A single‐point mutation enables lactate dehydrogenase from Bacillus subtilis to utilize NAD+ and NADP+ as cofactor

Abstract

AbstractThe NAD+‐dependent lactate dehydrogenase from Bacillus subtilis (BsLDH) catalyzes the enantioselective reduction of pyruvate to lactate. BsLDH is highly specific to NAD+ and exhibits only a low activity with NADP+ as cofactor. Based on the high activity and good stability of LDHs, these enzymes have been frequently used for the regeneration of NAD+. While an application in the regeneration of NADP+ is not sufficient due to the cofactor preference of the BsLDH. In addition, NADP+‐dependent LDHs have not yet been found in nature. Therefore, a structure‐based approach was performed to predict amino acids involved in the cofactor specificity. Methods of site‐saturation mutagenesis were applied to vary these amino acids, with the aim to alter the cofactor specificity of the BsLDH. Five constructed libraries were screened for improved NADP+ acceptance. The mutant V39R was identified to have increased activity with NADP+ relative to the wild type. V39R was purified and biochemically characterized. V39R showed excellent kinetic properties with NADP(H) and NAD(H), for instance the maximal specific activity with NADPH was enhanced 100‐fold to 90.8 U/mg. Furthermore, a 249‐fold increased catalytic efficiency was observed. Surprisingly, the activity with NADH was also significantly improved. Overall, we were able to successfully apply V39R in the regeneration of NADP+ in an enzyme‐coupled approach combined with the NADP+‐dependent alcohol dehydrogenase from Lactobacillus kefir. We demonstrate for the first time an application of an LDH in the regeneration of NADP+.