Expansion of application of biocatalysts by introduction of organic solvents and non-natural substrates

通过引入不寻常的条件，如有机溶剂和非天然底物，挖掘生物催化剂的潜在能力，扩大生物催化剂的用途是很重要的。本研究发现卤代苯丙氨酸作为嗜热分解酶的非天然底物，成功地进行了含卤代苯丙氨酸的非天然多肽的对映选择性合成。这些非天然多肽有望具有新的生理活性。此外，我们尝试了在有机合成中起重要作用的有机硅化合物的生物转化，并成功地实现了含有立体硅原子的伯醇的光学拆分。研究还表明，伯羟基硅烷的脱氢反应仅在乙醇脱氢酶(HLADH)的作用下进行，并通过NAD^+的原位再生催化了一定量的NAD^+。也就是说，β-羟基硅烷被HLADH脱氢为β-羰基硅烷，后者自发降解为醛，为HLADH再生NAD^+提供底物。这些结果表明，有机硅化合物的特殊特性的应用使得构建高效的生物转化系统成为可能，而这是传统底物不可能实现的。另外，在分批加料体系中使用非天然酸酸酐作为酰基供体可以避免水的积聚，这是加氢催化酯化的一大问题。我们还成功地构建了一种新型的非载体生物反应器，由于生物催化剂不溶于有机溶剂，因此引入了有机溶剂作为特殊条件，不需要传统的固定化。

It is important to draw out the latent abilities and expand the use of biocatalysts by introducing unusual conditions such as organic solvents and non-natural substrates. We found in this study that halophenylalanines served as non-natural substrates of thermolysin, and the enantioselective synthesis of non-natural peptides containing halophenylalanines was successfully carried out. These non-natural poptides are expected to have novel physiological activities. Furthermore, we tried the bioconversion of organosilicon compounds which play important roles in organic synthesis, and succeeded in the optical resolution of primary alcohols containing stereogenic silicon atom. It was also demonstrated that the dehydrogenation of primary beta-hydroxysilanes proceeded enantioselectively with only alcohol dehydrogenase (HLADH) and a catalytic amount of NAD^+ due to in-situ NAD^+ regeneration. That is, beta-hydroxysilanes were dehydrogenated by HLADH to beta-carbonylsilanes, which were spontaneously degraded into aldehydes, which served substrates for HLADH to regenerate NAD^+. These results indicated that the application of specific features of organosilicon compounds enabled the construction of the efficient bioconversion systems, being impossible with conventional substrates. In addition, it was found that the use of non-natural acid anhydrides as acyl donors in fed-batch system could avoid the accumulation of water which is a big problem of hydrolasecatalyzed esterification. We were also successful in constructing a novel non-support bioreactor needing no conventional immobilization by introducing organic solvent as unusual condition because biocatalysts are not soluble in organic solvents.