Study On The Enzymatic Properties Of Extracellular Protease From Halobacteriaceae Sp. And Optimization Of Enzyme-producing Conditions

Halophilic archaea grow in brine,natural Saline Lake or high salt pickled foods such as high salt environment,there is a special need and adaptability of NaCl.The major metabolites secreted during microbial growth are extracellular proteases.Extracellular protease secreted by halophilic archaea in the growth process has a certain degree of salt tolerance.The catalytic reaction can also be carried out under high salt conditions.In this paper,we studied the growth and enzyme production characteristics of Halobacteriaceae sp.,and made a preliminary study on the enzymatic properties of extracellular proteases.And the required for the growth of medium was optimized.On this basis,the single factor method and response surface method were used to optimize the culture conditions of extracellular protease.The preliminary study on the hydrolysis ability of extracellular protease was carried out to further determine the enzymatic hydrolysis ability.The main research contents are as follows:1.In this paper,the characteristics and the production process of an extracellular protease secreted by Halobacteriaceae sp.were studied.The strains grew slowly and secreted the maximum protease activity in Exponential phase.The optimum temperature was 60 ?.Further the stability experiments showed that this protease was stable at 40-80 ?.The protease activity was stable at a pH range of6.5-8.5 with an optimum pH of 7.5.Activity of the protease decreased with NaCl.Ca2+ was induced the activity of the protease as well as enhancesd its thermal stability.Mn2+ was inhibited the activity of the protease.The activity of the protease was not afected by K+ and Mg2+.Slight inhibition in the protease activity was observed with1.31 mol/L isopropanol and 1 mmol/L DTNB.Compared with control,the metallo protease inhibitor EDTA inhibited the 57% protease activity when compared with control.The protease activity was completely inhibited by 1 mmol/L PMSF.The results suggesting that the protease is a metal dependency serine-protease.2.The growth and fermentation medium components of Halobacteriaceae sp.were optimized by single factor experiment.The final choice of 0.1% glucose and0.1% peptone as nitrogen source and carbon source fermentation.The optimal concentration was 22% in the range of NaCl concentration by single factor experiment.On the basis of single factor experiment,interaction between the 3 factors of glucose,peptone,sodium chloride are analyzed by using the response surface method.Therefore,the optimal culture conditions for the growth and metabolism of Halobacteriaceae sp.is glucose 0.11%,peptone F403 0.09%,sodium chloride concentration of 23.58%.The predicted maximum enzyme production will reach22.107 U/mL,and the results show that the extracellular protease production reaches19.86U/mL,and the difference is expected to be 0.11%,which indicates that the model is feasible.3.The extraction of myofibrillar protein and myosin solution using spent carp back muscles,and as a substrate for enzymatic hydrolysis of 1h on 60?.After the end of the reaction,boiling bath termination reaction,The solution of the degree of hydrolysis was determined.The results showed that the extracellular protease of Halobacteriaceae sp.could hydrolyze the myofibrillar protein and myosin solution of silver carp,and the crude enzyme has a stronger ability to hydrolyze myosin than myofibrillar protein.Amino acid analysis showed that the content of amino acids in the hydrolysate was increased,which improved the flavor and nutritional value of the food.The results showed that the enzyme can be used in the food fermentation industry,and has potential value for improving the taste and flavor of food.