5427
Hebat-Allah A. Salem
Production and Characterization of Selective Fungal Tannase
tannase, Mucor circinilloides isolate, multificatorial design (Placket-Burman design and Box-Behnken design), partial purification, chemical modification, glycosylation, kinetic parameters, thermodynamic properties.
A survey of three isolated fungal strains was carried out for the production of fungal tannases. Mucor Cercinilloides isolate F6-3-12 was the strain with the highest tannase activity. Tannase enzyme was produced by Mucor circinilloides isolate utilizing different agricultural plants: green tea leaves, sumac leaves and pomegranate rind under solid state fermentation technique and green tea or tannic acid under submerged fermentation technique. The highest tannase production was obtained by solid state fermentation in the following order pomegranate rind > green tea leaves and sumac leaves. Enhancement of tannase production was achieved by two statistically based experimental designs to optimize the fermentation medium. The Plackett-Burman multifactorial design was first employed to investigate the effect of 7 factors of tannase production. The most significant factors found to be moisture level, incubation period and sodium nitrate concentration. Further optimization was done using Box-Behnken design in which the effect of the three factors in three levels could be tested. The Box-Behnken design showed that the maximum yield of tannase was produced when the concentration of sodium nitrate was 8g/l with moisture level 1:6 with three days of incubation. Under optimal conditions enzyme production was increased by 5.91 fold compared to non optimized medium. The crude Mucor circinilloides isolate F6-3-12 was partially purified by precipitation with solvent at concentration 75%. The best way for partial purification was by acetone 75% in which 65.8% and 49.9% of the total activity and protein were recovered respectively. The partially modified tannase was chemically modified by glycosylation to increase the stability of the enzyme. Glycosylation of the enzyme was done by covalent coupling of the partially purified enzyme to sodium periodate activated agar. Then a comparison was set between the native (partially purified) and conjugated tannases. The activation energy values of native and conjugated enzymes were 29.48 and 12.77KJ/mol respectively. It was observed that the glycosylation process had a highly significant impact on improving the values of Vmax, Km, turn over number (Kcat), specificity constant (Vmax/Km), catalytic efficiency (Kcat/Km) and thermodynamic parameters for tannase hydrolysis (∆H, ∆G, ∆S). Compared to the native enzyme, the conjugated preparation revealed lower deactivation constant rate (kD), higher deactivation energy for irreversible thermal inactivation (Ead), higher half life (t 1\2) , and higher D values (decimal reduction time).
2017
Ph.d
Cairo
Pharmacy