Production, purification, properties and application of the cellulases from a wild type strain of a yeast isolate [Elektronische Ressource] / Mohamed Korish

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Production, Purification, Properties and Application of the Cellulases from a Wild type Strain of a Yeast isolate Dissertation for attaining the Degree of Doctor of Natural Sciences At the Faculty of Biology of the Johannes Gutenberg-University Mainz Mohamed Korish Born in Kafr Elsheikh, Egypt Mainz 2003 These investigations were performed at the Institute of Microbiology and Wine Research at the Johannes Gutenberg-University, Mainz, Germany, from December 1999 to May 2003 under the supervision of Prof. Dr. Helmut König. Dean: Prof. Dr. Harald Paulsen st1 Referee: Prof. Dr. Helmut König nd2 Referee: Prof. Dr. Wolfgang Wernicke Date of oral examination: July 16, 2003 CONTENTS 1 INTRODUCTION………………………………………..…… 1 2 MATERIALS………………………………………………...... 9 2.1 Organism……………………………………………………….. 9 2.2 Chemicals………………………………………………………. 9 2.3 Media............................................................................................ 12 2.3.1 CMC agar medium……………………………………………... 13 2.3.2 Maintenance medium…………………………………………... 13 2.3.3 GYP-medium ………………………………………………….. 13 2.3.4 Basal salt medium……………………………………………… 14 2.3.

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Production, Purification, Properties and Application of
the Cellulases from a Wild type Strain of a Yeast isolate


Dissertation for attaining the Degree of
Doctor of Natural Sciences


At the Faculty of Biology of the
Johannes Gutenberg-University Mainz


Mohamed Korish
Born in Kafr Elsheikh, Egypt


Mainz 2003



These investigations were performed at the Institute of Microbiology and
Wine Research at the Johannes Gutenberg-University, Mainz, Germany,
from December 1999 to May 2003 under the supervision of Prof. Dr.
Helmut König.























