芳香杂环酮还原酶的基因挖掘及催化性质  

Gene mining and characterization of an aromatic heterocyclic ketone reductase from Modestobacter roseus

作　　者：薛佳钰 许国超[1] 倪晔[1] XUE Jiayu;XU Guochao;NI Ye(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《微生物学通报》2024年第11期4416-4428,共13页Microbiology China

基　　金：国家自然科学基金(22378169)。

摘　　要：【背景】(R,S)-2-氯-1-(6-氟-3,4-二氢-2H-1-苯并吡喃-2-基)-乙醇[NEB-8(R,S)]是奈必洛尔的重要手性中间体,由芳香杂环酮还原酶不对称合成。目前的天然酮还原酶催化效率低、对映选择性差,而商业酮还原酶来源不明确、反应转化率低。【目的】研究具有高对映选择性、稳定性良好、催化效率优异的酮还原酶实现NEB-8(R,S)的高效不对称合成。【方法】基于已有的酮还原酶的蛋白质序列,通过基因挖掘,获得了来源于玫瑰谦虚杆菌(Modestobacter roseus)的酮还原酶基因mradh,在大肠杆菌(Escherichia coli)BL21(DE3)中进行了表达,测定其对映选择性,表征其酶学性质,并优化其在高浓度底物下的反应。【结果】筛选获得了一种新型酮还原酶MrADH,其属于中链醇脱氢酶超家族,兼具氧化和还原能力。MrADH的氧化、还原活性的最适pH值分别为9.5和7.0;对于酮酯类底物、间位取代芳香酮、杂环酮和仲醇等底物,MrADH具有良好的还原氧化活性;此外,在40℃条件下,MrADH半衰期可长达207.0h,显示出良好的热稳定性。MrADH能够高效地催化还原600 mmol/L的(S)-2-氯-1-(6-氟-3,4-二氢-2H-1-苯并吡喃-2-基)-乙酮,以接近100%的对映体过量(e.e.)和99.7%的转化率合成产物醇。【结论】来源于M.roseus的酮还原酶可以高效合成高光学纯度的盐酸奈必洛尔中间体,为奈必洛尔的合成奠定了基础。[Background](R,S)-2-chloro-1-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)-ethanol[NEB-8(R,S)]is an important chiral intermediate for the synthesis of nebivolol.It is synthesized by asymmetric reduction of aromatic heterocyclic ketones under the catalysis of ketoreductase.However,the low catalytic efficiency and poor enantioselectivity of natural ketoreductases and the unclear source of commercial ketoreductases result in low conversion rates.[Objective]To develop a new method for the asymmetric synthesis of NEB-8(R,S)with high enantioselectivity,good stability,and high catalytic efficiency.[Methods]We mined a ketoreductase gene mradh from Modestobacter roseus based on the existing protein sequences of ketoreductases,expressed this gene in Escherichia coli BL21(DE3),and determined the enantioselectivity of the protein.Furthermore,we characterized the enzymatic properties and optimized its reaction conditions under high concentrations of substrate.[Results]In this study,a novel ketoreductase MrADH was identified which belongs to the medium-chain alcohol dehydrogenase superfamily and exhibits both oxidative and reductive activities.The oxidation and reduction activities of MrADH were the highest at pH 9.5 and pH 7.0,respectively.MrADH had good reduction and oxidation activities for ketoesters,meta-substituted aromatic ketones,heterocyclic ketones,and secondary alcohols.In addition,it had a half-life of up to 207.0 h at 40℃,demonstrating good thermal stability.MrADH efficiently catalyzed the reduction of 600 mmol/L(S)-2-chloro-1-(6-fluoro-3,4-dihydro-2H-1-benzopyran-2-yl)-ethanone to synthesize the product alcohol with the enantiomeric excess(e.e.)near 100%and a conversion rate of 99.7%.[Conclusion]The ketoreductase from M.roseus can efficiently synthesize high-optical purity intermediate of nebivolol hydrochloride,laying a foundation for the synthesis of nebivolol hydrochloride.

关 键 词：基因挖掘 芳香杂环酮还原酶 高立体选择性 MrADH 奈必洛尔 

分 类 号：Q78[生物学—分子生物学]