Nawaz, Haq (2004) KINETIC AND THERMODYNAMIC CHARACTERIZATION OF NATIVE AND CHEMICALLY MODIFIED GLUCOAMYLASE FROM FUSARIUM SOLANI. PhD thesis, University of Agriculture, Faisalabad.
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 (22.56ÃÂÃÂÃÂÃÂ±1.80 U g-1). Different physical and chemical parameters were optimized. The maximum enzyme activity under optimum conditions was 61.35ÃÂÃÂÃÂÃÂ±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 35ÃÂÃÂÃÂÃÂ±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 ÃÂÃÂ¢ÃÂÃÂÃÂÃÂ 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 ÃÂÃÂ¢ÃÂÃÂÃÂÃÂ 5.5 to 4.0 ÃÂÃÂ¢ÃÂÃÂÃÂÃÂ 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 (ÃÂÃÂ¢ÃÂÃÂÃÂÃÂG*) 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 (ÃÂÃÂ¢ÃÂÃÂÃÂÃÂG*) 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.
|Item Type:||Thesis (PhD)|
|Uncontrolled Keywords:||glucoamylase, fusarium solani, solid state fermentation, wheat bran, rice bran, green gram bran, maize bran, black green bran|
|Subjects:||Physical Sciences (f) > Chemistry(f2)|
|Deposited By:||Mr. Muhammad Asif|
|Deposited On:||09 Sep 2006|
|Last Modified:||04 Oct 2007 21:01|
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