) di atas media Czapek-Dox yang dibentuk telah diperolehi.

Tiga strain telah dikenalpasti sebagai penghasil enzim pengurai kanji mentah

berdasarkan keupayaan enzim-enzim tersebut menguraikan kanji mentah. Kanji sagu,

kentang dan beras telah dikenalpasti sebagai substrat yang sangat baik untuk

menghasilkan enzim. Tiga kulat yang telah dikenalpasti adalah Gibbere//a pu/icaris

menggunakan jujukan 1 8S rRNA, Acremonium sp. dan Synnematous sp. secara

taksonomi. Perbandingan aktiviti pengurai menggunakan kanji mentah dan kanji

yang telah digelatinisasi, kespesifikan substrat, pH optimum dan hasil hidrolisis

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menunjukkan Acremonium sp. mempunyai aktiviti yang luas bagi kanji yang

berukuran kecil dan besar sementara enzim yang dihasilkan oleh G. pulicaris dan

Synnematous sp. bergantung kepada ukuran dan struktur butiran, manakala enzim

yang dihasilkan oleh Acremonium sp. adalah unik dan tidak bergantung pada ukuran

dan struktur butiran. Keputusan yang diperoleh, menunjukkan enzim yang

dihasilkan dari kulat G. pu/icaris dan Acremonium sp. adalah glukoamilase dan

hanya menghasilkan glukosa manakala enzim pemecah kanji mentah dari

Synnematous sp. adalah a-amilase yang menghasilkan campuran glukosa dan

maltosa. Enzim pengurai kanji glukoamilase didapati lebih efisien berbanding enzim

pengurai kanji a-amilase. G1ukoamilase dan a-amilase menunjukkan kadar

pemecahan yang tinggi ke atas kanji mentah beras. Berdasarkan kespesifikan

substrat, corak pemecahan dan produk yang dihasilkan, Acremonium sp. telah

dipilih dan dikaji dengan lebih lanjut untuk penghasilan enzim pengurai kanji

mentah. Keadaan optimum untuk penghasilan enzim dengan baik telah diteliti

dengan menggunakan sumber nitrogen dan karbon. Keputusan menunjukkan aktiviti

pengurai kanji mentah paling tinggi apabila Acremonium sp. ditumbuhkan dalam

kelalang berpengaduk pada 120 rpm, 30°C selama 5 hari dan menghasilkan aktiviti

glukoamilase 67 U/ml. Enzim ini diaruh dalam media cecair dengan penambahan

kanji sagu mentah sebagai sumber karbon, pepton dan sodium nitrat sebagai sumber

nitrogen. Acremonium sp. menunjukkan aktiviti maksimum apabila dihidupkan pada

pH 5.0, suhu 30°C selepas pengeraman selama 5 hari. Jumlah kandungan kanji sagu

mentah yang ditambahkan ke dalam medium kultur didapati mampu bertindak

sebagai agen penggeluaran enzim pengurai kanji mentah. Enzim pengurai kanji

mentah daripada Acremonium sp. ditulinkan sehingga homogen dengan kombinasi

penurasan ultra, DEAE-Toyopearl-650 S penukar ion dan Sephadex G-150 gel

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penurasan berturus. Enzim tulin pengurai kanji mentah telah dianggarkan

mengandungi dua sub-unit iaitu 22 dan 39 kDa dengan menggunakan gel

elektroforesis sodium dodesil sulfat poliakrilamid dan satu jalur tunggal yang

diperolehi dengan menggunakan gel elektroforesis poliakrilamid-natif Hidrolisis

kanji mentah adalah optimum pada pH 5.5 dan suhu 55°C. Enzim didapati stabil

pada julat pH 3. 0 hingga 7.0 dan suhu sehingga 60°C. Enzim pengurai kanji mentah

tulin mempunyai nilai Km 10.0 and 3. 8 mglml dan nilai Vnuu 195 IJ.moles/ml/min dan

391 IJ.moles/ml/min untuk amilose dan amilopektin, berturut-turut. Perlakuan awal

kanji sagu mentah pada pH rendah dan pemanasan di bawah suhu penggelatinan

boleh meningkatkan hidrolisis secara enzimatik.

