Asymmetric synthesis of L-tert-leucine and L-3-hydroxyadamantylglycine using branched chain aminotransferase
Asymmetric synthesis of L-tert-leucine and L-3-hydroxyadamantylglycine using branched chain aminotransferase
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DOI：
10.1016/j.molcatb.2010.05.014
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发表时间：
2010-09-01
影响因子：
--
通讯作者：
Kim, Byung-Gee
中科院分区：
文献类型：
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作者：
Hong, Eun Young;Cha, Minho;Kim, Byung-Gee
L-叔亮氨酸（Tle）和L-3-羟基金刚烷基甘氨酸（HAG）是多种药物类别的重要中间体。它们是使用支链氨基转移酶（SCAT）以L-谷氨酸（Glu）作为氨基供体从相应的酮酸不对称产生的。对于L-Tle和L-HAG的生产，分别使用来自肠杆菌属TL 3（BCATen）的BCAT和来自大肠杆菌K12（ilvE，新命名为BCATes）的BCAT。在我们目前的研究中，我们的特点是BCATen和BCATes的基本性质，如底物特异性，对映选择性，和动力学参数。L-谷氨酸脱氨基产物α-酮戊二酸对BCATen和BCATes的活性有明显的抑制作用。在10 mM α-酮戊二酸存在下，两种酶的活性均降低至80%。为了克服α-酮戊二酸的产物抑制和转氨反应的平衡问题，用L-谷氨酸脱氢酶（GDH）/甲酸脱氢酶（FDH）和AspAT进行偶联反应。偶联反应大大提高了两种目标化合物的产率。在BCATen/GDH/FDH偶联反应中由150 mM相应的酮酸以90%转化率产生135 mM的L-Tle（> 99%ee）。此外，使用重组全细胞在BCATes/AspAT偶联反应中从100 mM相应的酮酸产生90.5 mM L-HAG（>99% ee）。(C)2010 Elsevier BV保留所有权利。
L-Tert-leucine (Tle) and L-3-hydroxyadamantylglycine of (HAG) are important intermediates for a variety of pharmaceutical classes. They were asymmetrically produced from corresponding keto acids using branched-chain aminotransferase (SCAT) with L-glutamate (Glu) as an amino donor. For the production of L-Tle and L-HAG, BCAT from Enterobacter sp. TL3 (BCATen) and BCAT from Escherichia coli K12 (ilvE, newly named as BCATes) were used, respectively. In our current study, we characterized the basic properties of BCATen and BCATes such as substrate specificity, enantioselectivity, and kinetic parameters. The activities of BCATen and BCATes were inhibited severely by alpha-ketoglutarate which is a deaminated product of L-Glu. In the presence of 10 mM alpha-ketoglutarate, both enzymes activities were reduced up to 80%. In order to overcome product inhibition by alpha-ketoglutarate and the problem of equilibrium of the transamination reaction, coupling reactions were carried out with L-glutamate dehydrogenase (GDH)/formate dehydrogenase (FDH) and AspAT. The coupling reaction dramatically increased the yields of both target compounds. 135 mM of L-Tle (>99% ee)was produced from 150 mM corresponding keto acid in BCATen/GDH/FDH coupling reaction with 90% conversion. In addition, 90.5 mM L-HAG (>99% ee) was produced from 100 mM corresponding keto acid in BCATes/AspAT coupling reaction using recombinant whole-cells. (C) 2010 Elsevier B.V. All rights reserved.