NADP+ production using thermostable NAD+ kinase of Corynebacterium flaccumfaciens AHU-1622

Abstract

A sonicate of Corynebacterium flaccumfaciens AHU-1622 had the highest NAD+ kinase activity (1.22 mU/mL culture broth) of the strains of bacteria we investigated. This enzyme was thermostable, with activity maintained at 50 °C for 1 h. This treatment inactivated phosphatase activity. Resting cells of the bacterium also had NAD+ kinase activity when treated at 60 °C for 30 min with 0.2% Triton X-100. NADP+ production was achieved using 8 μmol NAD+, 8 μmol ATP, 16 μmol MgCl2, 1.6 μmol NaN3, and 12 mU NAD+ kinase (0.1 g of permeabilized wet cells) in 2 mL of 0.1 M phosphate buffer, pH 7.5. The conversion ratio of NADP+ from NAD+ was 75% after 10 h of incubation at 50 °C, and the amount of accumulated NADP+ was 3 μmol/mL of reaction mixture. The NAD+ kinase activity of the permeabilized cells was stable and did not decrease after repeated use.