Purification, characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata
Purification, characterization, and substrate specificity of a glucoamylase with steroidal saponin-rhamnosidase activity from Curvularia lunata
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新月弯孢中具有甾体皂苷-鼠李糖苷酶活性的葡糖淀粉酶的纯化、表征和底物特异性
DOI：
10.1007/s00253-007-1117-3
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发表时间：
2007-10-01
影响因子：
5
通讯作者：
Qian, Xiao-hong
中科院分区：
文献类型：
--
作者：
Feng, Bing;Hu, Wei;Qian, Xiao-hong
以前曾报道，来自新月弯孢菌的葡糖淀粉酶能够水解甾体皂苷C-3位糖链的末端1，2-连接的鼠李糖残基。本研究采用凝胶过滤和离子交换层析等柱层析技术对该酶进行分离纯化，并对其进行了鉴定。通过MALDI-TOF/TOF（TM）蛋白质组学分析仪分析蛋白质片段，表明该酶是1，4-α-D-葡聚糖葡糖水解酶EC 3.2.1.3，GA，并且与来自Aspergillus sp.的葡糖淀粉酶具有相当大的同源性。本研究首次发现该葡萄糖淀粉酶是由C. lunata，能够水解甾体皂苷的末端鼠李糖基。该酶具有葡萄糖淀粉酶的一般特性，能水解淀粉。其分子量为66 kDa，在50 ℃、pH 4下具有最佳活性，在使用薯蓣皂苷元-3-O-α-L-吡喃鼠李糖基（1 -> 4）-[α-L-吡喃鼠李糖基（1 -> 2）]-β-D-吡喃葡萄糖苷（化合物II）作为底物的条件下，其比活性为12.34 U mg总蛋白（-1）。此外，本文还对四种商业化的尼日尔曲霉葡糖淀粉酶进行了研究，发现它们对甾体皂苷末端鼠李糖残基的水解活性相似。
It has been previously reported that a glucoamylase from Curvularia lunata is able to hydrolyze the terminal 1,2-linked rhamnosyl residues of sugar chains at C-3 position of steroidal saponins. In this work, the enzyme was isolated and identified after isolation and purification by column chromatography including gel filtration and ion-exchange chromatography. Analysis of protein fragments by MALDI-TOF/TOF (TM) proteomics Analyzer indicated the enzyme to be 1,4-alpha-D-glucan glucohydrolase EC 3.2.1.3, GA and had considerable homology with the glucoamylase from Aspergillus oryzae. We first found that the glucoamylase was produced from C. lunata and was able to hydrolyze the terminal rhamnosyl of steroidal saponins. The enzyme had the general character of glucoamylase, which hydrolyze starch. It had a molecular mass of 66 kDa and was optimally active at 50 degrees C, pH 4, and specific activity of 12.34 U mg of total protein(-1) under the conditions, using diosgenin-3-O-alpha-L-rhamnopyranosyl(1 -> 4)-[alpha-L-rhamnopyranosyl (1 -> 2)]-beta-D-glucopyranoside (compound II) as the substrate. Furthermore, four kinds of commercial glucoamylases from Aspergillus niger were investigated in this work, and they had the similar activity in hydrolyzing terminal rhamnosyl residues of steroidal saponin.