Aspergillus sp.RSD生淀粉糖化酶的分离纯化及酶学性质  

Purification and characterization of a raw starch-digesting glucoamylase from Aspergillus sp. RSD

作　　者：韦荣霞 张梁[1,2] 石贵阳[1,2] 

机构地区：[1]江南大学工业生物技术重点实验室,江苏无锡214122 [2]粮食发酵工艺与技术国家工程实验室,江苏无锡214122

出　　处：《微生物学通报》2014年第1期17-25,共9页Microbiology China

基　　金：国家863计划项目(No.2011AA100905;2012AA021201);教育部"新世纪优秀人才支持计划"项目(No.NCET-11-0665)

摘　　要：【目的】纯化得到一种生淀粉糖化酶,并对其酶学性质进行分析。【方法】从曲霉RSD发酵液中,经过硫酸铵分级盐析,HiPrep DEAE FF16/10弱阴离子交换层析,凝胶过滤层析,Hiprep 16/10 source 30S阳离子交换层析最终纯化出一种电泳纯的生淀粉酶。【结果】粗酶液纯化倍数为12.65倍,活力回收率为9.02%,SDS-PAGE结果显示该酶的相对分子质量约为82 kD。对该酶的酶学性质分析结果表明,该酶最适作用温度为50°C,在50°C以下稳定性很好,对高温较为敏感;最适作用pH为4.5,在pH 3.5-7.0范围内酶活力较为稳定,在40°C、pH 4.6条件下以可溶性淀粉为底物时的Km值和Vmax值分别为7.44 g/L和1.45 g/(L·min);金属离子对酶活性的影响试验表明,Fe2+对该酶具有显著激活效果,EDTA、Cu2+、K+对该酶酶活力有不同程度的抑制作用;底物特异性研究表明该酶对麦芽糊精具有较高酶活力。【结论】与市售糖化酶及生淀粉糖化酶相比,该酶对生淀粉的降解能力更高,在工业应用上有较好的前景。[Objective] A raw starch-digesting glucoamylase was got, and its characterizations were studied. [Methods] A raw starch-digesting glucoamylase was purified from the fermentation broth of Aspergillus sp. RSD using the combination of ammonium sulfate fractionation, HiPrep DEAE FF16/10 anion exchange, Gel filtration chromatography and Hiprep 16/10 source 30S cation exchange. [Results] The crude enzyme was purified 12.65 times with 9.02% recovery of enzyme activity. The molecular weigh of the purified raw starch glucoamylase was about 82 kD identified by SDS-PAGE. The enzyme properties showed that it’s optimal reaction temperature was 50 °C, and the enzyme had a good stability under 50 °C as well as unstable under high temperature; the optimal reaction pH of this enzyme was 4.5, and there was a good stability between pH 3.5 and pH 7.0. At the condition of 40 °C and pH 4.6, its Km and Vmax for soluble starch were 7.44 g/L and 1.45 g/（L·min）. The effects of different metal ions on enzyme activity showed that the enzyme was strongly activated by Fe2＋, however, EDTA, Cu2＋ and K＋ inhibited the enzyme activity at various extents. The substrate specificity of raw starch-digesting glucoamylase showed that the enzyme had higher enzyme activity on malt dextrin. [Conclusion] Compared with commercially available glucoamylase and raw starch-digesting glucoamylase, this enzyme has higher degradation ability on raw materials, and has a good application prospect in industry.

关 键 词：生淀粉糖化酶 分离纯化 酶学性质 

分 类 号：TQ925[轻工技术与工程—发酵工程]