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Title of Thesis

Haq Nawaz
Institute/University/Department Details
University of Agriculture, Faisalabad/Department of Chemistry
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
glucoamylase, fusarium solani, solid state fermentation, wheat bran, rice bran, green gram bran, maize bran, black green bran

The project was planned to study the kinetic and thermodynamic characterization of native and chemically modified glucoamylase from Fusarium solani. Production of glucoamylase employing Fusarium solani under Solid State Fermentation (SSF) was optimized. Different substrates like wheat bran, rice bran, green gram bran, maize bran and black green bran were studied to optimize the best substrate. Wheat bran showed the highest activity (22.56±1.80 U g-1). Different physical and chemical parameters were optimized. The maximum enzyme activity under optimum conditions was 61.35±3.69 U g-1 dry wheat bran. The optimum conditions were: fructose as additive 1%(w/w), urea as additive 1% (w/w), initial moisture content of solid substrate 70%, incubation period 96h, inoculum size 15%, incubation temperature 35±10C, and pH 5.0

Crude dialyzed glucoamylase was purified to homogeneity using Fast Protein Liquid Chromatography (FPLC) unit and five step purification procedure, comprised of ammonium sulfate precipitation, HiLoad anion-exchange, hydrophobic interaction, Mono-Q anion exchange and gel filtration. The onset of glucoamylase precipitation occurred at 30% and completed at 65% saturation of ammonium sulfate at 00C. The five sep purification protocol for glucoamylase resulted into 26.2 fold purification having specific activity of 602.6 Umg-1 proteins with 31.8% recovery. The purity of glucoamylase was confirmed on 10% SDS-PAGE which displayed a single band.

The native and sub-unit molecular masses of glucoamylase from Fusarium solani were determined on Pharmacia FPLC system using gel filtration chromatography and 10% SDS-PAGE respectively. The native and sub-unit molecular masses were almost same i.e. 41 kDa and 40 kDa, respectively, indicating that glucoamylase was monomeric in nature. The purified glucoamylase from Fusarium solani was chemically modified by cross-linking with aniline and ethylenediamine as nucleophiles in the presence of 1-ethy1-3 (3-diamethy laminopropy1) carbodimide EDC) and abbreviated as ACG-1, ACG-7, ACG-13, ECG-2, ECG-11 and ECG-17 respectively. All the chemically modified forms of glucoamylase showed a decrease in activity and more critically there was a slight rising and falling trend in the activity.

The coupling of glucoamylase with aniline shifted pH optima from 3.0- 5.5 to 4.0 – 6.5 and temperature from 400C. to 600C. All the aniline coupled glucoamylases possessed low energy of activation (Ea) to form the enzyme-substrate complex except ACG-1, as compared to native (35.99 kj Mo1-1). Similarly coupling with ethylenediamine also resulted in the expansion of pH optima from 3.0 – 5.5 to 4.0 – 7.0 and temperature from 400C. to 550C.. Moreover, all the ethylenediamine coupled glucoamylases also exhibited low energy of activation profile as compared to native. Maximum decrease was observed for ECG-11 and ECG-17 (19.70 kj Mo1-1)

The effect of additional aromatic and hydrophobic interactions on thermophilicity was evaluated by determining the Michaelis constants (Vmax, Km, Kcat and Kcat/Km). All the aniline coupled glucoamylases showed high value of Vmax, Kcat and Kcat/Km at 550C as compared to native enzyme. The affinity of soluble starch at 550C decreased for all the modified forms except ACG-7. Thermodynamic data for soluble starch hydrolysis indicated that additional aromatic interactions resulted into a decrease in free energy (∆G*) for hydrolysis. Maximum decrease was observed for ACG-7 (40.91 kj Mo1-1). The coupling of ethylenediamine with glucoamylase also improved the kinetic and thermodynamic properties. Vmax, and Kcat and Kcat/Km Values were high for modified glucoamylases as compared to native at 550C.

Kinetic and thermodynamic studies of native and chemically modified glucoamylases revealed that additional aromatic and hydrophobic interactions resulted into stabilization of glucoamylase fromFusarium solani. Both the aniline and ethylenediamine coupled glucoamylases exhibited longer half-lives (t1/2) as compared to native enzyme at high temperature. Moreover, all the modified forms possessed high free energy (∆G*) for thermal unfolding indicating that they required more energy for thermal denaturation as compared to native. Besides temperature, all the modified forms also showed stability towards 4M urea and proteolytic nicking by proteases.

In the light of present finding, it is concluded that increasing aromatic-aromatic interactions and hydrophobization resulted into thermophilization and thermostabilization of glucoamylase from Fusarium solani which could be used for hydrolysis of starch in the industry.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 0 Contents
155.75 KB
2 1 Introduction 1
63.84 KB
3 2 Review of Literature 9
281.68 KB
  2.1 Optimization of conditions for the production of glucoamylase under solid State fermentation. 9
  2.2 Purification, kinetic and thermodynamic studies 17
  2.3 Chemical modification and thermal stability. 34
4 3 Materials and Methods 42
218.34 KB
  3.1 Microorganism 42
  3.2 Glucoamylase assay 44
  3.3 Protein estimation 45
  3.4 Production of glucoamylase under solid-state fermentation. 46
  3.5 Isolation of glucoamylase 49
  3.6 Purification of glucoamylase 50
  3.7 Carboxyl Group Modification 53
  3.8 Molecular mass determination 55
  3.9 Carboxyl group modification for alteration of characteristics. 62
  3.10 pH optima 62
  3.11 Effect of substrate K max , K m, K cat
  3.12 Irreversible thermal denaturation 65
  3.13 Determination of stability 66
  3.14 Statistical analysis 67
5 4 Results and Discussion 68
997.62 KB
  4.1 Optimization of conditions under solid state fermentation 69
  4.2 Purification of glucoamylase 83
  4.3 Molecular mass determination of glucoamylase 90
  4.4 Chemical modification of glucoamylase 92
  4.5 Monitoring of carboxyl group modification 98
  4.6 Effect of chemical modification on temperature optima 100
  4.7 Effect of chemical modification on pH optima 106
  4.8 Effect of additional aromatic interactions on kinetics of soluble starch hydrolysis 114
  4.9 Effect of additional hydrophobic interactions on kinetic of soluble Starch hydrolysis 119
  4.10 Effect of aniline coupling on the thermodymaics of soluble starch Hydrolysis by glucoamylase . 122
  4.11 Effect of ethylenediamine coupling on the thermodynamics of soluble startch Hydrolysis by glucoamylase 125
  4.12 Effect of chemical modification on thermostability of glucoamylase 127
6 5 Summary 156
38.93 KB
7 6 Literature Cited 160
214.15 KB