Milker Sofia, Goncalves Leticia C P, Fink Michael J, Rudroff Florian
Institute of Applied Synthetic Chemistry, TU Wien, Vienna, Austria.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, United States.
Front Microbiol. 2017 Nov 13;8:2201. doi: 10.3389/fmicb.2017.02201. eCollection 2017.
This paper describes the measurement and analysis of activity and stability of cyclohexanone monooxygenase from sp. NCIMB 9871 (CHMO), a model Baeyer-Villiger monooxygenase, in the recombinant host . This enzyme was often described as poorly stable , and has recently been found to deactivate rapidly in the absence of its essential cofactors and antioxidants. Its stability was scarcely studied, so far. Under conditions common for the overexpression of CHMO we investigated the ability of the host to support these properties using metabolomics. Our results showed that failed to provide the intracellular levels of cofactors required to functionally stabilize the enzyme, although the biocatalyst was produced in high concentration, and was invariably detected after protein synthesis had stopped. We thus infer that biotechnological applications of CHMO with this host relied on a residual activity of approximately 5-10%. Other microorganisms might offer a more efficient solution for recombinant production of CHMO and related enzymes.
本文描述了来自菌株NCIMB 9871的环己酮单加氧酶（CHMO，一种典型的拜耳-维利格单加氧酶）在重组宿主中的活性和稳定性的测定与分析。这种酶常被描述为稳定性较差，最近发现它在缺乏必需辅因子和抗氧化剂的情况下会迅速失活。到目前为止，其稳定性几乎未被研究。在CHMO过表达的常见条件下，我们使用代谢组学研究了宿主支持这些特性的能力。我们的结果表明，尽管生物催化剂以高浓度产生，并且在蛋白质合成停止后始终能检测到，但宿主未能提供功能上稳定该酶所需的细胞内辅因子水平。因此我们推断，使用这种宿主进行CHMO的生物技术应用依赖于大约5 - 10%的残余活性。其他微生物可能为CHMO及相关酶的重组生产提供更有效的解决方案。
