Characterization of a novel thermotolerant NAD+-dependent formate dehydrogenase from hot climate plant cotton (Gossypium hirsutum L.)

NAD+-dependent formate dehydrogenases (FDH, EC 1.2.1.2), providing energy to the cell in methylotrophic microorganisms, are stress proteins in higher plants and the level of FDH expression increases under several abiotic and biotic stress conditions. They are biotechnologically important enzymes in NAD(P)H regeneration as well as CO2 reduction. Here, the truncated form of the Gossypium hirsutum fdh1 cDNA was cloned into pQE-2 vector, and overexpressed in Escherichia coli DH5 alpha-T1 cells. Recombinant GhFDH1 was purified 26.3-fold with a yield of 87.3%. Optimum activity was observed at pH 7.0, when substrate is formate. Kinetic analyses suggest that GhFDH1 has considerably high affinity to formate (0.76 +/- 0.07 mM) and NAD+ (0.06 +/- 0.01 mM). At the same time, the affinity (1.98 +/- 0.4 mM) and catalytic efficiency (0.0041) values of the enzyme for NADP(+) show that GhFDH1 is a valuable enzyme for protein engineering studies that is trying to change the coenzyme preference from NAD to NADP which has a much higher cost than that of NAD. Improving the NADP specificity is important for NADPH regeneration which is an important coenzyme used in many biotechnological production processes. The Tm value of GhFDH1 is 53.3 degrees C and the highest enzyme activity is measured at 30 degrees C with a half-life of 61 h. Whilst further improvements are still required, the obtained results show that GhFDH1 is a promising enzyme for NAD(P) H regeneration for its prominent thermostability and NADP(+) specificity

Discovery of an acidic, thermostable and highly NADP+ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929

WOS: 000434458100013PubMed ID: 29777512To identify a robust NADP(+) dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties. A new NADP(+) dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6 x histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8-6.2). Its optimum temperature was 60 A degrees C and 50 A degrees C with NADP(+) and NAD(+), respectively and its T-m value was 78 A degrees C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K-M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP(+)), NADP(+) and NAD(+), respectively. An NADP(+) dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures.Research Fund of the Yildiz Technical University [FDK-2018-3331]This work was supported by Research Fund of the Yildiz Technical University (Project Number: FDK-2018-3331) and special thanks to Dr. Siqing Liu from USDA ARS for providing chromosomal DNA of Lactobacillus buchneri NRRL B-30929