Studies On Fermentation Optimization,Purification And Enzyme Characteristics Ofα-amylase From Aspergillus Oryzae

The fermentation medium of Aspergillus oryzae FS3-11producing a-amylase was optimized. The single-factor test of fermentation was carried out. The result indicated that the optimum carbon source was flour and starch, organic nitrogen source was soybean flour and beef extract, and the inorganic nitrogen source was NaNO3. Then the P-B design was taken to evaluate these factors of fermentation medium.3factors which have the most significant influence on the production of the amylase fermentation were found. They were starch, beef extract and NaN03. Then through the response surface methodology of design, the composition of optimum fermentation medium was gained. The fermentation medium (g/L) were flour50, soybean flour20, starch24.25, beef extract10.54, NaNO36.89, K2HPO43, MgSO41, FeSO40.01. And the optimum parameters of fermentation condition were studied. The optimum initial pH of fermentation was7.0, The optimal amount of inoculation was7.5%, and the optimal liquid volume were60mL culture in250mL shake flasks, the fermentation temperature was33.5℃. The fermentation time was80hours. The fermentation level of α-amylase was3254.60U/mL, increased by78.98%more than that of the initial level.The a-amylase from FS3-11was purified by using ammonium sulfate twice· column chromatography on DEAE-Sepharose F.F. and S-200. The specific activity of the purified enzyme reached298064.7U/mg, the purification multiple reached9.29, and the rate of recovery was14.35%. Its fineness was checked to be about a single band by SDS-PAGE and its molecular weight was50.43KD.Enzyme characteristics of a-amylase was researched. Studies indicated that the optimum temperature was55℃, the enzymatic showed less temperature stability. The optimum pH was5.5. The enzyme showed that it had good pH stability.The different effectors have effect on a-amylase. The result as this:1. Heavy metal ion showed inhibition to the enzyme, but Ca2+ion showed stimulation for the enzyme activity.2. Different organic solvent have different effect on the enzyme.3. The N-bromo-succinimide had inhibition on enzyme activity. It was estimated that tryptophan was very importment to keep configuration of activity center.