新型R-扁桃酸脱氢酶的基因挖掘及表达鉴定

Gene Mining,Expression and Characterization of Novel R-mandelate Dehydrogenases

R-扁桃酸脱氢酶在苯乙酮酸的生物合成中起着关键的作用,挖掘具有高催化活性及稳定性的新型R-扁桃酸脱氢酶具有重要的意义。为了获得理想的R-扁桃酸脱氢酶,采用了基因组挖矿技术从Lactobacillus harbinensis菌株中获得了一个新型的R-扁桃酸脱氢酶LhDMDH,重组LhDMDH的比酶活高达1264.3 U/mg,约为探针的4倍,在已报道的R-扁桃酸脱氢酶中处于领先水平。同时,考察了4个重组酶主要的酶学特性,它们的最适反应温度在25-30℃,最适反应pH在9.0-9.5。动力学参数的结果表明,LhDMDH对底物的K_（cat）值为30.28 S^-1,明显高于其它重组酶。此外,底物谱分析的结果也表明LhDMDH在外消旋扁桃酸的手性拆分及苯乙酮酸的生物合成中更具优势。在R-扁桃酸脱氢酶基因挖掘方面取得了较为理想的结果,为进一步的改造及应用奠定了坚实的基础,也为其它酶的挖掘提供了可资借鉴的经验。

R-mandelate dehydrogenase plays a key role in the biosynthesis of phenylglyoxylic acid, thus exploiting nove R-mandelate dehydrogenase with higher catalytic activity and stability has significant economic value. In order to obtain the perfect R-mandelate dehydrogenase, a novel R-mandelate dehydrogenase was obtained by genome mining, named as LhDMDH, which was from Lactobacillus harbinensis. The specific activity of LhDMDH was 1：264.3 U/mg, which was near to four times that of the probe and leading in the reported enzyme. Meanwhile, the main enzymatic characterizations of the four recombinant enzymes were researched. Their temperature optima were 25 to 30 ~C, and their pH optima were 9.0 to 9.5. The Kcat of LhDMDH is 30.28 S1, which is obviously higher than the others. In addtion, the results of substrate spectrum of R-mandelate dehydrogenases indicated that the LhDMDH could have advantages over other enzymes in chiral resolution of racemic mandelic acid and the biosynthesis of phenylglyoxylic acid. This gained ideal results in genome mining of R-mandelate dehydrogenases, established a solid foundation for further transformation and application, and provided a useful experience for the exploiting of other enzymes.