Purification And Characterization Of Amylase And Lipase From Ascosphaera Apis ND-2

The culture media in the flask-shaking for amylase and lipase production of the entomopathogenic fungus Ascosphaera apis were optimized to increase the yield of both enzymes using the Plackett-Burman design combined with the response surface method(RSM).Amylase The optimized components of culture medium for amylase production included 45.99 g�1-1 maltose,1.51 g�l-1 CaCl2,and pH 6.6.The highest amylase activity peaked at 46.25 U/ml.The optimized media could increase amylase production by 10.52-fold than the non-optimized basal media.The cell supernatant was obtained by removing the mycelia and vacuum filtration.The enzyme was purified to homogeneity with the aid of ammonium sulphate precipitation,dialysis,PEG20000,DEAE-cellulose A-52 and Sephadex G-100 column chromatography,with 36.53%yield and 2.3-fold purification.Amylase appeared homogeneous on SDS-PAGE with a molecular weight of 75 kDa.The optimal temperature and pH for amylase was found to be at 55? and 7.5,respectively.The amylase was stable when the temperature was from 30? to 55?and pH was in the range of 6-10.Fe2+ could significantly enhance the amylase activity.K+,Fe3+and Na+had slight inhibitions on amylase.Mg2+,Ca2+ and Zn2+ expressed significant inhibitory effects on amylase.Cu2+,Mn2+and Ba2+were found to be no inhibition on the enzyme.All the organic solvents were found to have inhibition on amylase but DMSO hardly had inhibition on the test amylase.The kinetic constants(km and Vmax)of amylase were 2.13 mg�ml-1 and 1.44 mg�ml-1�min-1,respectively.The amylase was determined to be a-amylase on the basis of Thin Layer Chromatography(TLC).The production of amylase in bioreactor was higher than that in the flask.Lipase The optimized culture medium involved in 50 g�l-1 olive oil,3.88 g�l-1 yeast extract powder,0.713 g�l-1 NaCl,and 0.34 g�-1 VB2.The lipase activity was up to 22.19 U/ml.The optimization of the medium contributed to 3.38-fold higher lipase production than that of the control experiments.Lipase in the culture filtrate was purified to homogeneity by ammonium sulphate precipitation,dialysis,PEG20000 and Sephadex G-100 column chromatography,with 33.00%yield and 34.15-fold purification.The purified lipase was homogeneous detected by SDS-PAGE and its molecular weight was estimated to be about 54 kDa.The optimal temperature and pH for lipase was found to be 55? and 7.5,respectively.The lipase was stable when the temperature ranged from 30? to 55? and pH values varied in the range of 3-10.Fe2+,Fe3+and K+could improve lipase activity.Na+,Mg2+,Ba2+,Zn2+,Ca2+,Mn2+and Cu2+displyed slight inhibitions on lipase activity.Ethanol and dichloromethane could slightly upgrade lipase activity,but the other organic solvents hardly had inhibitory impact on lipase.The kinetic values(Km and Vmax)of lipase were 8.21 mg�ml-1 and 23.81 mg�ml-1�min-1,respectively.The production of lipase using bioreactor was higher than that in the flask.