Characterization Analysis And Cloning, Expression Of Elastase Of Aeromonas Hydrophila

Elastase is a kind of protease characteristic of degrading elastin specially. For its special enzymatic characteristic, the enzyme has broad application in medical therapy, food processing and functional cosmetics. Furthermore it is paid close attention in sewage treatment. At present elastase mostly comes from animal pancreas. Elastase is costly for limited material. Elastase production from microbe culture is promising because of its advantage in high productivity , simple production process and so on.1. The optimal condition of elastase production in Aeromonas hydrophila strain J-1 (AhJ-1) was conducted. The effects of carbon sources, nitrogen sources, temperature, inatial pH, metal ion and surface activity reagent on the elastase production of AhJ-1 were investigated. The optimal condition was obtained by orthogonal experiment of three factors and three levels. The optimal medium was as follows: 5g/L tryptose, 5g/L surose, 2.5g/L NaCl, 2.0g/L K₂HPO₄, Tween-80 1.0g/L. In this case, AhJ-1 was cultured at 28℃ for 48h on a shaker at a speed of 100r·min^-1.2. Elastase produced by Aeromonas hydrophila strain J-1was purified and characterized.Elastase was successfully purified by ultrafiltration through 10kDa nominal molecular weight cut-off membrane ,30%-60% ammnium sulfate fractionation, anion-exchange chromatography and sephaceryl chromatography. SDS-PAGE analysis showed that elastase from Aeromonas hydrophila J-1 was 33kDa monomeric protein. The optimum pH and temperature of purified elastase was pH 8.5 and 37℃,respectively. Presence of Ca²⁺ did not have the distinct influence on the themostability of enzyme activity. The enzyme degraded broadly elastin,casein,gelatin,BSA, but not keratin. The activity of enzyme could be inhibited by metalloprtease inhibitor, such as EDTA and OPA, but not by serine protease inhibitor PMSF. Additionally, the activity of enzyme inhibited by EDTA could not be restored by adding Ca²⁺. The results suggested the enzyme was Zn²⁺/Fe²⁺ metallo -protease.