【摘要】 短链脱氢酶(SDRs)是一大类可以还原羰基为手性醇的催化剂.为了从无色杆菌JA81中筛选可能的羰基还原酶,首先将它的基因组信息与COG等5个数据库比对后得到19个假定短链脱氢酶基因.通过分析保守序列和进化关系,选择16个具有活性位点保守序列的基因进行克隆,并在大肠杆菌中进行异源表达.以4-苯基-3-丁炔-2-酮为底物,检测重组菌的整细胞转化活性和对映体选择性,发现多个假定短链脱氢酶具有羰基还原活性,其中SDR3067全细胞催化活性最高,产物为(S)-醇,光学纯度>99%ee.对SDR3067进行纯化后发现该酶是NADPH依赖型还原酶,其最适反应pH为6,最适温度为25℃,当1g/L底物转化10 min时,底物即可转化24.9%.研究结果表明基因组数据挖掘是获得新型高效高选择性短链脱氢酶的有效途径.图7表1参23更多还原

【Abstract】 Short-chain dehydrogenases/reductases(SDRs) are a group of catalysts that can catalyze carbonyl compounds to form chiral alcohols.To obtain potential ketone reductases from Achromobacter sp.JA81,analysis of its genome was performed using COG,NR,TrEMBL,KEGG and SwissProt databases and 19 putative SDRs genes were revealed.Among them,16contained the conservative sequences of typical ketone reductases.They were cloned and expressed in Escherichia coli.The whole cells containing the recombinant protein were tested for the activity and enantioselectivity in the reduction of 4-pheny1-3-butyn-2-one.Several putative SDRs were active toward the substrate,with SDR3067 displaying the highest activity in whole-cell assays and producing(S)-alcohol in >99%ee.It was purified and further characterized to be an NADPH-dependent reductase with the optimal pH of 6,and optimal reaction temperature of 25℃.When the substrate concentration reached 1 g/L,SDR3067 converted 24.9%substrate to(S)-alcohol in 10 minutes under the optimized conditions.The results demonstrated that genome mining is an efficient approach to obtain novel short-chain dehydrogenases/reductases with high activity and stereoselectivity.Fig 7,Tab 1,Ref 23更多还原