College of Material, Chemistry and Chemical Engineering, Hangzhou Normal University, No. 1378, Wenyixi Road, Hangzhou, 311121, People's Republic of China.
J Ind Microbiol Biotechnol. 2014 Nov;41(11):1609-16. doi: 10.1007/s10295-014-1502-8. Epub 2014 Sep 5.
A novel aldo-keto reductase (LEK) from Lodderomyces elongisporus NRRL YB-4239 (ATCC 11503) was discovered by genome database mining for carbonyl reduction. LEK was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity and the catalytic properties were studied. Among the substrates, ethyl 4-chloro-3-oxobutanoate was converted to ethyl (R)-4-chloro-3- hydroxybutanoate ((R)-CHBE), an important pharmaceutical intermediate, with an excellent enantiomeric excess (e.e.) (>99 %). The mutants W28A and S209G obtained by site-directed mutation were identified with much higher molar conversion yields and lower Km values. Further, the constructed coenzyme regeneration system with glucose as co-substrate resulted in a yield of 100 %, an enantioselectivity of >99 %, and the calculated production rate of 56.51 mmol/L/H. These results indicated the potential of LEK for the industrial production of (R)-CHBE and other valuable chiral alcohols.
通过对基因组数据库进行挖掘，发现了一种来自 Lodderomyces elongisporus NRRL YB-4239（ATCC 11503）的新型醛酮还原酶（LEK），可用于羰基还原。通过在大肠杆菌 BL21（DE3）中过表达、纯化至均相，并研究了其催化特性。在底物中，用乙基 4-氯-3-氧代丁酸酯转化为乙基（R）-4-氯-3-羟基丁酸酯（（R）-CHBE），这是一种重要的医药中间体，具有出色的对映体过量（ee）（>99%）。通过定点突变获得的突变体 W28A 和 S209G 的摩尔转化率和 Km 值均明显提高。此外，构建的以葡萄糖为共底物的辅酶再生系统的产率为 100%，对映选择性>99%，计算的生产速率为 56.51mmol/L/h。这些结果表明，LEK 具有在工业上生产（R）-CHBE 和其他有价值的手性醇的潜力。
