Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes

Abstract

A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60 degrees C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30 degrees C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.

Ethanol Production from Enzymatically Treated Dried Food Waste Using Enzymes Produced On-Site

“…The rich and diverse composition of food wastes-which includes proteins, starch, insoluble carbohydrates, and lipids-can facilitate the production of a diverse range of enzymes. Some characteristic categories of enzymes that have already been produced from food wastes are amylases [23,24], proteases [25,26], ligninocellulolytic enzymes [27,28], lipases [29,30], and pectinolytic enzymes [31,32].…”

Optimization of cultural conditions for the production of an extracellular protease by Pseudomonas species

“…Proteases producing bacteria are widely isolated from soil with high protein content (Sing et al, 2001). Genus Pseudomonas a gram-negative bacterium that predominantly produces alkaline proteolytic enzymes and the proteases has been purified (Jellouli et al, 2008).…”

Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes

“…PCR with the primer pair ME4F/ME4R yielded a 1,988 bp fragment carrying a 1,497 bp ORF encoding a polypeptide composed of 498 amino acid residues (AB618054). The N-terminal amino acid residue of mature ME-4 protease (Cheng et al 2009) is located at position 198 of the deduced amino acid sequence, which suggested that ME-4 protease is synthesized as a pre-pro-protein, as previously observed for elastases from P. aeruginosa (Schad et al 1987;Jellouli et al 2008). The sequence of the first 20 N-terminal residues of the 197 amino acid pre-pro-region was similar to the signal peptide sequences of Pseudomonas elastases (BAB79621, AAG07111, and AAZ78231), and was followed by a 177 amino acid pro-peptide (Bever and Iglewski 1998;Gupta et al 2008).…”

“…The probe hybridized to 3.2 kb PstI, 5.0-kb EcoRI, and 6.0 kb ClaI fragments (data not shown), which indicated that strain ME-4 carries one gene encoding pre-pro-LasB_ME4. These findings indicate that native ME-4 protease might be identical to one of the Pseudomonas elastases (Gupta et al 2008;Jellouli . 2008).…”