Dean: Prof. Dr. Harald Paulsen

st1 Referee: Prof. Dr. Helmut König
nd2 Referee: Prof. Dr. Wolfgang Wernicke

Date of oral examination: July 16, 2003

CONTENTS

1 INTRODUCTION………………………………………..…… 1
2 MATERIALS………………………………………………...... 9
2.1 Organism……………………………………………………….. 9
2.2 Chemicals………………………………………………………. 9
2.3 Media............................................................................................ 12
2.3.1 CMC agar medium……………………………………………... 13
2.3.2 Maintenance medium…………………………………………... 13
2.3.3 GYP-medium ………………………………………………….. 13
2.3.4 Basal salt medium……………………………………………… 14
2.3.5 Bacto Yeast Nitrogen Base…………………………………….. 14
2.4 Solutions and reagents…………………………………………. 14
2.4.1 Protein stain solutions…………………………………………. 14
2.4.2 CMC agarose…………………………………………………… 15
2.4.3 Sample buffer………………………………………………….. 15
2.4.4 Electrophoresis buffer………………………………………….. 15
2.4.5 SDS-gel stain solution………………………………………….. 15
2.4.6 DNS-reagent……………………………………………………. 16
2.4.7 Bradford reagent………………………………………………... 16
2.4.8 TBE buffer……………………………………………………… 16
2.5 Equipment……………………………………………………… 17
3 METHODS…………………………………………………..... 18
3.1 Identification of isolated yeast by PCR ………………………... 18
3.1.1 DNA extraction………………………………………………… 18 3.1.2 PCR amplification …………………………………………… 19
3.1.3 DNA sequencing……………………………………………… 20
3.2 Cellulases screening test ……………………………………… 20
3.3 Determination of cellulolytic activities………………………. 21
3.4 Isoelectric focusing (IEF)……………………………………. 21
3.5 Polyacrylamide gel electrophoresis…………………………… 22
3.6 Protein determination………………………………………….. 25
3.7 Preparation of phosphoric acid-swollen avicel………………… 25
3.8 Preparing dialysis tubing……………………………………… 26
3.9 Optimization of culture conditions……………………………. 26
3.9.1 Inoculum preparation…………………………………………. 26
3.9.2 Experimental media …………………………………………… 26
3.9.3 Incubation temperature………………………………………… 27
3.10.4 Selection of carbon source…………………………………….. 27
3.9.5 CMC concentration……………………………………………. 27
3.9.6 Selection of nitrogen source……………………………………. 27
3.9.7 Peptone concentration…………………………………………. 28
3.9.8 Medium pH value……………………………………………… 28
3.9.9 Surfactants ……………………………………………………. 28
3.9.10 Tween 80 concentration………………………………………. 29
3.9.11 Induction ………………………………………………………. 29
3.9.12 Lactose concentration…………………………………………. 29
3.9.13 Culture agitation ………………………………………………. 29
3.9.14 Cultivation time ………………………………………………. 30
3.10 Purification of cellulase………………………………………… 30
3.10.1 Preparation of crude enzyme ………………………………….. 30 3.10.2 Chromatography………………………………………………. 31
3.10.2.1 Separation by anion-exchange chromatography……………… 31
3.10.2.2 Fractionation by hydrophobic interaction chromatography HIC. 32
3.10.2.3 Rechromatography……………………………………………. 32
3.11 Characterization of cellulase………………………………….. 32
3.11.1 pH dependence……………………………………………….. 33
3.11.2 pH stability……………………………………………………. 33
3.11.3 Temperature optimum……………………………………......... 33
3.11.4 Thermal stability……………………………………………….. 33
3.11.5 Chemical compounds………………………………………… 34
3.11.6 Metal ions ……………………………………………………. 34
3.11.7 Organic solvents ……………………………………………….. 34
3.11.8 Inhibition by oligosacchrides ………………………………….. 34
3.11.9 Substrate concentration ……………………………………… 35
3.11.10 Activity towards different substrate………………………….. 35
3.11.11 Saccharification of cellulosic materials ……………………….. 36
4 RESULTS……………………………………………………… 38
4.1 Morphology of the yeast isolate………………………………. 38
4.2 Identification of yeast isolate ………………………………… 39
4.3 Cellulolytic ability of yeast isolate…………………………… 41
4.4 Factors affecting cellulase production………………………… 43
4.4.1 Effect of incubation temperature………………………………. 43
4.4.2 Effect of carbon source on cellulase production ……………… 44
3.4.3 Effect of CMC concentration………………………………….. 44
4.4.4 Effect of various nitrogen sources……………………………… 44
4.4.5 Effect of peptone concentration……………………………….. 46 4.4.6 Effect of medium pH value ……………………………………. 46
4.4.7 Effect of surfactants………………………………………....... 48
4.4.8 Effect of Tween 80 concentration……………………………… 48
4.4.9 Induction of cellulase by different saccharides………………… 49
4.4.10 Induction of cellulase by lactose ………………………………. 50
4.4.11 Effect of agitation on cellulase production……………………. 51
4.4.12 Time course of cellulase production…………………………… 51
4.5 Isoelectric point………………………………………………… 54
4.6 Apparent molecular mass……………………………………… 55
4.7 Purification of cellulase………………………………………… 56
4.7.1 Crude cellulase preparation……………………………………. 56
4.7.2 Anion-exchange chromatography……………………………… 57
4.7.3 Hydrophobic interaction chromatography (HIC)……………… 57
4.7.4 Rechromatography…………………………………………… 59
4.8 Physical and chemical properties of purified cellulase (I)…… 62
4.8.1 Effect of pH on enzyme activity………………………………. 62
4.8.2 Effect of pH on cellulase I stability……………………………. 63
4.8.3 Effect of temperature on cellulase I activity………………….. 63
4.8.4 Effect of temperature on enzyme stability…………………….. 65
4.8.5 Effect of various chemicals on enzyme activity………………. 66
4.8.6 Effect of metal ions on enzyme activity……………………….. 66
4.8.7 Effect of organic solvents on enzyme activity…………………. 69
4.8.8 Inhibitory effect of oligosaccharides …………………………... 70
4.8.9 Substrate specificity……………………………………………. 71
4.8.10 Reaction kinetics ………………………………………………. 72
4.8.11 Saccharification products ……………………………………… 73 5 DISCUSSION………………………………………………… 77
5.1 Optimal conditions for cellulase production………………… 77
5.1.1 Optimal temperature………………………………………… 77
5.1.2 Optimal carbon source………………………………………. 78
5.1.3 Carbon source concentration…………………………………… 80
5.1.4 Optimal nitrogen source………………………………………. 80
5.1.5 Concentration of nitrogen source ……………………………… 82
5.1.6 Optimum pH value of culture………………………………… 82
5.1.7 Surfactants effect……………………………………………… 84
5.1.8 Induction of cellulase by lactose……………………………… 85
5.1.9 Inducer concentration…………………………………………. 87
5.1.10 Culture agitation………………………………………………. 88
5.1.11 Cultivation time ……………………………………………… 89
5.2 Isoelectric point pI…………………………………………… 90
5.3 Summary of the purification steps…………………………… 91
5.4 Characteristics of purified of cellulase I ……………………… 93
5.4 .1 pH optimum……………………………………………………. 93
5.4.2 pH stability…………………………………………………… 95
5.4.3 Temperature optimum…………………………………………. 95
5.4.4 Temperature stability………………………………………… 98
5.4.5 Various compounds as activators or inhibitors ……………… 100
5.4.6 Metal ions as activators or inhibitors………………………. 102
5.4.7 Inhibition by organic solvents ……………………………… 104
5.4.8 Substrate specificity………………………………………… 106
5.5 Mode of action and synergism of cellulases ……………… 107
5.6 Systematic position of the yeast isolate………………………… 108 6 SUMMARY ………………………………………………….. 109
7 ABSTRACT................................................................................ 112
8 KURZZUSAMMENFASSUNG……………………………… 113
9 REFERENCES……………………………………………….. 114




