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ACKNOWLEDGEMENTS

I wish to express my sincere gratitude and thanks to my supervisor, Dr. Nazamid

Saari and members of the supervisory committee, Dr. Zaiton Hassan and Associate

Professor Dr. Son Radu for their guidance and encouragement during the course of

this study and the preparation of this thesis.

I gratefully acknowledge the financial support by Emeritus Prof Ryoichi

Ohgushi of Ohgushi Yoshika Foundation, Kanazawa Japan, and the Chancellor of

Andalas University Prof Dr. Martis Rahman for an opportunity given and to

Universiti Putra Malaysia for granting the research facilities.

I would like to acknowledge Prof Fusao Tomita from Faculty of Agriculture,

Hokkaido University, Japan and Dr. Katsuhiko Ando from Tokyo Research

Laboratories, Kyowa Hakko, Japan for their assistance in the identification of the

endophytic fungi.

My deepest appreciation to my parents, Haji Dalmi and Maini and my son

Pramana Yuda Sayeti, my daughter Ameliora Reski Sayeti (Aisyah), brother Salmi

for the enonnous amount of love, support and sacrifice they had given.

Most of all I am grateful to my husband Syahril Amiruddin for being

supportive, understanding and loving all the way and for spending his time helping

me type and print this thesis.

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Finally, I wish to say thanks, to all my friends and mates in Dr. Nazamid

Saari's, Dr. Azizah Hamid's and Dr. Suraini's laboratories for their help throughout

the course of this investigation.

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I certify that an Examination Committee met on 22th May 2001 to conduct the final examination of Yetti Marlida on her Doctor of Philosophy thesis entitled "Isolation and Purification of Raw Starch Degrading Enzyme from Endophytic Fungi and Its Application for Glucose Production" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

Suraini Abd Aziz, Ph.D. Faculty of Food Science and Biotechnology, Universiti Putra Malaysia (Chairperson)

Nazamid Saari, Ph.D. Faculty of Food Science and Biotechnology, Universiti Putra Malaysia (Member)

Zaiton Hasan, Ph.D. Faculty of Food Science and Biotechnology, Universiti Putra Malaysia (Member)

Son Radu, Ph.D. Associate Professor Faculty of Food Science and Biotechnology, Universiti Putra Malaysia (Member)

Othman Omar, Ph.D. Professor Faculty of Science and Technology Universiti Kebangsaan Malaysia (Independent Examiner)

M��HAYIDIN'Ph.D. ProfessorlDeputy Dean of Graduate School, Univer���sia Date: � � tJUN �UUl

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This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the Degree of Doctor of Philosophy.

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AINI IDERIS, Ph.D. Professor Dean of Graduate School, University Putra Malaysia Date:

1 2 JIJL 2001

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any degree at UPM or other institutions.

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YETTI MARLIDA �a JUHe d.OD/

TABLE OF CONTENTS

DEDICATION ABSTRACT ABSTRAK ACKNOWLEDGEMENTS APPRO V AL SHEETS DECLARATION FORM LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS

CHAPTER

I INTRODUCTION

II LITERATURE REVIEW Definition of Raw Starch Degrading Enzyme

Classification of Raw Starch Degrading Enzyme Distribution of Raw Starch Degrading Enzyme Reaction Mechanism of Raw Starch Degrading Enzyme Screening of Raw Starch Degrading Enzyme Microbiology of Endophytic Fungi

Production of Raw Starch Degrading Enzyme Regulation of Raw Starch Degrading Enzyme Cultural Conditions Catabolite Repression

Factors Affecting Production of Raw Starch Degrading Enzyme Effect of pH Effect of Temperature Effect of Media Composition

