文献类型：期刊文献

中文题名：微小根毛霉耐热α-淀粉酶基因的克隆与高效表达(英文)

英文题名：Cloning and high-level expression of thermostable α-amylase gene from Rhizomucor pusillus

作者：何正贵[1];尹燕辰[1];毛佑志[1];周思行[1];潘琪[1];魏东芝[1];高蓓[1]

机构：[1]华东理工大学生物反应器国家重点实验室/鲁华生物技术研究所,上海200237

年份：2014

卷号：45

期号：2

起止页码：165

中文期刊名：南方农业学报

外文期刊名：Journal of Southern Agriculture

收录：CSTPCD;;北大核心:【北大核心2011】；CSCD:【CSCD_E2013_2014】；

基金：National High Technology Research and Development Program of China(2013AA102109);Fundamental Research Fundsfor the Central Universities

语种：中文

中文关键词：微小根毛霉;α-淀粉酶;表达;麦芽糖;毕赤酵母

外文关键词：Rhizomucor pusillus;α-amylase;expression;maltose;Pichia pastoris

摘要：【目的】开发具有高表达活力的耐热耐酸新型真菌α-淀粉酶,研究其酶学性质和催化活性,探讨其潜在工业应用潜力。【方法】采用RT-PCR从嗜热真菌微小根毛霉（Rhizomucor pusillus）中克隆耐热α-淀粉酶基因,并命名为RpAmy。以质粒pPIC9K为表达载体,将α-淀粉酶基因（RpAmy）转入毕赤酵母（Pichia pastoris）表达宿主KM71,进行异源表达,并测定动力学参数和酶学性质,分析水解淀粉的产物。【结果】克隆得到嗜热真菌微小根毛霉（Rhizomucor pusillus）耐热α-淀粉酶基因,其开放阅读框全长1416 bp,编码471个氨基酸,与米根毛霉α-淀粉酶的氨基酸序列相似性最高（56.0%）。RpAmy在毕赤酵母（Pichia pastoris）KM71中表达,最高酶活达到32100.0 U/mL,蛋白表达量为1.5 mg/mL;测定纯化后RpAmy比活力、Km和Vmax,分别为6745.9 U/mg、2.26 mg/mL、0.2875 mg/mL·min;最适pH和最适温度分别为pH 4.0、70℃;具有较宽的pH耐受性（4.0~9.0）和较高的温度耐受性（60℃）;水解可溶性淀粉产物主要为麦芽糖（69.0%,w/w）和葡萄糖（22.0%,w/w）。【结论】从微小根毛霉（Rhizomucor pusillus）中克隆得到α-淀粉酶基因（RpAmy）,其可在毕赤酵母（Pichia pastoris）KM71中高效异源表达并得到α-淀粉酶RpAmy。该酶具有较好的耐酸耐热性质,并能水解淀粉产生高麦芽糖含量的糖浆,在工业应用方面具有很大的潜力。
【Objective】The present experiment was conducted to develop new thermostable and acid-stable fungalα-amylase with high-level expression in heterologous expression systems of Pichia pastoris, study their enzymatic property and catalytic activity in order to evaluate its potential application prospects in starch process industry. 【Method】RT-PCR was performed to clone α-amylase gene from Rhizomucor pusillus. The obtained α-amylase gene, designated as RpAmy was inserted into vector pPIC9K and transformed into Pichia pastoris KM71 to study heterologous expression.Enzymatic properties and kinetic parameters of RpAmy were studied and soluble starch hydrolyzed products by α-amylase was analyzed by TCL and HPLC. 【Result】Rhizomucor pusillus α-amylase gene RpAmy showed 1416 bp of ORF full length, encoded a protein of 471 amino acid residues, which shared the highest（56.0%） similarity to the α-amylase from Rhizopus oryzae. The α-amylase gene（RpAmy） was functionally expressed in Pichia pastoris and achieved the highest activity of 32100.0 U/mL（1.5 mg/mL total protein） in shaking flask. The specific activity, Km and Vmax for purified RpAmy were 6745.9 U/mg, 2.26 mg/mL and 0.2875 mg/mL·min towards soluble starch, respectively. The optimum pH and temperature of RpAmy were 4.0 and 70 ℃, respectively. It was stable in a wide pH range of 4.0-9.0and higher temperature（60 ℃）. High level of maltose（69.0%, w/w） and glucose（22.0%, w/w） were generated as the main hydrolysis products of soluble starch that catalyzed by RpAmy. 【Conclusion】In this study, the α-amylase gene（RpAmy） from Rhizomucor pusillus was cloned and expressed high level in Pichia pastoris KM71 with high activity.The obtained RpAmy exhibited superior pH and thermo stability and could produce high content of maltose from soluble starch, thus illustrating a great potential application in starch processing.