新型环氧化物水解酶AuEH1的表达及酶学性质

Expression of a Novel Epoxide Hydrolase AuEH1 from Aspergillus usamii and Its Enzymatic Properties

DOI：10.3969/j.issn.1673-1689.2017.11.006

中文关键词: 宇佐美曲霉 环氧化物水解酶 表达 酶学性质 对映选择性 （S）-环氧苯乙烷

英文关键词: Aspergillus usamii,epoxide hydrolase,expression,enzymatic properties,enantioselec- tivity,（S）-styrene oxide

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中文摘要:

采用RT-PCR技术从宇佐美曲霉 （Aspergillus usamii） E001总RNA中扩增了一种新型环氧化物水解酶AuEH1的编码基因Aueh1。将该基因与表达质粒pET-28a（+） 连接，转化大肠杆菌 （Escherichia coli） BL21（DE3），获基因工程菌E. coli/Aueh1，IPTG诱导表达重组AuEH1 （reAuEH1）。菌体经超声波破碎，收集上清作为reAuEH1酶液。分析结果表明，reAuEH1活性为8.12 U/mL；其最适温度为40 ℃，在40 ℃及以下稳定；最适pH为6.5，在pH 5~9范围内稳定；所测金属离子 （除Ag+和Cu2+外） 及EDTA对reAuEH1活性影响不大。在40 mmol/L磷酸钾缓冲液（pH 6.5）、20 mmol/L外消旋环氧苯乙烷 （（R，S）-SO） 和0.8 U/mL reAuEH1体系（终体积6 mL） 中，35 ℃反应50 min，所获手性产物 （S）-SO的摩尔产率为30.8%、对映体过量 （e.e.） 值＞99%。

英文摘要:

A gene encoding a novel epoxide hydrolase （AuEH1），Aueh1，was amplified from the total RNA of Aspergillus usamii by RT-PCR technique and ligated with pET-28a（+），followed by transforming the recombinant plasmid，pET-28a-Aueh1，into E. coli BL21（DE3），forming an engineering strain E. coli/Aueh1. The recombinant AuEH1 （reAuEH1） was expressed in E. coli by IPTG induction. After the cells were disrupted by sonication，the supernatant was collected and used as the reAuEH1 solution. The analytical results indicated that the activity of reAuEH1 was 8.12 U/mL. The temperature and pH optima of reAuEH1 were 40 ℃ and 6.5，respectively. It was stable at a temperature of 40 ℃ or below，and at a pH range of 5~9. Its activity was not significantly influenced by EDTA and metal ions tested except for Ag+ and Cu2+. Under the conditions of 40 mmol/L potassium phosphate buffer （pH 6.5），20 mmol/L racemic styrene oxide （（R，S）-SO） and 0.8 U/mL reAuEH1，in a final volume of 6 mL，the hydrolytic resolution of （R，S）-SO was conducted at 35 ℃ for 50 min. The chiral product （S）-SO was obtained with a molar yield of 30.8% and an enantiomeric excess （e.e.） value more than 99%.

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