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,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。以右旋糖酐70 kDa为底物,对右旋糖酐酶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。酶促反应的最适温度为35℃,最适pH为5.0,酶在pH 4.0-7.0和50℃以下稳定。酶的最适底物浓度为3%,酶催化水解右旋糖酐的主产物为异麦芽糖,确定该酶为内切右旋糖酐酶,并且只对连续的α-1,6葡萄糖苷键起作用。酶的Km值为3.55×10-5 mol/L,Vmax=4.29×10-2 mol(葡萄糖)/min.L。Cu2+和Zn2+对酶活有较强的促进作用,低浓度的Cu2+使酶活力提高到134.7%,Mn2+对酶催化活力抑制作用较强。【结论】筛选得到一株新的右旋糖酐酶的产生菌F1001,产生的右旋糖酐酶活性高、稳定性较好。为该酶进行催化反应和工业应用提供了重要参数。
[Objiective]To obtain new fungi producing dextranase,we screened and identified a strain F1001 showing high dextranase activities.We provided a new strain with dextranase activity for producing clinical dextran.[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,and activities of dextranase were measured with dextran 70 kDa as the substrate.[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.The optimal reaction temperature was 35℃,and the optimum pH was 5.0,it was stable in the condition of pH 4.0-7.0 and under the temperature of 50℃.The optimum substrate concentration was 3%（w /v）.The final dextranase hydrolysis product was isomaltose,which 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 stable.These results may provide an important basis for industrial applications.
出处
《微生物学报》
CAS
CSCD
北大核心
2011年第4期495-503,共9页
Acta Microbiologica Sinica
基金
安徽省长三角科技联合攻关项目(10140702001)
合肥工业大学大学生创新性实验计划项目(CXSY10067)~~
关键词
右旋糖酐酶
棘孢青霉
分离纯化
酶学性质
dextranase
Penicillium aculeatum
purification
enzyme property