Purification of Raw Starch Degrading Enzyme Properties of Raw Starch Degrading Enzyme (RSDE)

Effect of Metals on RSDE Activity Specificity Optimum pH and Stability Product of Hydrolysis

Application of Starch Degrading Enzyme in Food Industry

III ISOLATION, SCREENING AND IDENTIFICATION OF RAW STARCH DEGRADING ENZYME FROM ENDOPHYTIC FUNGI Introduction Materials and Methods

Materials Isolation of Endophytic Fungi

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29 29 29 30 30 34 36 40 40 42 44 45 47 49 50 58 60 63 64 65 66 68

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73 74 74 74

Qualitative Measurement ofRSDE 75

Quantitative Measurement of RSDE 75

Measurement ofRSDE Activity 76 Identification of Isolates 76

Results and Discussion 77 Qualitative Analysis 77 Quantitative Analysis 80 Identification of Endophytic Fungi 85 rRNA Gene Analysis 85 Taxonomic Analysis 85

Conclusions 86

IV RAW STARCH DEGRADING ACTIVITIES OF SELECTED 98 ENDOPHYTIC FUNGI IN LIQUID CULTURE MEDIUM Introduction 98 Materials and Methods

Materials 99 Organisms 99 Medium Cultivation 99 Effect of pH on Enzymatic Hydrolysis 100 Effect of Different Raw Starches as Substrate 100 Estimation of Raw Starch Degrading Ability (RSDA) 101 Measurement of Activity 101 Optimum pH and Temperature 101 Determination of End Product 102

Results and Discussion 103 . Enzyme Production Profiles 103

Effect of pH of Reaction Medium on Hydrolysis 105 Substrate Specificity 105 Ratio of De grad ability of Raw to Gelatinized Starch 109 Identification of End product by TLC 111 Selected Strains 111 Optimum of Reaction Conditions 113

Conclusions 113

V COMPARISON OF DEGRADATIVE PATTERNS OF RAW 117 STARCH DEGRADING ENZYMES FROM NEWLY ISOLATED ENDOPHYTIC FUNGI Introduction 117 Materials and Methods 118

Materials 118 Cultivation of Microorganisms 118 Enzyme Preparation 119 Enzymatic Hydrolysis of Raw Starches 119 Identification of Hydrolysis Product 120

Results and Discussion 120 Hydrolysis Patterns ofRSDE from Gibbere/Ia pu/icaris 120 Hydrolysis Patterns ofRSDE from Acremonium sp. 123 Hydrolysis Patterns ofRSDE from Synnematous sp. 125

Conclusions 128

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VI IMPROVEMENT OF RAW STARCH DEGRADING ENZYME 129

PRODUCTION BY ACREMONIUM SP. ENDOPHYTIC FUNGUS Introduction 129 Materials and Methods 130

Endophytic Fungus/Culture Microorganism 130 Cultivation 130 Raw Starch Degrading Activity and Protein Assay 131 Measurement of Growth 131 Effect of Initial pH, Temperature and Culture 131 Statistical Analyses 132

Results and Discussion 132 Effect oflnitial pH 134 Effect of Temperature 136 Effect of Carbon Sources 136 Effect of Nitrogen Sources 139

Conclusions 145

VI PURIFICATION AND CHARACTERIZATION OF RAW STARCH 146 DEGRADING ENZYME BY ACREMONIUM SP. ENDOPHYTIC FUNGUS Introduction 146 Materials and Methods 147 Materials 147 Organism and Culture Conditions

Preparation of Crude Extract 148 Purification Procedure 148 DEAE-ToyopearJ650 S 148 Sephadex G-150 148 Enzyme Activity and Protein Assay 149 Characterization of Enzyme 149 Optimum pH and Stability 149 Optimum Temperature and Heat Stability 150 Effect of Metal Ion 150 Polyacrylamide Gel Electrophoresis 150 Substrate Specificity 151 Determination of Kinetic Parameters 151 Determination of End Product 151

