Enzymes in organic synthesis: use of subtilisin and a highly stable mutant derived from multiple site-specific mutations
作者:Chi Huey Wong、S. T. Chen、William J. Hennen、Jeffrey A. Bibbs、Y. F. Wang、Jennifer L. C. Liu、Michael W. Pantoliano、Marc Whitlow、Philip N. Bryan
DOI:10.1021/ja00159a006
日期:1990.1
wild-type enzyme to organic synthesis has been demonstrated in the regioselective acylation of nucleosides in anhydrous dimethylformamide (with 65-100% regioselectivity at the 5'-position), in the enantioselective hydrolysis of N-protected and unprotected common and uncommon amino acid esters inmore » water (with 85-98% enantioselectivity for the L-isomer), and in the synthesis of di- and oligopeptides
发现通过六个位点特异性突变(Met50Phe、Gly169Ala、Asn76Asp、Gln206Cys、Tyr2l7Lys 和 Asn2l8Ser)衍生自枯草杆菌蛋白酶 BPN' 的枯草杆菌蛋白酶突变体(枯草杆菌蛋白酶 8350)在水溶液中比野生溶液中的稳定性高 100 倍在无水二甲基甲酰胺中比野生型稳定 50 倍。使用酯、硫酯和酰胺底物以及过渡态类似物抑制剂 Boc-Ala-Val-Phe-CFsub 3} 的动力学研究表明,野生型和突变型酶具有非常相似的特异性和催化作用特性。野生型酶的抑制常数 (Ki = 5.0 mu}M) 是突变酶 (Ki = 1.1 mu}M) 的约 5 倍,表明突变酶与反应过渡态的结合比野生型酶。该结果与观察到的相应酯和酰胺底物的速率常数一致;即,突变体的 ksub cat}/Ksub m} 值大于野生型酶的值。突变酶和野生型酶在有机合成中的应用已在无