| Higher trehalase-producing strains were screened from twenty-five isolates of entomopathogenic fungi, with the method of transparent zones and trehalase activity measurement of culture filtrate.The effects of the carbon source, nitrogen source and vitamins on production of trehalase were studied using single-factor analysis. The results showed that soluble starch, peptone, vitaminB2, pH6 and K+ promoted enzyme produce. Optimum medium for trehalase produce was obtained by orthogonal optimization. The culture medium consisted of 2.5g/L soluble starch, 5g/L peptone, 4×10-4 mol/L K+, 0.05 g/L vitaminB2 and pH5.5. The best conditions for producing enzyme were 15 mL of inoculation amount, 50 mL of liquid volume, mycelial age 7 days, culture time 3 days were employed, respectively. Under optimal culture conditions, maximum trehalase activity reached 26.28U mL?1.The strong active enzyme was purified to electrophoretic homogeneity using the combination of various chromatographic steps: DEAE-cellulose, Sephadex G-100. The purified enzyme was a monomer with an apparent molecular weight of 40 kDa measured by SDS-PAGE.The enzyme characteristics of trehalase were detected. The results showed that the optimum temperature for its activity was 30℃, and it is stability in 40℃. The optimal pH for its activity is 6-7, and it is stability at pH 6.5. Na+, Mn2+ and Fe3+ activated its activity but Mg2+ and Al3+ inhibited enzyme activity. Methanol, pantanol, acetone and dioxane solutions activated the enzyme activity while glycol, methanal and glutaraldehyde inhibited its activity. Moreover, butanol and glycerol activated the enzyme, while they showed an inhibitory effect when they reached to a certain concentration. The inhibitory kinetics of Mg2+ on the enzyme showed that Mg2+ was a reversible competitive inhibitor of the enzyme. Mg2+ couldn't affect pH stability of the enzyme. When the temperature was above 60℃, the thermostability of enzyme increased with the increasing of the concentration of Mg2+.
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