Studies on Fermentation Optimization, Purification and Enzyme Characteristics of Lipase from Aspergillus Oryzae FS-1
|School||Fujian Normal University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Aspergillus oryzae lipase fermentation optimization purification enzyme characteristics|
The fermentation medium of Aspergillus oryzae FS-1 producing lipase was optimized.The single-factor test was carried out and the result indicated that the optimum carbon source was corn flour, nitrogen source was peptone. Then the Plackett-Burman design was taken to investigate which factors contributed to lipase fermentation most and 3 most significant influence factors were found, those were peptone, olive oil and Na2HPO4. In the following experiments, Response surface methodology of Box-Behnken design was carried out to optimize the 3 significant factors and the optimum levels of those were found. The formulation of the optimum fermentation medium(g/L) was corn flour 0.5, peptone 50, olive oil 15, NaNO3 10, Na2HPO42.5, KCl 0.5, MgSO4 0.5, FeSO4 0.01. The optimum fermentation condition was obtained: the optimal amount of inoculation was 2%, the optimum initial pH was 6.8 and the optimal liquid volume were 50mL culture in 250mL shake flasks, the fermentation temperature was 28℃, the optimum initial fermentation pH was 8.0, the fermentation time was 48 hours. After the optimization, the fermentation level of lipase was increased from 3.25U/mL to 18.75U/mL.The purification of FS-1 lipase was carried out. Collecting fermentation supern atant fluid, The proteinase was purified by using ammonium sulfate, dialysis and DEAE Sepharose Fast Flow. After these purification processes, the purification mul-tiple reached 9.21 and the rate of recovery was 26.39%. Its fineness was checked to be about a single band by SDS-PAGE.Enzyme characteristics research indicated that the best enzymatic reaction temperature was 36℃, the enzymatic reaction was relative stable under 40℃; the optimum pH of enzymatic reaction was 7.0, enzymatic reaction was stable between pH 3.0-9.0. Olive oil and glycerol tributyrate were used as the substrate and the enzymatic reaction fit the Michealis equation, the maximum reaction initial velocity Vm were 23.70μmol/mL/min and 24.33μmol/mL/min. Michaelis constant (Km) were 48.44μmol/mL and 20.43 u mol/mL each. The activation energy(Ea) were 41.78 kJ/mol and 31.68kJ/mol.Research on the effects of several effectors on the FS-1 liapse activity, (1)the metal ion’s influence on the enzyme activity indicated that Na+, K+, Li+, Ba2+ion had no any effects on the enzyme activity, Ca2+ion had a few stimulative effects on the enzyme activity, while Mg2+, Cu2+, Fe2+, Hg2+, Fe3+, Al3+inhibited the enzyme.(2) Organic solvent influence on the enzyme activity indicated that methonal, ethanol, formaldehyde and isopropyl alcohol inhibited the enzyme. Formaldehyde is most strong inhibition. N-pentane had no effect on the enzyme.(3)Chemically modified agents, include cis-butenedioic, monobromo-acetic, acet-imidazole,β-mercaptoethanol, acetylacetone, chloramines-T, N-bromo-succinimide and benzyl sulfonicacid fluoride, influence on the enzyme activity showed thatβ-mercaptoethanol and benzyl sulfonicacid fluoride inhibited the enzyme. Preliminary judgment was that methionine residues and serine residues closely related with catalytic activity.