生淀粉糖化酶的菌种筛选及酶学研究

Enzymatic Study on Species Isolation from Nature to Secrete Active Raw-Starch Glycoamylase

摘要从自然界分离到一株高酶活的生淀粉糖化酶菌株黑曲霉Sx。在最适产酶条件:固体培养基pH6.5,固水比1∶1.6,30℃发酵24h酶活达382u/g。经纸层析鉴定酶解产物为葡萄糖。粗酶液在贮藏时,当酶液中的SDS浓度达6mg/ml时,30℃保藏10天,酶活保持不变。分析经初步分离提纯的酶的性质得:以玉米淀粉为底物时,该酶最适pH4.5,最适酶反应温度为60℃。pH4.0～7.0范围内酶活力稳定。酶具有较强的热稳定性,80℃保温1h以后,酶活力仍能保持15%。金属离子等对酶的影响为:Ag+、Sn2+对酶有强烈抑制作用;Hg2+、Zn2+、Mn2+、Cu2+有抑制作用;EDTA、Ca2+、Fe2+对酶无影响;Co2+、K+有一定的激活作用。

Aspergillus niger Sx,isolated from nature,could secrete raw-starch glucoamylase (RSAG)with high enzyme activity at level of 382u/g.After fermentation for 24 hours at 30℃in solid wheat bran culture ,the maximum secretion of RSAG was achieved at pH6.5 with the ratio of solid to water as 1∶1.6.Further,the enzymatic production was confirmed by paper chromatography as glucose.Interestingly,it was discovered that SDS could keep RSGA in its initial state.When the concentration in the crude enzyme was 6mg/ml,the activity of enzyme was kept intact and active after 10days storage at 30℃.Additionally,RSGA was purified initially by means of ammonium sulfate fractionation and dialysis.The optimum hydrolysis conditions for the purified enzyme were pH4.5,60℃.Amazingly,it preserved a high stabillity at pH between 4.0and 7.0,After incubation at 80℃ for 1 hour,the enzyme still retained 15% activity.Metal ions such as Ag+、Sn2+ and Hg2+ could inhibit the enzyme to different extents,where as EDTA、Ca+ showed effect on the activity of the enzyme.In contrast to the forenamed cation,Co2+ and K+ could stimulate the activity of enzyme slightly.