Isolation And Identification Of Protease-producing Marine Microorganisms And The Fermentation Conditions For Production Of Alkaline Protease

Protease is a protein catalytic hydrolysis of the enzyme, the enzyme catalytic reaction speedfaster, no industrial pollution, are widely used in leather depilation, drink clearing,detergents, cosmetics, and medical treatment and other industries. Protease is widely existsin animal internal organs, in the plant leaf, fruit, and microorganisms. Due to the animaland plant resources are limited, industrial production protease preparations mainly usingmicrobial fermentation preparation, strains currently used in industrial production is mainlyfor the mold, bacteria and a small number of yeast and actinomyces. Marinemicroorganisms have unique physical characteristics, such as salt tolerance, resistance tohigh pressure, low temperature, etc., so the Marine microorganisms will become theimportant source of new bioactive substances. So from Marine microorganisms in thescreening of a new type of high yield strains protease is very meaningful.In this paper a new strain of protease strain has been screened, identification, optimizationof fermentation conditions, the product purification, determination of product propertiessuch as the experimental process and the problems are illustrated. Water samples takenfrom QinHuangdao coastal strains filtration, after early screening and compound screeningto determine one of the strongest capacity of producing protease strain-D5.16s rDNAsequencing analysis of D5and physiological and biochemical identification determine itsfalse alternating belongs to the Pseudoalteromonas sp. Then the D5strain culture mediumof single factor experiment, determined the suitable strains enzyme production of carbonsource, nitrogen source, inorganic species. For the final medium：Peptone10g/L, BeefExtract3g/L, Glucose5g/L、NH4NO30.8g/L、FeCl30.2g/L、MnCl20.2g/L、MgCl20.2g/L、CaCl20.2g/L; The fermentation conditions30oC,24h,150r/min. In this condition toincrease the enzyme activity to1140U/ml。Then with salting out, chromatography andother methods of protease in fermented liquid purification. After purification, D5fungusprotease than to live by the initial65.23U/mg increased to441.87U/mg, increased6.77times, the yield of40.3%. Studied the properties of the purified product, and the resultsshowed that the protease from the D5bacteria as the alkaline protease, the molecularweight of about26kDa, optimum pH9, optimum temperature of35, thermal stability andaccording to the control determine the reaction should be a kind of serine protease.