Elimination of a Free Cysteine by Creation of a Disulfide Bond Increases the Activity and Stability of Candida boidinii Formate Dehydrogenase

Abstract

NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) is an industrial enzyme widely used for NADH regeneration. However, enzyme inactivation caused by the oxidation of cysteine residues is a flaw of native FDH. In this study, we relieved the oxidation of the free cysteine of FDH from Candida boidinii (CboFDH) through the construction of disulfide bonds between A10 and C23 as well as I239 and C262. Variants A10C, I239C, and A10C/I239C were obtained by the site-directed mutagenesis and their properties were studied. Results showed that there were no significant changes in the optimum temperature and pH between variants and wild-type CboFDH. However, the stabilities of all variant enzymes were improved. Specifically, the CboFDH variant A10C (A10Cfdh) showed a significant increase in copper ion resistance and acid resistance, a 6.7-fold increase in half-life at 60°C, and a 1.4-fold increase in catalytic efficiency compared with the wild type. Asymmetric synthesis of l-tert-leucine indicated that the process time was reduced by 40% with variant A10Cfdh, which benefited from the increase in catalytic efficiency. Circular dichroism analysis and molecular dynamics simulation indicated that variants that contained disulfide bonds lowered the overall root mean square deviation (RMSD) and consequently increased the protein rigidity without affecting the secondary structure of enzyme. This work is expected to provide a viable strategy to avoid the microbial enzyme inactivation caused by the oxidation of the free cysteine residues and improving their performances.IMPORTANCE FDH is widely used for NADH regeneration in dehydrogenase-based synthesis of optically active compounds to decrease the cost of production. This study highlighted a viable strategy that was used to eliminate the oxidation of free cysteine residues of FDH from Candida boidinii by the introduction of disulfide bonds. Using this strategy, we obtained a variant FDH with improved activity and stability. The improvement of activity and stability of FDH is expected to reduce its price and then further to decrease the cost of its application.

A Novel 3-Phytosterone-9α-Hydroxylase Oxygenation Component and Its Application in Bioconversion of 4-Androstene-3,17-Dione to 9α-Hydroxy-4-Androstene-3,17-Dione Coupling with A NADH Regeneration Formate Dehydrogenase

“…The optimal temperature for KshB activity is 30 °C, and the pH optimal of KshB is 7.0. As reported in a previous work, the optimal pH and temperature conditions of FDH used in this work were approximately from pH 6.5 to 8.5 and 37 °C, respectively [29]. To make the enzymes play more comparative roles in the coupled reaction, the biocatalytic conditions should be optimized.…”

“…The plasmids were then transformed into E. coli BL21 (DE3) cells using the calcium chloride method. Modified FDH was obtained from our laboratory [29]. Luria-Bertani (LB) medium containing peptone (10 g/L), yeast extract (5 g/L), and NaCl (10 g/L) was used for strain cultivation, and kanamycin (Km, 100 mg/mL) or ampicillin (Amp, 200 mg/mL) was added when needed.…”

Asp305Gly mutation improved the activity and stability of the styrene monooxygenase for efficient epoxide production in Pseudomonas putida KT2440

“…Three clear bands with a molecular weight of about 45 kDa for StyA, 19 kDa for StyB and 43 kDa for formate dehydrogenase (FDH: EC 1.2.1.2), respectively, were observed after the 6× His tagged purified protein was analyzed by 15% SDS-PAGE. The NAD + -dependent FDH was expressed in this study for NADH regeneration in order to avoid the need for addition of external NADH which is required for the efficient conversion of the substrate by SMO [34]. On the other hand, this homodimeric enzyme could catalyze the oxidation of formate ion to carbon dioxide.…”

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli

“…Among them, the specific activity of leucine dehydrogenase from Planifilum fimeticola are also significantly better than those of other enzymes, which has great application potential. In addition, using whole cells as a biocatalyst, the formate dehydrogenase ( Cb FDH) from Candida boidinii ( Slusarczyk et al, 2000 ; Zheng et al, 2016 ) and Pf LeuDH are coexpressed to achieve the regeneration of reducing coenzyme (NADH). As shown in Scheme 1 , the process of preparing L -Tle by whole-cell catalysis of TMP was preliminarily studied, which laid a solid foundation for the realization of low-cost and large-scale biosynthesis of L -Tle.…”