Results and Discussion 152 Purification ofRSDE 152 Properties ofRSDE 152 Inhibitor and Activator 158 Substrate Specificity 162 Kinetic Constant 162 End product 164

Conclusions 164

VIII IMPROVEMENT OF GLUCOSE PRODUCTION IN ENZYME 166 REACTION SYSTEM UTILIZING ACID-TREATED SAGO STARCH AS SUBSTRATE Introduction 166

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Materials and Methods 167 Preparation of RSDE Solution 167 Preparation of Acid-Treated Sago Starch 167 Measurement of Physical Properties of Sago Starch 168 Enzymatic Hydrolysis of Starch Granules 168 Determination of Glucose Conversion 168

Results and Discussion 169 Effect of Acid-Temperature Pretreatment on the 169 Rheological Properties of Sago Starch Enzymatic Hydrolysis of Acid-Treated Sago Starch 171 Glucose Production 173

Conclusions 178

IX CONCLUSIONS 179

BIBLIOGRAPHY 181

APPENDICES 198

APPENDIX A 199 APPENDIXB 202 APPENDIXC 208 BIODATA OF THE AUTHOR 215

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Table

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LIST OF TABLES

Distribution of microorganisms producing raw starch degrading enzymes

Mechanism of action of different raw starch degrading enzymes and basis for classification

Several important producers of raw starch degrading enzyme and growth conditions

Various models for the purification of raw starch degrading enzyme from microorganisms

Properties of purified raw starch degrading enzymes

Qualitative analysis of RSDE ability of different strains of endophytic fungi on raw starches

Data blast of 18S rRNA of Gibberella pulicaris (EP7) as subject compared to the published of Gibberella pulicaris (AB003) with 1740 nucleotide

Morphological characteristic of Gibberella puiicaris, Acremonium sp. and Synnematous sp. of endophytic fungi

Ratio of raw starch degrading activity to gelatinized starch activity (RDA) on raw sago and potato starches by three selected endophytic fungi

Maximum RSDE activity, dry weight of mycelium and protein concentrations of crude extract from the fermentation of Acremonium sp. at 27 °C grown at different pHs.

Maximum RSDE activity, dry weight of mycelium and protein concentrations of crude extract from the fermentation ofAcremonium sp. at pH 5.0 grown at different temperatures

Maximum RSDE activity, dry weight of mycelium and protein concentration of crude extract from the fermentation of Acremonium sp. at 30°C, initial pH 5.0 in the presence of different carbon sources

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110

135

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13 Effect of various combinations of nitrogen sources on 141

cell growth and RSDE activity at 30°C pH 5.0,20 gil raw sago starch as carbon source

14 Purification ofRSDE from Acremonium sp. 155 endophytic fungus

15 Effect of various reagents on raw starch degrading 161 enzyme (RSDE) activity of Acremonium sp. endophytic fungus

16 Substrate specificity ofRSDE from Acremonium sp. 163 endophytic fungus

17 Rheological properties of acid-treated and untreated 170 sago starch

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LIST OF FIGURES

Figure Page

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A typical clear zone fonned by isolate EP23 of endophytic 81 fungus after 5 days incubation on Czapek-Dox agar using raw sago as carbon source

A typical clear zone fonned by isolate EP23 of endophytic fungus after 5 days incubation on Czapek-Dox agar using raw potato as carbon source

A typical clear zone fonned by isolate EP23 of endophytic fungus after 5 days incubation on Czapek-Dox agar using raw tapioca as carbon source

A typical clear zone fonned by isolate EP23 of endophytic fungus after 5 days incubation on Czapek-Dox agar using raw com as carbon source

A typical clear zone fonned by isolate EP23 of endophytic fungus after 5 days incubation on Czapek-Dox agar using raw wheat as carbon source

A typical clear zone fonned by isolate EP23 of endophytic fungus after 5 days incubation on Czapek-Dox agar using· raw rice as carbon source

Quantitative analysis ofRSDE activity of different strains of endophytic fungi grown on various raw starches

Phylogenetic relationships of Gibbere//a pu/icaris (EP7) and references based on 18S rRNA.