ABBREVIATIONS
Aps Ammonium persulphate
bp Base pair
BSA Bovine serum albumin
CMC Carboxymethylcellulose
dNTP Deoxyribonucleotide 5’-triphsphate (N= A,T,G,C)
DNS Dinitrosalisylic acid
FPLC Fast protein liquid chromatography
HPLC High performance liquid chromatography
PAGE Polyacrylamide gel electrophoresis
PCR Polymerase chain reaction
PI Isoelectric point
rpm Round per minute
SDS Sodium dodecyl sulphate
Taq Thermus aquaticus
TBE Tris-boric acid-EDTA
TEMED N,N,N,N-Tetramethyl ethylenediamine
TRIS 2-Amino-2-hydroxymethylpropane-1,3-diol
O.D. Optical density
UV Ultraviolet
v Volume
wt Weight


INTRODUCTION


1 INTRODUCTION
Cellulases refer to a group of enzymes which act together to hydrolyze
cellulose into soluble sugars. They are distributed throughout the biosphere
such as plants, animals and microorganisms. However, cellulases from
higher plants such as Lantana camara and Cuscuta reflexa are mostly
involved in fruit ripening and senescence (Chatterjee and Sanwal, 1999).
Few animals such as the blue mussel Mytilus edulis (Bingze et al., 2000),
the green mussel (Marshall, 1973), the edible snail Helix pomatia
(Rebeyrotte et al., 1967; Maeda et al., 1996) the marine mollusc Littorina
brevicula (Purchon, 1977), termites and protozoa (König et al., 2002) were
reported as cellulase producers. Protozoa such as Epidinium caudatum and
Eudiplodinium ostracodinium. Archaea such as Sulfolobus solfataricus
(Moracci et al., 2001) and Pyrococcus furiosus (Voorhorst et al., 1999) are
also cellulases producers. However, microorganisms are considered to be the
main source for cellulases with novel and high specific activities. Microbial
cellulases are the most economic and available sources, because
microorganisms can grow on inexpensive media such agriculture and food
industries by-products.
It is now well established that the cellulolytic activity is a
multicomponent enzyme system and consists of three major components;
endo-ß-glucanase (EC 3.2.1.4), exo-ß-glucanase (EC 3.2.1.91) and ß -
glucosidase (EC 3.2.1.21).


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