祖先羰基还原酶的挖掘及酶法合成(S)-乙偶姻的研究

An ancestral carbonyl reductase:mining and application in the synthesis of(S)-acetoin

【背景】(S)-乙偶姻是一种重要的精细化学品,开展其绿色高效合成具有重要的应用价值。【目的】祖先酶可能具有较好的热稳定性。论文以筛选高活性和稳定性的祖先羰基还原酶为目标,并探究其酶学性质,实现(S)-乙偶姻的高效酶法合成。【方法】基于祖先序列重建法的基因挖掘策略筛选双乙酰还原酶,并对其进行了酶学性质表征,构建不对称还原双乙酰合成(S)-乙偶姻的反应体系。【结果】获得了一种可以不对称还原双乙酰合成(S)-乙偶姻的祖先羰基还原酶AncBDH1,并在Escherichia coli中实现可溶性表达。序列保守性分析显示,AncBDH1含有短链醇脱氢酶家族的特征基序,且与已报道羰基还原酶的同源性均较低。对AncBDH1进行了镍柱纯化和酶学性质表征,显示其仅可利用NADH为辅酶,最适反应pH值为7.0,最适反应温度为60℃,催化活性不依赖金属离子,并且表现出对二甲基亚砜等有机溶剂的良好耐受性。Anc BDH1可以在12 h内将200 mmol/L双乙酰完全转化为(S)-乙偶姻。【结论】发现了一个具有较高的最适温度及有机溶剂耐受性的祖先羰基还原酶AncBDH1,并实现了(S)-乙偶姻的高效酶法合成,为(S)-乙偶姻的酶法合成奠定了一定的研究基础。

[Background](S)-Acetoin is a class of important fine chemicals with wide applications.Efficient and sustainable synthesis of(S)-acetoin is of special interest.[Objective]Due to the high thermostability,ancestral carbonyl reductases are promising in industrial biocatalysis.This study aims to mine an ancestral carbonyl reductase with high activity and thermostability,characterize its enzymatic properties,and establish an efficient enzymatic catalysis system for the synthesis of(S)-acetoin.[Methods]The gene mining strategy based on ancestral sequence reconstruction was adopted to identify ancestral carbonyl reductases.The enzyme properties were characterized,and the purified ancestral carbonyl reductase was used for the establishment of the(S)-acetoin synthesis system.[Results]An ancestral carbonyl reductase AncBDH1 was identified and heterogeneously expressed in Escherichia coli in the soluble form,and it displayed a high activity in the asymmetric reduction of diethyl into(S)-acetoin.The sequence of AncBDH1 contained the conserved motifs of short-chain dehydrogenase/reductase superfamily and exhibited low similarity with the sequences of other reported carbonyl reductases.AncBDH1 was purified by nickel-affinity chromatography for enzyme characterization.It displayed high dependence on NADH instead of NADPH as a cofactor,with the optimum performance at pH 7.0 and 60℃.The activity of AncBDH1 was not dependent on metal ions and AncBDH1 could tolerate organic solvents such as dimethyl sulfoxide.AncBDH1 completely reduced 200 mmol/L diethyl into(S)-acetoin within 12 h.[Conclusion]This study provides a novel ancestral carbonyl reductase AncBDH1 with tolerance to high temperature and organic solvents and capable of catalyzing the synthesis of(S)-acetoin,laying a certain research foundation for the biocatalytic synthesis of(S)-acetoin.