Increasing specific activity of NAD+-dependent Q105R mutant of candida methylica formate dehydrogenase

NAD+-dependent FDHs (EC 1.2.1.2) use sodium formate as the substrate and catalyze convertion of sodium formate ion into CO2. At the same time, NAD+ is reduced into NADH form. Because of low redox potential, NAD+-dependent FDHs are mostly used enzymes in the industrial area and they provide NADH regeneration for the synthesiz of optical chiral compounds. NAD+-dependent FDHs have some disadvantages such as low specific activity and they can be inactivated at high temperatures especially when they are used in the industrial area. Recently, protein engineering studies are aimed to overcome these disadvantages. In this study, we used site-directed mutagenesis method for increasing specific activity of Q105R mutant Candida methylica FDH whose thermostability was increased. In the previous studies, K328V mutation has been introduced into the Q105R mutant of Candida methylica FDH due to the specific activity of K328V mutation was increased at Candida boidinii FDH. First we have designed primer that codes valine residue at 328th position. Site directed mutagenesis method has been performed by using the designed primers. Then PCR products have been digested by DpnI enzyme which cuts methylated adenine site. Digested DNA fragments carrying the required double mutant (K328V on Q105R) have been cloned into pQE-2 vector then transformed into BL-21 chemical competent cells. Three random colonies have been chosen and purified at the end of the transformation. Purified plasmids have been sent for DNA sequence analysis. The accuracy of mutation has been checked by comparing with DNA sequence. Mutant K328V cells have been cultivated for kinetic measurement and then protein purification has been carried out for cultivation of the cells. Purified protein has been run on SDS-PAGE and chosen fractions have been collected for kinetic measurement analysis. The kinetic measurement has been carried out at 25 oC at 340 nm wavelength with constant NAD+ concentration (4 mM) and changing substrate concentrations (0-80 mM). To calculate Km and kcat value, Hyper32 programme has been used. Calculated Km and kcat value reveals that K328V mutation decreases specific activity and Km value.