Department of Bioengineering, Gebze Technical University, 41400, Gebze, Kocaeli, Turkey; Enzyme Consultancy and Identification Center (ETDAM), Gebze Technical University, 41400, Gebze, Kocaeli, Turkey.
Department of Chemistry, Gebze Technical University, 41400, Gebze, Kocaeli, Turkey; Enzyme Consultancy and Identification Center (ETDAM), Gebze Technical University, 41400, Gebze, Kocaeli, Turkey.
Enzyme Microb Technol. 2020 Jun;137:109552. doi: 10.1016/j.enzmictec.2020.109552. Epub 2020 Mar 10.
Nowadays, the use of formate dehydrogenase (FDH, EC 1.17.1.9) is well established as a means of NADH regeneration from NAD via the coupled conversion of formate into carbon dioxide. Recent studies have been reported that specifically Chaetomium thermophilum FDH (CtFDH) is the most efficient FDH catalyzing this reaction in reverse (i.e. using CO as a substrate to produce formate, and thereby regenerating NAD). However, to date the production of active CtFDH at high protein expression levels has received relatively little attention. In this study, we have tested the effect of batch and high cell density fermentation (HCDF) strategies in a small stirred fermenter, as well as the effect of supplementing the medium with casamino acids, on the expressed level of secreted CtFDH using P. pastoris. We have established that the amount of expressed CtFDH was indeed enhanced via a HCDF strategy and that extracellular protease activity was eliminated via the addition of casamino acids into the fermentation medium. On this basis, secreted CtFDH in an active form can be easily separated from the fermentation and can be used for subsequent biotechnological applications.
如今，甲酸脱氢酶（FDH，EC 1.17.1.9）已被广泛应用于通过将甲酸偶联转化为二氧化碳，从 NAD 再生 NADH。最近的研究报告称，特别是嗜热毛壳菌 FDH（CtFDH）是催化此反应（即使用 CO 作为底物生成甲酸，从而再生 NAD）的最有效的 FDH。然而，迄今为止，高表达水平的活性 CtFDH 的生产相对较少受到关注。在本研究中，我们测试了分批发酵和高密度发酵（HCDF）策略在小型搅拌发酵罐中的效果，以及在发酵培养基中添加水解酪蛋白对毕赤酵母表达分泌型 CtFDH 的影响。我们已经确定，通过 HCDF 策略确实可以提高表达的 CtFDH 量，并且通过向发酵培养基中添加水解酪蛋白可以消除细胞外蛋白酶的活性。在此基础上，可从发酵液中轻易分离出具有活性的分泌型 CtFDH，并可用于后续的生物技术应用。
