Investigation of novel, nicotinamide adenine dinucleotide phosphate dependent formate dehydrogenase enzymes

Abstract

NADPH dependent oxidoreductases are remarkable biocatalysts for industrial production of chiral chemicals. For in situ regeneration of required expensive cofactors, Formate dehydrogenases (FDHs) are quite important. It is a significant necessity to determine the FDHs which have NADP+ dependency and also multi-directional stability for cofactor recycling process. In this study, it was aimed to get two, new NADP+ dependent FDHs via recombinant DNA technologies and protein engineering methods from natural sources, a pathogenic bacterium Burkholderia dolosa PC543 (BdFDH) and ethanol tolerant bacterium Lactobacillus buchneri NRRL B-30929 (LbFDH), and also investigate the effect of N- and C- terminus His tag extensions on solubility and activity of BdFDH. Two novel NADP+ dependent FDHs from these microorganisms were cloned, purified and characterized with their unique features. The first enzyme is a new, DMSO tolerant formate dehydrogenase, which has dual cofactor specificity and tolerance to acidic conditions and obtained from B. dolosa PC543 (BdFDH). The expression of N- and Cterminus His-tagged BdFDHs were performed, separately and it was determined that the C-terminus His-tagged enzyme was active and soluble whereas the N-terminal version of enzyme was not. The other putative NADP+ dependent formate dehydrogenase gene was obtained from L. buchneri NRRL B-30929. It was determined that LbFDH has high activity at an acidic pH range (pH 4.8-6.2) and at high temperatures. Its Tm value has been obtained as 78 ◦C by differential scanning calorimetry (DSC) as an unusual value for equivalent enzymes. The specific activity of LbFDH (24.6 U/mg) with NADP+ is a beneficial feature among all NADP+ dependent FDHs at 60 ◦C. If these enzymes are compared in terms of its kinetic constants, it can be said that BdFDH has a great interaction with formate ion and LbFDH has a beneficial affinity towards NADP+, due to these capabilities, both of FDHs can be utilized as model enzymes for protein engineering studies. In conclusion, these two novel enzymes with their performance in acidic pH values, DMSO tolerance and coenzyme preference, have great potential to recycle cofactors in biocatalysis of chiral intermediates in various industries.