The Development of Leucine Dehydrogenase and Formate Dehydrogenase Bifunctional Enzyme Cascade Improves the Biosynthsis of L-tert-Leucine
The Development of Leucine Dehydrogenase and Formate Dehydrogenase Bifunctional Enzyme Cascade Improves the Biosynthsis of L-tert-Leucine
复制标题
亮氨酸脱氢酶和甲酸脱氢酶双功能酶级联的开发提高了L-叔亮氨酸的生物合成
DOI：
10.1007/s12010-016-2160-2
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发表时间：
2016-11-01
影响因子：
3
通讯作者：
Fang, Baishan
中科院分区：
文献类型：
--
作者：
Lu, Jixue;Zhang, Yonghui;Fang, Baishan
亮氨酸脱氢酶(LDH)和甲酸脱氢酶(FDH)是基于迷你咖啡因中两种不同的粘附素与LDH和FDH中相应的Dockerin之间的高亲和力相互作用而组装在一起的。用再生的无定形纤维素(RAC)在体外进一步吸附含有两种酶的迷你咖啡因，形成双功能酶复合体(迷你咖啡酚与乳酸脱氢酶(LDH)和脱氢酶(FDH)分别被RAC、RSLF吸附)。系统比较了双功能酶复合体和游离酶混合物的酶学性质。在不同浓度的酶、辅酶和底物条件下，比较了RSLF和游离酶混合物催化合成L-叔亮氨酸的效果。与游离混合酶相比，RSLF的初始L-叔亮氨酸产量提高了2倍。L-叔亮氨酸每升91克，对映体纯度为99%e.E.经RSLF多酶催化得到。结果表明，基于粘附素-Dockerin相互作用的双功能酶复合体在合成L-叔亮氨酸方面具有很大的潜力。
Leucine dehydrogenase (LDH) and formate dehydrogenase (FDH) were assembled together based on a high-affinity interaction between two different cohesins in a miniscaffoldin and corresponding dockerins in LDH and FDH. The miniscaffoldin with two enzymes was further absorbed by regenerated amorphous cellulose (RAC) to form a bifunctional enzyme complex (miniscaffoldin with LDH and FDH adsorbed by RAC, RSLF) in vitro. The enzymatic characteristics of the bifunctional enzyme complex and free enzymes mixture were systematically compared. The synthesis of L-tert-leucine by the RSLF and free enzyme mixture were compared under different concentrations of enzymes, coenzyme, and substrates. The initial L-tert-leucine production rate by RSLF was enhanced by 2-fold compared with that of the free enzyme mixture. Ninety-one grams per liter of L-tert-leucine with an enantiomeric purity of 99 % e.e. was obtained by RSLF multienzyme catalysis. The results indicated that the bifuntional enzyme complex based on cohesin-dockerin interaction has great potential in the synthesis of L-tert-leucine.