Study On Optimization Of Production Conditions And Properties Of Low Temperature Protease From Chilling Tolerance

The cold-adapted protease has been widely studied for its high activity in lower temperature as well as potential high values for application on detergence, food, milk, medicine and cosmetics et al, which needs enzymatic catalysis at low temperature. Therefore, cold-adapted protease has aroused wide research and development by scientists in the world.In this paper, low-temperature protease-producing bacteria were screened from25strains of psychrotroph which were preserved in laboratory by using the method of flat proteolysis casein, and4strains were identified as protease-producing bacteria with high capability of producing cold-active protease, which were named H8, W7, Z5and Z4. The protease activity produced by strain H8, W7, Z5and Z4were respectively54.6±1.6U-mL-1、88.1±1.2U·mL-1、64.6±2.1U·mL-1and32.5±1.9U·mL-1. The strain W7were proved to be with the highest protease activity, which was identified as a strain of Pseudomonas sp. based on morphological, physiological characteristics and phylogenetic analysis of16S rDNA. The strain W7was named as Pseudomonas sp. W7.In order to optimize the emzyme producing condition of protease-producing bacteria W7, the influence of the various carbon and nitrogen sources, complex nitrogen sources and inorganic salts on the W7protease-production was determined by the single factor experiment. Then the Plackett-Burman design was used to select3important factors from7factors which can affect protease-production. They were glucose, peptone and MgSO4in culture medium. The optimum culture components obtained by the response surface methodology (RSM) were glucose4.25g·L-1, peptone7.00g·L-1and MgSO40.14g·L-1. After the optimization, the yield of the protease increased significantly and reached to183.9U·mL-1. Finally, components of the optimized medium were determined as glucose4.25g·L-1, peptone7.00g·L-1, casein4.00g·L-1, K2HPO45.00g·L-1, KH2PO41.00g·L-1, CaCl20.26g·L-1, MgSO40.14g·L-1. The fermentation condition of protease-producing bacteria W7was determined by the orthogonal experiment. The optimized fermentation conditions were established as follow:cultural temperature20℃, the initial pH7.0, the volumes size3%, the aeration50mL, and the rotate speed100rpm. Under the optimum conditions, the maximum protease activity was193.2U`mL-1after48h. The strain W7had a relatively stable protease-production from the fourth generation to the tenth generation.The protease characteristic was primarily studied. The results showed that the optimal reaction temperature was35℃, and the protease can remain higher activity between0℃and40℃. The optimal pH of the protease is9.0, it was stable at range from pH5.0to pH10.0. Mn2+and Mg2+can promote the enzyme activity, Cu2+, Hg2+, Al3+, Pb2+and Ca2+can partly inhabit its activity. The EDTA can obviously inhabit its activity. But the5%of the acetone can activate the enzyme activity.To psychrotrophic bacteria W7, the change of temperature was detected to influent enzyme production. Strain W7was cultured and zymography analysized at different temperature of15℃,20℃and25℃The results showed that the psychrotroph W7secreted two different proteases at20℃and25℃. They were named as cold-adapted protease I (Pro I) and cold-adapted protease II (Pro II). The molecular of Pro I was larger than Pro II. But only the cold-adapted protease I was screted at15℃. And Pro I played a predominantly role at low-temperature.