Saadet Alpdağtaş1,2, Sevil Yücel2, Handan Açelya Kapkaç3, Siqing Liu4, Barış Binay5. 1. Department of Biology, Faculty of Science, Van Yuzuncu Yil University, Tusba, 65080, Van, Turkey. 2. Department of Bioengineering, Faculty of Chemistry and Metallurgy, Yildiz Technical University, Esenler, 34210, Istanbul, Turkey. 3. Department of Biology, Anadolu University, Tepebaşı, 26470, Eskişehir, Turkey. 4. U.S. Department of Agriculture, Renewable Product Technology Research Unit, National Centre for Agricultural Utilization Research, Peoria, IL, USA. 5. Department of Bioengineering, Gebze Technical University, Gebze, 41400, Kocaeli, Turkey. binay@gtu.edu.tr.
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.
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.
Authors: Mehmet M Çakar; Jouni Ruupunen; Juan Mangas-Sanchez; William R Birmingham; Deniz Yildirim; Ossi Turunen; Nicholas J Turner; Jarkko Valjakka; Barış Binay Journal: Biotechnol Lett Date: 2020-06-16 Impact factor: 2.461
Authors: Liliana Calzadiaz-Ramirez; Carla Calvó-Tusell; Gabriele M M Stoffel; Steffen N Lindner; Sílvia Osuna; Tobias J Erb; Marc Garcia-Borràs; Arren Bar-Even; Carlos G Acevedo-Rocha Journal: ACS Catal Date: 2020-06-08 Impact factor: 13.084