I= PURIFICATION AND CHARACTERIZATION OF NATIVE, PROTEOLYTICALLY NICKED AND CHEMICALLY MODIFIED B-GLUCOSIDASE FROM ASPERGILLUS NIGER
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Title of Thesis
PURIFICATION AND CHARACTERIZATION OF NATIVE, PROTEOLYTICALLY NICKED AND CHEMICALLY MODIFIED B-GLUCOSIDASE FROM ASPERGILLUS NIGER

Author(s)
Muhammad Hamid Rashid
Institute/University/Department Details
Department of Zoology/ Punjab University
Session
1997
Subject
Zoology
Number of Pages
147
Keywords (Extracted from title, table of contents and abstract of thesis)
b-glucosidase, aspergillus niger, kallar grass, leptochloa fusca, polysaccharides, proteolytic nicking

Abstract
β-glucosidase production was maximum on untreated kallar grass (Leptochloa fusca) compared to other substrates (wheat bran and sigma cell-20 cellulose). But in the presence this substrate maximum amount of polysaccharides were also produced. Three types of polysaccharides (high, low and intermediate molecular weight), non covalently attached to glucosidase, were observed when Aspergillus niger NIAB 280 was grown on kallar gray wheat bran and salicin in combination. High and intermediate molecular weight polysaccharioc were alpha linked to B-glucosidase. The low molecular weight polysaccharide streak was to kallar grass and could be hemicellulose-lignin complex. Polysaccharides non-covalent attached to B-glucosidase were removed by specially designed compartmental electrophore apparatus. The enzyme was partially purified and concentrated. The non-covalently attached polysaccharides were directly associated with the B-glucosidase stability. The polysaccharide glucosidase complex was extremely resistant to proteases and far more stable against urea a temperature as compared with polysaccharide free β-glucosidase.

β -glucosidase was purified to homogeneity (46 fold) after ammonium sulfa precipitation, hydrophobic interaction, ion-exchange and gel filtration chromatography. The native and sub-unit molecular weights of purified β -glucosidase were 330 and 110 k.. respectively. The optimum temperature for β -glucosidase was 70°C. It gave maximum active within the pH range of 4.6-5.3. km for p-nitrophenyl β -D-glucopyranoside (pNPG) at 40°C pH 5.0 was 1.11 mM. While kcat for pNPG at 40°C, pH 5.0 was 4000 min1. NaCI and Mno activated the β -glucosidase at low (0.1 mM & 25 uM respectively) and inhibited it at his concentrations (1 M & 250 uM respectively). β -glucosidase was also inhibited by ammonite sulfate at 500 mM concentration.

The role of carboxyl groups in the catalytic activity of β -glucosidase was found chemically modifying it with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide in the present of glycinamide as nucleophile under various conditions. One mole of EDC per mole of glucosidase inactivated β -glucosidase with second-order rate constant of 4.77x10-2 mM mil Glucose partially protected the active-site carboxyl against chemical modification. The pro donating group-was found to be a carboxyl. The pKa's of acidic and basic limbs of nat enzyme were 2.9 and 6.5, respectively. The effects of neutralization with Glycinam modification (GAM) and reversal with Ethylenediamine dihydrochloride (EDAM) of negate charges of surface carboxyl groups on the kinetic properties of the enzyme were a determined.

A simple and sensitive Native Enzyme Mobility Shift Assay (NEMSA) for determine the number of modified carboxyl groups has been described. This assay gives addition information on the heterogeneity of modified enzyme at each step.

The neutralization and reversal of negative charges had a dramatic effect on thermostability of modified β -glucosidase. The half-lives of GAM and EDAM at 1 temperatures (55 and 60°C) were significantly reduced, whereas at higher temperatures (64 and 67°C) half-lives were enhanced as compared to native β -glucosidase. Both enthalpies (ˆ†H*) and entropies of activation (ˆ†S*) for denaturation of GAM and EDAM were decreased as compare with native enzyme.

β -glucosidase when treated with proteases (אּ-chymotrypsin, subtilisin and thermolys gave periodic loss and gain of enzyme activity. Further more, when β -glucosidase was tread with thermolysin, 110 kDa subunit of β -glucosidase was degraded into ten small molec weight bands. The smallest band was of 35 kDa. Surprisingly all bands were active.

Download Full Thesis
2305.82 KB
S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
193.6 KB
2 1 Abstract 2
158.42 KB
  1.1 Introduction 2
  1.2 Materials and Methods 2
  1.3 Results and Discussion 7
  1.4 Conclusions 9
  1.5 References 9
3 2 Separation of Polysaccharides from B-glucosidase 13
619.28 KB
  2.1 Abstract 13
  2.2 Introduction 13
  2.3 Materials and Methods 14
  2.4 Staining of Native Gels : 18
  2.5 Staining of SDS -denaturing Gels : 21
  2.6 Results and Discussion 22
  2.7 Conclusions 28
  2.8 Non-destructive removal of polysaccharides from B-glucosidase 29
  2.9 Introduction 29
  2.10 Materials and Methods 29
  2.11 Results and Discussion 32
  2.12 Conclusions 37
  2.13 References 37
4 3 Abstract 44
193.54 KB
  3.1 Introduction 44
  3.2 Materials and Methods 44
  3.3 Results and Discussion 47
  3.4 References 53
5 4 Purification and Characterization of Native B-glucosidase 58
502.88 KB
  4.1 Abstract 58
  4.2 Introduction 58
  4.3 Materials and Methods 58
  4.4 Results and Discussion 73
6 5 Chemical Modification of B-glucosidase 86
509.32 KB
  5.1 Abstract 86
  5.2 Active-Site Residues Identification and Alteration of Kinetic Properties of B- glucosidase by Chemical Modification 87
  5.3 Introduction 87
  5.4 Materials and Methods 92
  5.5 Results and Discussion 95
  5.6 Stability of Chemically Modified B-glucosidase 105
  5.7 Introduction 105
  5.8 Materials and Methods 105
  5.9 Results and Discussion 109
  5.10 References 114
7 6 Proteolytic Nicking of b-glucosidase 120
113.78 KB
  6.1 Abstract 120
  6.2 Introduction 120
  6.3 Materials and Methods 121
  6.4 Results and Discussion 121
  6.5 References 123
  6.6 Appendix 124
  6.7 Publications 127