I= ISOLATION, PURIFICATION AND CHARACTERIZATION OF NATIVE AND CHEMICALLY MODIFIED GLUCOAMYLASES FORM ARACHENIOTUS CITRINUS
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Title of Thesis
ISOLATION, PURIFICATION AND CHARACTERIZATION OF NATIVE AND CHEMICALLY MODIFIED GLUCOAMYLASES FORM ARACHENIOTUS CITRINUS

Author(s)
Mubashir Niaz
Institute/University/Department Details
Botany University of Agriculture, Fasisalabad.
Session
2003
Subject
Botany
Number of Pages
154
Keywords (Extracted from title, table of contents and abstract of thesis)

Abstract

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
113.23 KB
2 1 Introduction 1
103.22 KB
3 2 Review of literature 13
83.59 KB
4 3 Materials and methods 22
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  3.1 Microbial Strain 22
  3.1.1 Slants Preparation 22
  3.1.2 1noculum preparation 22
  3.13 Solid phase growth studies of arachniotus citrinus 23
  3.1.4 Extraction of gluconamylasses 23
  3.1.5 Protein Estimation 24
  3.1.6 Glucoamylase assay 25
  3.1.7 Calculations for glucoamyases activity units: 26
  3.2 Purification of Glucoamylase 27
  3.2.1 ammonium sulfate precipitation 27
  3.2.2 Fast protein liquid chromatography( FPLC) 28
  3.2.3 Hiload anioin -exchange chromatography 28
  3.2.4 Hydrophobic-interaction chromatography 29
  3.2.5 Mono-Q anio - exchange chromatography 29
  3.3 Cargoxy1 group modification of glucoamylase 29
  3.3.1 Native enzyme mobility shift assay (NEMSA) 31
  3.3.2 Hirachin - 7% page 31
  3.3.3 Native molecular weight 32
  3.4 Carboxy group modificaito for alteration of characteristics 38
  3.4.1 Temperature optimum 39
  3.4.2 activation energy (Ea) 39
  3.5 pH optimum 39
  3.6 Effect of substrate( V max , K m , K eat ) 40
  3.6.1 Thermodynamics of K eat 40
  3.6.2 Thermodynamic of soluble starch hydrolysis 42
  3.7 Irreversible thermal inactivation 43
  3.7.1 activation energy of thermal denaturation 44
  3.7.2 Thermodynamics of irreversible thermal inactivation 44
  3.8 Determination of stability 45
  3.8.1 Effect of Urea 46
  3.82 Effect of Urea 46
5 4 Results and discussions 47
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  4.1 Production of glucoamylase 47
  4.2 Purification glucoamylase 47
  4.2.1 Hiload ammonium sulfate precipitation 48
  4.2.2 Hiload anion exchange chromatography 49
  4.2.3 Hydrophobic Interaction chromatography 51
  4.2.4 Mono-Q anoin exchange chromatography 52
  4.3 Chemical modification of glucoamylase 53
  4.4 Impact of carboxy1 group Modification on temperature optima 61
  4.5 Effect of chemical modification on pH optima of glucoamylases 68
  4.6 Thermohilicity: 76
  4.6.1 Effect of additional aromatic interactions on kinetics of soluble starch hydrolysis 76
  4.6.2 Effect of hydrophobization on kinetics of soluble starch hydrolysis 81
  4.7 Effect of chemical modifications on thermostability of glucoamylase 95
  4.7.1 Irreversible thermal stability 96
  4.8 Stability to proteases 114
  4.9 Stability to urea 118
6 5 Summary 124
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7 6 Literature cited 136
692.02 KB
8 7 Appendices 136
75.22 KB