一株产右旋糖酐酶青霉的分离及酶的纯化和性质

Purification, characterization of an extracellular dextranase from an isolated Penicillium sp

摘要:

摘要:【目的】从土壤中筛选到一株新的产右旋糖酐酶的真菌F1001，对菌株进行鉴定并研究酶的分离纯化及酶学性质。为酶法制备药用级右旋糖酐提供新的右旋糖酐酶产生菌株。【方法】通过形态特征和ITS rDNA序列分析方法鉴定菌株。利用硫酸铵盐析、Sepharose 6B 凝胶柱纯化，得到纯度较高的酶蛋白，并对其酶学性质及催化机理进行研究。【结果】经过鉴定确定菌株F1001为棘孢青霉（Penicillium aculeatum）。通过SDS?PAGE测得棘孢青霉右旋糖酐酶的分子量为66 kDa左右，以右旋糖酐70

Abstract:

Abstract: [Objective] To obtain new fungi producing dextranase, we identified a strain F1001 showing high dextranase activities. [Methods] Morphological and ITS rDNA sequences homology analysis were performed to identify the strain F1001. The enzyme was purified to electrophoretic homogeneity by the steps of ammonium sulfate precipitation and Sepharose 6B column chromatography. We studied the catalytic properties and the mechanism of the dextranase. [Results]The isolated strain F1001 was identi?ed as Penicillium aculeatum precisely by ITS rDNA sequences homology analysis. Its molecular mass was estimated to be about 66 kDa by SDS-PAGE. Activities of dextranase were measured with dextran 70 kDa as substrate, the optimal reaction temperature was 35℃, and the optimum pH was 5.0, it was relatively stable in the condition of pH 4.0 ? 7.0 and under 50℃. The optimum substrate concentration was 3% (w/v). The final dextranase hydrolysis product was isomaltose, it was proved that the enzyme was endodextranase and only had activity with dextran joined mainly by continual α,1-6 glucosidic linkages. The Km for dextranase was calculated to be 3.55×10-5 mol/L, and the Vmax was 4.29×10-2 mol（Glu）/min?L. The enzyme activity was enhanced by Zn2+and Cu2+, and the low concentration of Cu2+ could improve the dextranase activity to 134.7%. However, the enzyme was strongly inhibited by Mn2+. [Conclusion]We isolated a new strain F1001 producing high dextranase activity and the enzyme was relatively stable. These results may provide an important basis for industrial applications.