evocatal GmbH, Merowingerplatz 1a, 40225 Düsseldorf, Germany.
Enzyme Microb Technol. 2011 May 6;48(6-7):472-9. doi: 10.1016/j.enzmictec.2011.02.004. Epub 2011 Feb 12.
A gene encoding an NADPH-dependent carbonyl reductase from Neurospora crassa (nccr) was cloned and heterologously expressed in Escherichia coli. The enzyme (NcCR) was purified and biochemically characterised. NcCR exhibited a restricted substrate spectrum towards various ketones, and the highest activity (468U/mg) was observed with dihydroxyacetone. However, NcCR proved to be very selective in the reduction of different α- and β-keto esters. Several compounds were converted to the corresponding hydroxy ester in high enantiomeric excess (ee) at high conversion rates. The enantioselectivity of NcCR for the reduction of ethyl 4-chloro-3-oxobutanoate showed a strong dependence on temperature. This effect was studied in detail, revealing that the ee could be substantially increased by decreasing the temperature from 40 °C (78.8%) to -3 °C (98.0%). When the experimental conditions were optimised to improve the optical purity of the product, (S)-4-chloro-3-hydroxybutanoate (ee 98.0%) was successfully produced on a 300 mg (1.8 mmol) scale using NcCR at -3 °C.
从粗糙脉孢菌（Neurospora crassa）中克隆并异源表达了编码 NADPH 依赖的羰基还原酶（nccr）的基因。该酶（NcCR）被纯化并进行了生化特性分析。NcCR 对各种酮具有受限的底物谱，并且在二羟丙酮中观察到最高活性（468U/mg）。然而，NcCR 在还原不同的α-和β-酮酯时表现出非常高的选择性。几种化合物以高对映体过量（ee）和高转化率转化为相应的羟基酯。NcCR 对乙基 4-氯-3-氧代丁酸酯还原的对映选择性强烈依赖于温度。详细研究了该效应，结果表明通过将温度从 40°C（78.8%）降低到-3°C（98.0%），ee 可以显著提高。当优化实验条件以提高产物的光学纯度时，在-3°C 下使用 NcCR 成功地在 300mg（1.8mmol）规模上生产出（S）-4-氯-3-羟基丁酸酯（ee 98.0%）。
