Huang Rui, Li Chuang, Zhao Shi-Guang, Liu Qing-Tao, Liu Yan, Xue Zheng-Lian
College of Biological and Food Engineering, Anhui Polytechnic University, Wuhu 241000, China.
College of Biological and Food Engineering, Anhui Polytechnic University, Wuhu 241000, China.
Enzyme Microb Technol. 2023 May;166:110228. doi: 10.1016/j.enzmictec.2023.110228. Epub 2023 Mar 17.
In order to excavate microbial epoxide hydrolases (EHs) with desired catalytic properties, a novel EH, SfEH1, was identified based on the genome annotation of Streptomyces fradiae and sequence alignment analysis with local protein library. The SfEH1-encoding gene, sfeh1, was then cloned and over-expressed in soluble form in Escherichia coli/BL21(DE3). The optimal temperature and pH of recombinant SfEH1 (reSfEH1) and reSfEH1-expressing E. coli (E. coli/sfeh1) were both determined as 30 ℃ and 7.0, also indicating that the influences of temperature and pH on reSfEH1's activities were more obvious than those of E. coli/sfeh1 whole cells. Subsequently, using E. coli/sfeh1 as catalyst, its catalytic properties towards thirteen common mono-substituted epoxides were tested, in which E. coli/sfeh1 had the highest activity of 28.5 U/g dry cells for rac-1,2-epoxyoctane (rac-6a), and (R)-1,2-pentanediol ((R)-3b) (or (R)-1,2-hexanediol ((R)-4b)) with up to 92.5% (or 94.1%) ee was obtained at almost 100% conversion ratio. Regioselectivity coefficients (α and β) displayed in the enantioconvergent hydrolysis of rac-3a (or rac-4a) were calculated to be 98.7% and 93.8% (or 95.2% and 98.9%). Finally, the reason of the high and complementary regioselectivity was confirmed by both kinetic parameter analysis and molecular docking simulations.
为了挖掘具有所需催化特性的微生物环氧化物水解酶（EHs），基于弗氏链霉菌的基因组注释和与本地蛋白质库的序列比对分析，鉴定出一种新型EH，即SfEH1。随后克隆了编码SfEH1的基因sfeh1，并在大肠杆菌/BL21(DE3)中以可溶形式进行过量表达。重组SfEH1（reSfEH1）和表达reSfEH1的大肠杆菌（大肠杆菌/sfeh1）的最佳温度和pH均确定为30℃和7.0，这也表明温度和pH对reSfEH1活性的影响比对大肠杆菌/sfeh1全细胞的影响更明显。随后，以大肠杆菌/sfeh1为催化剂，测试了其对13种常见单取代环氧化物的催化特性，其中大肠杆菌/sfeh1对rac - 1,2 - 环氧辛烷（rac - 6a）的活性最高，为28.5 U/g干细胞，并且在几乎100%的转化率下获得了ee高达92.5%的(R)-1,2 - 戊二醇((R)-3b)（或ee高达94.1%的(R)-1,2 - 己二醇((R)-4b)）。计算得出在rac - 3a（或rac - 4a）的对映收敛水解中显示的区域选择性系数（α和β）分别为98.7%和93.8%（或95.2%和98.9%）。最后，通过动力学参数分析和分子对接模拟证实了高区域选择性和互补区域选择性的原因。
