Enzymatic Characterization Of Deep-sea Strain Pseudomonas Aeruginosa And Hydrolysis Application Of Elastase

Ptorease have already been widely applied to food industry,medical treatment,light industry,and other fields, being able to effectively catalyze the hydrolysis of protein. People tend to develop microbial protease with more varieties and functions thanks to the shortage of protease from plants or animals which can not meet our demands anymore. Marine microbes successfully attracted our attention with its tremendous amount and categories, thus, people tend to ask the sea for kinds of new materials. This article was aimed at to isolate novel protease producing bacteria from the deep-sea sediments, and study on the enzyme characteristics and hydrolysis specificity of the protease.Separated a protease-producing strain with high tolerance to salty conditions from the deep sea mud by the method of enrichment with high concentration of sodium chloride. Based on the result of phisical biochemical test and 16 s rDNA sequencing, the strain SWJSS3 was identified as Pseudomonas aeruginosa. Enzymatic activity of the protease produced by the strain was 230 U/m L, which decreased to 40.70% when being applied within the solvent with 15% NaCl for 3 h.Single factor and orthogonal tests were used to improve the enzymatic activity of the protease, which climbed up to 750 U/mL after optimization. The optimized conditions are as followed, inoculation rate 1%, medium volume 10%,original pH 7.5, temperature 30℃, incubation period 32 h;Glucose 0.4%, glycerol 0.8%, yeast extract 1.6%, K2HPO4 0.02%, Tween-80, 0.05%, NaCl1.0%.The crude protease was then purified by ultrafiltration, DEAE-Sepharose fast flow column and Sephadex G-75 column.The protease was finally purified 16.64 times, with the enzymatic activity recovery of 14.86%. The purified protease obtained a single band in the SDS-PAGE analysis, stating that the protease has been electophoretic pure. This protease was testified to be an elastase coded by gene Las B, with the molecular weight of 33.08 kDa. Result of analysis on the optimum conditions for the enzymatic activity of the protease are pH 7.5 and temperature 60℃. It could also be stable within the temperature of 20-50℃, and the pH of 5-10. But it would be effectively inhitied by 5 mM EDTA, DTT, Zn²⁺, Cu²⁺, Fe³⁺, Al³⁺, while be improved by 5 mM Mn²⁺ or being exposed to the organic solvent with logP value greater than 3.9.Analysis result of free amino acids showed that there is a difference on the release regularity of free amino acids between the elastase from microorganism and the commercail. When the time of enzymolysis reached 22 h, the total amount of free amino acids and that of hydrophobic aminoacids were 11707.08 mg/L and 11309.09 mg/L separately, while those of commercial elastase were 6665.75 mg/L and 6384.26 mg/L, stating that both the elastase exhibit good affinity to hydrophobic amino acids（eg. Val, Pro, Leu, Ile） while the effect of enzymolysis by microorganism elastase was better than that of the commercial one. Besides, amount of Leu, Val and Ala increased rapidly during the process, stating that Leu, Val and Ala would be the key locus of the enzymolysis by microorganism elastase.