화학공학소재연구정보센터
Current Microbiology, Vol.47, No.1, 40-45, 2003
Overexpression, purification, and characterization of the recombinant leucine aminopeptidase II of Bacillus stearothermophilus
For expression of Bacillus stearothermophilus NCIB 8924 leucine aminopeptidase II (LAP II) in Escherichia coli regulated by a T5 promoter, the gene was amplified by polymerase chain reaction and cloned into expression vector pQE-32 to generate pQE-LAPII. The His(6)-tagged enzyme was overexpressed in IPTG-induced E. coli M15 (pQE-LAPII) as a soluble protein and was purified to homogeneity by nickel-chelate chromatography to a specific activity of 425 U/mg protein with a final yield of 76%. The subunit molecular mass of the purified protein was estimated to be 44.5 kDa by SDS-PAGE. The temperature and pH optima for the purified protein were 60degreesC and 8.0, respectively. Under optimal condition, the purified enzyme showed a marked preference for Leu-p-nitroanilide, followed by Arg- and Lys-derivatives. The HiS(6)-tagged enzyme was stimulated by Co2+ ions, but was strongly inhibited by Cu2+ and Hg2+ and by the chelating agents, DTT and EDTA. The EDTA-treated enzyme could be reactivated with Co2+ ions, indicating that it is a cobalt-dependent exopeptidase. Taking the biochemical characteristics together, we found that the recombinant LAP II exhibits no important differences from those properties described for the native enzyme.