Cloning and expression in E. coli of an organic solvent-tolerant and alkali-resistant glucose 1-dehydrogenase from Lysinibacillus sphaericus G10.

ABSTRACT

The gene gdh encoding an organic solvent-tolerant and alkaline-resistant NAD(P)-dependent glucose 1-dehydrogenase (LsGDH) was cloned from Lysinibacillus sphaericus G10 and expressed in Escherichia coli. The recombinant LsGDH exhibited maximum activity at pH 9.5 and 50 °C. LsGDH displayed high stability at a wide pH ranging from 6.5 to 10.0 and was stable after incubation at 30 °C for 1 week in 25 mM sodium phosphate buffer (pH 6.5) in the absence or presence of NaCl. The activity of LsGDH was enhanced by Li+, Na+, K+, NH4+, Mg2+, and EDTA at pH 8.0. LsGDH exhibited high tolerance to 60% DMSO, 30% acetone, 30% methanol, 30% ethanol, 10% n-propanol, 30% isopropanol, 60% n-hexanol and 30% n-hexane. The relationship between stability and chain length of the alcohols fit a Gaussian distribution model (R2≥0.94), and demonstrated lowest enzyme stability in C4-alcohol. The results suggested that LsGDH was potentially useful for coenzyme regeneration in organic solvents or under alkaline conditions.