Purification And Enzymatic Characterization Of Metarhizium Anisopliae Esterase

Metarhizium anisopliae,a species of entomogenous fungi widely used at home and abroad,plays an important role in biological control of insects.Higher esterase-producing strain Ma-3 was screened from thirty-two isolates of entomogenous fungi,with the method of transparent zones and esterase activity measurement of culture filtrate.To test the effects of the carbon source,nitrogen source,metal ions and vitamins on production of esterase,single-factor analysis were used in the study.The results showed that D-Sorbitol,yeast extract powder,Ba2+ and VB4 promoted enzyme production.Optimum medium for esterase production was obtained by orthogonal design and response surface method.The optimized culture medium included 21.40 g/L of D-Sorbitol,11.45 g/L of yeast extract powder,2.60 g/L of Bat+,1 g/L of VB4,and pH 6.9.The optimum fermentation conditions for esterase production by Ma-3 were 25 mL of inoculation amount,125 mL of liquid volume,4 days of mycelial age,and 54 h of culture time.Under such optimized culture conditions,maximum esterase activity reached 46.19 U/mL.The esterase activity was increased 2.4 fold in the end.The esterase was purified to electrophoretic homogeneity using the combination of various chromatographic steps including DEAE-cellulose followed by Sephadex G-100.The purified enzyme have three bands with molecular weight of 40 KDa,45 KDa and 49 KDa measured by SDS-PAGE.The results indicated that the optimum temperature and pH for its activity was 50 ? and 7.0,and the esterase was relatively stable in pH range of 6-9 and at 20-50 ?.Mg2+ and Ca2+increased enzyme activity but Aril and Mn2+ reduced its activity.Esterase acivity was stimulated by acetone but inhibited by chloroform.The apparent Km and Vmax of esterase were calculated to be 0.875 mg/mL and 208.33 ?mol/min,respectively.