耐有机溶剂氨基酸脱氢酶基因挖掘与非天然氨基酸的非水相合成

Genome mining of organic solvent tolerant amino acid dehydrogenase for biosynthesis of unnatural amino acids in non-aqueous system

可在非水相体系高效催化不对称还原反应制备手性化合物的氧化还原酶具有重要科学意义与工业应用前景。基于基因挖掘技术,获得了17个耐盐氨基酸脱氢酶的基因,并分析了其进化同源性和蛋白质稳定性热力学参数。选取来源于Natranaerobius thermophilus的苯丙氨酸脱氢酶(PheDH),进行了基因合成和表达、分离和纯化,获得了耐盐氨基酸脱氢酶,并检测了其有机溶剂耐受性。结果表明,对于催化L-苯丙氨酸的氧化脱氨体系,反应的最适温度为60℃,最适pH为12。在含有30%的二甲亚砜反应体系中,催化活性是水相体系的1.2倍。而对于催化还原胺化制备L-高苯丙氨酸的体系,最适温度为70℃,最适pH为8.5。在含30%的甲基叔丁基醚和二甲亚砜反应体系中,催化活性分别是原始活性的101.3%和99.2%。研究表明,该耐盐酶具有较好的耐热、耐有机溶剂等抗逆性能。

The oxidoreductase,which can efficiently catalyze asymmetric reduction reactions to prepare chiral compounds in non-aqueous systems,has important scientific significance and industrial application prospects.Based on gene mining technology,17 salt-tolerant amino acid dehydrogenase genes were obtained,and their evolutionary homology and protein stability thermodynamic parameters were further analyzed.Bioinformatic identification combined with structure classification and thermodynamic parameters calculation led to a promising phenylalanine dehydrogenase from Natranaerobius thermophiles.Catalytic characteristics of phenylalanine dehydrogenase in the non-aqueous system were studied.This PheDH was highly stable for oxidative deamination of phenylalanine with optimal temperature of 60℃and optimal pH of 12,which is rarely reported in such alkaline environment.Enzyme activity was enhanced by 1.2 folds with 30%dimethylsulfoxide.For biosynthesis of L homophenylalanine by reductive ammoniation,the optimal reaction condition is 70℃and pH 8.5.With 30%methyl tert-butyl ether and dimethylsulfoxide,the relative activity is 101.3% and 99.2%, respectively. The results indicated that this halotolerant PheDH has better resistance to heat and organic solvents. This paper offers a novel strategy for mining of halotolerant amino acid dehydrogenase based on sequenced-driven approaches, and thus provides robust key enzymes to biochemists.