In vitro biosynthesis of optically pure d-(-)-acetoin from meso-2,3-butanediol using 2,3-butanediol dehydrogenase and NADH oxidase
In vitro biosynthesis of optically pure d-(-)-acetoin from meso-2,3-butanediol using 2,3-butanediol dehydrogenase and NADH oxidase
复制标题
使用 2,3-丁二醇脱氢酶和 NADH 氧化酶从内消旋 2,3-丁二醇体外生物合成光学纯 d-(-)-乙偶姻
DOI：
10.1002/jctb.6050
复制
发表时间：
2019-08-01
影响因子：
3.4
通讯作者：
Chen, Tao
中科院分区：
文献类型：
--
作者：
Cui, Zhenzhen;Zhao, Yujiao;Chen, Tao
背景
乙偶姻，尤其是手性的L-(+)-或D -(-)-对映体，是一种高价值的工业产品，可用作药物中间体。然而，通过传统化学合成生产光学纯乙偶姻成本高昂。经典的微生物发酵符合绿色化学的标准，但存在诸多缺点，例如工艺工程复杂、效价或生产率不理想，以及由于不可避免地产生副产物和培养基过于复杂而导致产品分离困难。相比之下，无细胞生物系统中的酶促生物催化在生产高纯度化学品方面具有众多优势，然而高效的生产光学纯乙偶姻的酶促方法却鲜有报道。
结果
构建了一种由2,3 -丁二醇脱氢酶和生成水的NADH氧化酶组成的高效偶联双酶系统，用于在不引入任何底物或副产物的情况下生产光学纯乙偶姻。对一种2,3 -丁二醇脱氢酶和三种NADH氧化酶进行了纯化、表征，并组装成不同的双酶系统。然后根据乙偶姻的效价选择了由LL - NOX和CG - BDH组成的最佳系统。在对反应条件进行优化后，94.0%的内消旋 - 2,3 -丁二醇转化为D -(-)-乙偶姻。最终，在6小时内生产出399.7 mmol/L的D -(-)-乙偶姻，对映体过量为91.2%，且未检测到任何副产物。
结论
据我们所知，这是首次报道在体外利用NADH氧化酶再生NAD⁺从内消旋 - 2,3 -丁二醇生产光学纯D -(-)-乙偶姻，也是酶促生物合成中底物转化率和乙偶姻效价最高的。因此，这项工作为体外生产D -(-)-乙偶姻提供了一种经济且绿色的替代方法。(c) 2019化学工业协会
BACKGROUND Acetoin, especially the chiral l-(+)- or d-(-)-enantiomer, is a high-value industrial product that can be used as a pharmaceutical intermediate. However, the production of optically pure acetoin via conventional chemical synthesis is expensive. Classical microbial fermentations satisfy the criteria of green chemistry, but have numerous disadvantages, such as complicated process engineering, unsatisfactory titers or productivities, and difficult product separation due to the inevitable generation of by-products and overly complex media. By contrast, enzymatic biocatalysis in cell-free biosystems has numerous advantages in production of chemicals with high purity, whereas efficient enzymatic methods for the production of optically pure acetoin have rarely been reported. RESULTS An efficient coupled two-enzyme system composed of 2,3-butanediol dehydrogenase and H2O-forming NADH oxidase was constructed to produce optically pure acetoin without introducing any substrate or by-product. A 2,3-butanediol dehydrogenase and three NADH oxidases were purified, characterized and assembled into different two-enzyme systems. The best system, composed of LL-NOX and CG-BDH, was then selected according to the acetoin titer. After optimization of the reaction conditions, 94.0% of meso-2,3-butanediol was converted to d-(-)-acetoin. Finally, 399.7 mmol L-1 d-(-)-acetoin with an enantiomeric excess of 91.2% was produced in 6 h without any detected by-products. CONCLUSIONS To our best knowledge, this is the first report on the production of optically pure d-(-)-acetoin from meso-2,3-butanediol in vitro using NADH oxidase to regenerate NAD(+), as well as the best substrate conversion and acetoin titer among enzymatic biosynthesis. This work therefore provides an economical and green alternative for the in vitro production of d-(-)-acetoin. (c) 2019 Society of Chemical Industry