Pseudomonas aeruginosa A2 elastase: Purification, characterization and biotechnological applications

• Published in: International journal of biological macromolecules 2012-04, Vol.50 (3), p.679-686
• Main Authors: Ghorbel-Bellaaj, Olfa, Hayet, Ben Khaled, Bayoudh, Ahmed, Younes, Islem, Hmidet, Noomen, Jellouli, Kemel, Nasri, Moncef
• Format: Article
• Language: English
• Subjects: agarose; Amino Acid Sequence; Animal Shells - chemistry; Animals; anion exchange chromatography; Biotechnology - methods; Cattle; chitin; Chitin - chemistry; Depilating; Deproteinizaton; dimethyl sulfoxide; DNA; EDTA (chelating agent); Elastase; enzyme activity; Enzyme Stability; ethyl ether; gel chromatography; Gene sequence; genes; Hair Removal; Hydrogen-Ion Concentration; hydrolysis; metalloproteinases; Metals - pharmacology; Molecular Sequence Data; molecular weight; nucleotide sequences; Organic Chemicals - pharmacology; Pancreatic Elastase - chemistry; Pancreatic Elastase - genetics; Pancreatic Elastase - isolation & purification; Pancreatic Elastase - metabolism; proteolysis; Pseudomonas aeruginosa; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Purification; Sequence Analysis; shrimp; solvents; Solvents - pharmacology; Temperature; ultrafiltration
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2012.01.038

► Purification of Pseudomonas aeruginosa A2 protease by a three-step procedure. ► Biochemical characterization of the purified enzyme. ► The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. ► The purified protease was tested for the deproteinization of shrimp shell waste in the process of chitin preparation. ► The application of the enzyme in depilating goat skin was also investigated. An extracellular protease from Pseudomonas aeruginosa A2 grown in media containing shrimp shell powder as a unique source of nutriments was purified and characterized. The enzyme was purified to homogeneity from culture supernatant by ultrafiltration, Sephadex G-100 gel filtration and Sepharose Mono Q anion exchange chromatography, with a 2.23-fold increase in specific activity and 64.3% recovery. The molecular mass of the enzyme was estimated to be 34kDa. Temperature and pH with highest activity were 60°C and 8.0, respectively. The protease activity was inhibited by EDTA suggesting that the purified enzyme is a metalloprotease. The enzyme is stable in the presence of organic solvents mainly diethyl ether and DMSO. The lasB gene, encoding the A2 elastase, was isolated and its DNA sequence was determined. The A2 protease was tested for shrimp waste deproteinization in the process of chitin preparation. The percent of protein removal after 3h hydrolysis at 40°C with an enzyme/substrate (E/S) ratio of 5U/mg protein was about 75%. Additionally, A2 proteolytic preparation demonstrated powerful depilating capabilities of hair removal from bovine skin. Considering its promising properties, P. aeruginosa A2 protease may be considered a potential candidate for future use in several biotechnological processes.