5254
Nermeen Hosni Ali Elzairy
Biotechnological Studies on Microbial Hemicellulases
Xylanase, Aspergillus flavus isolate, multifactorial design, Plackett-Burman design, Taguchi design, Partial purification, Ultrafiltration, Chemical modification, Glycosylation, Kinetic parameters, Thermodynamic properties.
A survey of ten isolated fungal strains was carried out for the production of fungal xylanases. Aspergillus flavus isolate NERMEEN12 was the strain with the highest xylanase activity. Xylanase enzyme was produced by Aspergillus flavus isolate utilizing different agricultural wastes rice straw, corn cobs, wheat bran, wheat straw and saw dust by solid state and submerged fermentation techniques. The highest xylanase production was obtained by solid state fermentation in the following order corn cobs> rice straw> wheat straw> wheat bran> saw dust. Enhancement of xylanase 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 19 factors on xylanase production. The most significant factors found to be, CaCl2.2H2O, CuSO4, CMC and NaCl. Further optimization was done using Taguchi L9 (3^4) orthogonal array design, by which the effect of four factors in three levels, could be tested. The Taguchi design showed that the maximum yield of xylanase was produced when CaCl2.2H2O, CuSO4, CMC and NaCl were present in the fermentation medium in concentrations (g/l): 1, 0.2, 10 and 10 respectively. Under optimal conditions enzyme production was increased by 2.4-fold compared to the non optimized medium. The crude Aspergillus flavus isolate NERMEEN12xylanse was partially purified by ultrafiltration of the enzyme followed by direct precipitation with solvent (at concentration 80%) or salting out with 80% ammonium sulfate solution. The best way for partial purification was byultrafiltration followed by salting out with 80% ammonium sulfate solution in which 57% and 49% of the total activity and protein were recovered respectively. The partially modified xylanase was chemically modified by glycosylation to increase the stability of the enzyme. Glycosylation of Aspergillus flavus isolate xylanase 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 xylanases. The activation energy values native and conjugated enzymes were 8.81 and 6.96 Kcal/mol, respectively.It was observed that the glycosylation process had a highly significant impact on improving the values of Vmax, Km , turnover number (kcat), specificity constant (Vmax/Km), catalytic efficiency (kcat/Km) and Thermodynamic parameters for xylan hydrolysis (ΔH, ΔG, ΔS). Native and conjugated enzymes were activated by addition of 0.2M and 0.3M Ca2+ ion solutions respectively. The inhibitory impacts of the other metal ions tested were more pronounced with the native xylanase than the conjugated enzyme. 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 (t1/2), and higher D values (decimal reduction time)
2016
M.Sc
Cairo
Pharmacy