Discovery of an acidic, thermostable and highly NADP+ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929
To 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 × 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 °C and 50 °C with NADP+ and NAD+, respectively and its Tm value was 78 °C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The KM 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.
KeywordsAcidic formate dehydrogenase Biochemical and kinetic characterization Highly NADP+ dependent formate dehydrogenase Lactobacillus buchneri NRRL B-30929 Solvent stable Thermostability
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.
Supplementary Fig. 1—MALDI-TOF MS analysis of His6-LbFDH
Supplementary Fig. 2—The Differential Scanning Calorimetry (DSC) result of LbFDH
Supplementary Fig. 3—Effect of metal ions on the activity of recombinant LbFDH.
Supplementary Fig. 4—Organic solvent stability of LbFDH in 20% DMSO and acetonitrile for 6 h.
Supplementary Fig. 5—Effect of different storage temperatures on the stability of LbFDH.
Supplementary Fig. 6 a, b, c, d—Michaelis–Menten curves for NAD+ and NADP+ dependent reaction of LbFDH.
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Conflict of interest
The authors declare that they have no conflict of interest.
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