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

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Abstract

Objectives

To identify a robust NADP+ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties.

Results

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.

Conclusions

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.

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Acknowledgements

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.

Supporting information

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.

Author information

Authors and Affiliations

  1. Department of Biology, Faculty of Science, Van Yuzuncu Yil University, Tusba, 65080, Van, Turkey

    Saadet Alpdağtaş

  2. Department of Bioengineering, Faculty of Chemistry and Metallurgy, Yildiz Technical University, Esenler, 34210, Istanbul, Turkey

    Saadet Alpdağtaş & Sevil Yücel

  3. Department of Biology, Anadolu University, Tepebaşı, 26470, Eskişehir, Turkey

    Handan Açelya Kapkaç

  4. U.S. Department of Agriculture, Renewable Product Technology Research Unit, National Centre for Agricultural Utilization Research, Peoria, IL, USA

    Siqing Liu

  5. Department of Bioengineering, Gebze Technical University, Gebze, 41400, Kocaeli, Turkey

    Barış Binay

Authors
  1. Saadet Alpdağtaş
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  2. Sevil Yücel
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  3. Handan Açelya Kapkaç
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  4. Siqing Liu
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  5. Barış Binay
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Corresponding author

Correspondence to Barış Binay.

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The authors declare that they have no conflict of interest.

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Alpdağtaş, S., Yücel, S., Kapkaç, H.A. et al. Discovery of an acidic, thermostable and highly NADP+ dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929. Biotechnol Lett 40, 1135–1147 (2018). https://doi.org/10.1007/s10529-018-2568-6

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  • DOI: https://doi.org/10.1007/s10529-018-2568-6

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