Giant colony of Gibberella pulicaris grown on potato dextrose agar medium after incubation at 27°C for 5 days

Giant colony of Acremonium sp. grown on potato dextrose agar medium after incubation at 27°C for 5 days

Giant colony of Acremonium sp. grown on potato dextrose

agar medium after incubation at 27°C for 7 days

Giant colony of Acremonium sp grown on potato dextrose

agar medium after incubation at 27°C for 10 days

Photomicrograph of conidiaphore of Acremonium sp. (enteroblastic type).

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14 Photomicrograph of hyphae and mycelia of Acremonium sp. 95 with septa.

15 Photomicrograph of hyaline hyphae and single cell of spore 96 of Acremonium sp.

16 Giant colony of Synnematous sp. grown on potato dextrose 96 agar medium after incubation at 27°C for 7 days

17 Photomicrograph of conidiophore from Synnematous sp. 97

18 Photomicrograph of conidiasphores from Synnematous sp. 97

19 Growth profiles of three selected endophytic fungi that 104 produced RSDE

20 Effect pH reaction ofRSDE on raw starches from selected 106 endophytic fungi

21 Substrate specificity ofRSDE on raw starches from selected 108 endophytic fungi

22 Analysis of end product by TLC 112

23 Optimum pH reaction ofRSDE produced by 115 Acremonium sp.

24 Optimum temperature reaction ofRSDE produced 116 by Acremonium sp.

25 Enzymatic hydrolysis of raw starches to glucose by 121 RSDE from Gibberella pulicaris

26 Enzymatic hydrolysis of raw starches to glucose by 124 RSDE from Acremonium sp.

27 Enzymatic hydrolysis raw starches to maltose by 126 RSDE from Synnematous sp.

28 Enzymatic hydrolysis of raw starches to glucose by 127 RSDE from Synnematous sp.

29 RSDE activity, dry weight and protein content of crude extract 133 produced by Acremonium sp. grown on original basal medium

30 Effect of different carbon sources on the production ofRSDE 140

31 Changes ofRSDE activity during growth of Acremonium sp. 143 at different concentrations of sodium nitrate as the nitrogen source

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32 Changes ofRSDE activity during growth of Acremonium 144

sp. at different concentrations of peptone as the nitrogen source

33 An ion-exchange chromatograph ofRSDE from 153 Acremonium sp. on DEAE-Toyopearl 650 S

34 Gel filtration chromatograph ofRSDE from Acremonium sp. 154 on Sephadex G-150

35 NATIVE and SDS-PAGE ofRSDE from Acremonium sp. 156

36 Standard curves showing relationship between relative 157 mobilities on polyacrylamide gel and relative molecular masses ofRSDE

37 Optimum pH and pH stability of purified RSDE from 159 Acremonium sp.

38 Optimum temperature and temperature stability of purified 160 RSDE from Acremonium sp.

39 HPLC chromatograph of the product degradation 165 of raw sago starch by purified RSDE

40 Effect of enzymatic hydrolysis on acid-treated sago starch 172

41 Effect of enzyme concentration on glucose production 175 using acid-treated sago starch

42 Effect of acid- treated sago starch on glucose 176 production using 100 unitlml enzyme

43 Glucose production using acid-treated and untreated sago starch 177

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LIST OF ABBREVIATIONS

MEA malt extract agar

RSDE raw starch degrading enzyme

RDA raw starch degrading ability

rpm rotation per minute

v/w volume per weight

mol.wt molecular weight

TLC Thin-layer chromatography

HPLC High liquid chromatography

mm minute

h hour

Gl glucose

G2 maltose

G3 maltotriose

M molar

mM millimolar

J..I.m micromol

g gram

mg milligram

m1 milliliter

cm centimeter

sp. spectes

PDA potato dextrose agar

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