Purification And Application Of Alkaline Protease From Strain NCB-01

Alkaline protease occupies a very important proportion in industrial enzymes,and is widely used in detergent,food,pharmaceutical,leather,and other industries.Marine is a huge biological treasure trove,and marine proteases have their own uniqueness and diversities because of the distinctiveness and openness of the marine environment.So,marine microorganisms are important sources of new enzyme preparations.The lab obtained a strain 7-5 of producing alkaline protease from Yantai Institute of Coastal Zone Research,CAS（named NCB-01）.It was identified that the strain was belonged to Bacillus of marine.On the basis of the previous studies,this paper purified the alkaline protease from the strain NCB-01,and studied its properties.The hydrolysis conditions were determined,gelatin was hydrolyzed at the optimal conditions of alkaline protease,which provided a basis for further applied research and industrial production.The main results are as follows:（1）This paper takes strain NCB-01 fermented broth as the research object,using the technology such as membrane separation and chromatography to separate of alkaline protease.The path of separation and purification process is:centrifuging the fermentation broth→the primary ultrafiltration separation→the secondary ultrafiltration separation→concentrate→DEAE-Sepharose ion exchange chromatography.After purification,the specific activity by the initial 838.36 U/mg increased to 2320.85 U/mg.The purification fold was 2.77,and the yield was 24.46%.（2）The result of enzymatic properties showed that the optimum temperature of the protease was 60℃and the optimum p H value was 8.It will be obtained a well activity low then 50℃and p H value 7-9.In addition,this protease could be activated by some metal ions such as Fe²⁺、K⁺、Na⁺、Mn²⁺、Ca²⁺、Mg²⁺and inhibited by serine-protease inhibitors（PMSF）,suggesting that this alkaline protease was a serine-protease.Moreover,this alkaline protease also inhibited towards cationic（CTAB）,non-ionic surfactants（Tween-80,Tween-20 and Triton X-100）.This alkaline protease exhibited extreme stability towards Na Cl concentration.The kinetic constants,Km and Vmax,were 1.11mg/m L and 91.74μg/（min·m L）,respectively,suggesting that the alkaline protease has good affinity and high specificity with casein.（3）The optimal hydrolysis conditions of alkaline protease from Bacillus NCB-01 was researched.The conditions of hydrolyzing gelatin were optimized by single factor experiment and the Box-Behnken experimental response surface methodology.The optimal hydrolysis conditions were determined as follows:temperature 55℃,p H 8.0,enzyme concentration 7200U/mg,hydrolysis time 2.5h,under those conditions,the degree of hydrolysis was 46.03%.In addition,through the model test of significance,the order of main effects of factors was:enzyme concentration>enzymolysis temperature>p H.