Abstract
An extracellular alkaline elastase was produced from Pseudomonas aeruginosa CTM50182. It was chromatographically purified using HPLC and Mono Q Sepharose column. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme (called AMPP) was a monomer with a molecular mass of 33,015.18Da. The N-terminal 29 amino acid sequence of AMPP showed high homology with those of Pseudomonas elastases. It showed optimal activity at pH 12 and 80°C and was stable at a pH range of 9–12 after 120h of incubation. Its thermoactivity and thermostability were upgraded in the presence of 5mM Co2+. Its half-life times at 70 and 80°C were 16 and 10h, respectively. It was completely inhibited by ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 1,10-phenanthroline, suggesting that it belongs to the metalloprotease family. AMPP also exhibited high catalytic efficiency, organic solvent-tolerance, and hydrolysis. The lasB gene encoding AMPP was cloned, sequenced, and expressed in Escherichia coli. The biochemical properties of the extracellular purified recombinant enzyme (rAMPP) were similar to those of native AMPP. This organic solvent-stable protease could be considered a potential candidate for application as a biocatalyst in the synthesis of enzymatic peptides.
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