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首页> 外文期刊>BMC Biotechnology >New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme
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New biotechnological perspectives of a NADH oxidase variant from Thermus thermophilus HB27 as NAD+-recycling enzyme

机译：嗜热栖热菌HB27的NADH氧化酶变异体作为NAD +循环酶的新生物技术观点

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Background The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH -oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds. Results The NADH -oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols. Conclusion We have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac -1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.
机译：背景技术使用烟酰胺再循环系统的生物转化的数量呈指数增长。因此，生物催化中的当前挑战之一是开发和优化更简单，有效的辅因子回收系统。一种再生NAD + 库的有前途的方法是使用NADH-氧化酶，将氧还原为过氧化氢，同时将NADH氧化为NAD + 。这类酶可用于手性底物的不对称还原，以获得对映体纯的化合物。结果此处介绍的NADH-氧化酶（NOX）是一种黄素酶，需要外源FAD或FMN才能达到其最大速度。有趣的是，通过在限制浓度的黄素辅因子中在高温（80°C）下孵育，该酶被6倍超活化，这种变化即使在低温（37°C）下也保持稳定。尽管与嗜热源分离，但该超活化形式在低温下仍具有较高的比活性（37.5 U / mg）。将NOX固定在由乙醛基激活的琼脂糖上可产生最稳定的酶制剂（比高活化可溶性酶稳定6倍）。固定化的衍生物在高溶剂浓度灭活后能够在生理条件下重新活化。灭活/再激活周期可以重复至少三遍，在所有情况下，在重新激活步骤之后，可以恢复全部的NOX活性。该固定化催化剂作为嗜热醇脱氢酶的循环伙伴存在，以进行动力学拆分仲醇。结论我们设计，开发和表征了一种多相且坚固的生物催化剂，该催化剂已被用作rac -1-苯基乙醇动力学拆分中的再循环伙伴。高稳定性及其可被重新活化的能力使得该生物催化剂在氧化还原生物转化中可高度可再利用以用于辅因子循环。

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