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

作　　者：李兴倩 胡蝶[2] 黄博梅 李雪婷 康雁君 邬敏辰 LI Xingqian;HU Die;HUANG Bomei;LI Xueting;KANGYanjun;WU Minchen(School of Pharmaceutical Science,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China;Wuxi Medical School,Jiangnan University,Wuxi 214122,China)

机构地区：[1]江南大学药学院,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]江南大学无锡医学院,江苏无锡214122

出　　处：《食品与生物技术学报》2017年第11期1157-1162,共6页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(21676117)

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Cu^(2+)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%。A gene encoding a novel epoxide hydrolase(AuEH1),Aueh1,was am plified from the total RNA o f Aspergillus usamii by RT-PCR technique and ligated w ith pET-28a(+),follow ed by transform ing the recombinant plasm id,pET-28a-A uehl,into B.coli BL21(DE3),form ing an engineering strain B.coli/Auehl.The recombinant AuEH1(reAuEH1)was expressed in B.coli by IPTG induction.A fter the cells were disrupted by sonication,the supernatant was collected and used as the reAuEH1solution.The analytical results indicated that the activity o f reAuEH1was8.12U/m L.The temperature and pH optima o f reAuEH1were40V and6.5,respectively.It was stable at a temperature o f40℃or below,and at a pH range o f5~9.Its activity was not significantly influenced by EDTA and metal ions tested except for Ag+and Cu2+.Under the conditions o f40m m ol/L potassium phosphate buffer(pH6.5),20m m ol/L racemic styrene oxide((E,F)-SO)and0.8U/mL reAuEHl,in a final volume of6mL,the hydrolytic resolution of(R,S)-SO was conducted at35D for50min.The chiral product(S)-SO was obtained with a molar yield of30.8%and an enantiomeric excess(e.e.)value more than99%.

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 

分 类 号：Q55[生物学—生物化学] Q78