Screening Of A Strain Producing Cold-adapted Protease And The Study On Fermentation Conditions And Enzymatic Properties

Protease is widely used in the industry. The mesophilic enzyme is used basically at present and its optimal activity temperature is about50℃. While the optimal activity temperature of cold-adapted protease is below40℃and it has thermal sensitivity, it has irreplaceable advantages than mesophilic enzyme. The meat could be tenderized in the process of refrigerated storage or at room temperature and the enzyme can be easily inactivated. Therefor, cold-adapted protease has a good potential for application in meat tenderized. A strain N-8producing cold-adapted protease had been isolated and identified from the frozen soil of Tianshan Mountains. The yield of cold-adapted protease was enhanced by UV and HNO2treatment. The conditions that producing cold-adapted protease were optimized and the properties of enzyme were researched. The results were reported as follows:By means of the morphological, physiologica, biochemical properties and16S rDNA sequence analysis, the strain N-8was identified as Pseudomonas sp. and named Pseudomonas sp. N-8. The optimal temperature of Pseudomonas sp. N-8. was20℃.A mutant strain UN-18producing the cold-adapted protease was gained from the initial strain N-8treated with UV and HNO2compound mutation. The results showed that the optimal conditions were as follows:Using30W UV lamp treated60s, following0.1mol/L nitrite for3min. A high and stable yield mutant UN-18was achieved. The enzyme productivity of the strain UN-18was43.9U/mL and it had increased by38.3%compared with that of the initial strain. After being propagated for ten generations, the yield of cold-adapted protease remained around90%and it proved UN-18had genetic stability.Medium and fermentation conditions were optimized by single factor and orthogonal experiments. The optimal culture medium components were determined(g/L):glucose15, yeast powder8, casein20, KH2PO40.4, pH7.0. The optimal culture conditions were inoculation time24h, initial pH7.0, inoculation volume4%, liquid volume30mL/250mL. Under the optimal conditions, the enzyme activity was47.1U/mL and it had increased by48.2%.Using ammonium sulfate precipitation step by step for enzyme purification, it showed that the optimal pH and temperature were7.0and25℃, respectively. For the thermal stabilty of the cold-adapted protease, it was stable over the range of25-45℃.The test of pH stable indicated that the enzyme was more stable within pH6.5-8.0. After55℃and30min treatment, the relative enzyme activity only retained24%. The protease activity was inhibited by K+、Cu2+、Ca2+and Mn2+, but was stimulated by Mg2+、Fe2+、Na+and Zn2+. The research on the salt resistance showed that the enzyme activity retained98%and89%under the NaCl of20g/L and40g/L, respectively.It proved that the enzyme had tolerance to NaCl.