Prokaryotic Expression and Characterization of a Thermo-stable Lysine Aminopeptidase

Abstract:

Aminopeptidase produced by Pseudomonas aeruginosa NJ-814 shows excellent thermo-stable feature. The gene encoding this peptidase was cloned and symbolized as lap in this paper. Sequencing results revealed lap is consists of 1461 nucleotides coding 486 amino acids. Recombinant gene pET-42a-lap was constructed by connecting lap with pET-42a（+） expression vector. Recombinant strain BLAP was generated by transferring pET-42a-lap into E.coil BL21（DE3），BLAP successfully express aminopeptidase after cultivated in proper condition. The recombinant aminopeptidase was purified 4.7-fold to homogeneity with a recovery of 83.5 % from cell free extract using Ni2+-NAT affinity column chromatography. The properties of the recombinant aminopeptidase were investigated and the results showed that the optimal reaction pH and temperature were pH 9.0 and 80 ℃ respectively，and it was extraordinary stable within pH 7.0～9.5 or below 70 ℃.According to substrate specificity analysis and enzymatic reaction kinetics，this recombinant enzyme belongs to lysine aminopeptidase，which is same as P. aeruginosa NJ-814 aminopeptidase reported before.