Screening Of Three Moderately Halophilic Bacteria Which Can Product Protease In Salt Field And Explotration Of Their Fementation Conditions Optimization

There are tens of thousands of large and small salt fields in the vast land of China.Because of its special environment,salt fields have bred a large number of halophilic bacteria adapted to high osmotic pressure and salinity.The unique physiological structure enables halophilic bacteria to secrete many secondary metabolites with special functions,and protease is one of them.Extracellular proteases secreted by halophilic bacteria can survive and catalyze directly in high salinity environment,and can be used in many extreme conditions of protease reaction.In this paper,three strains of protease-producing moderate halophilic bacteria Salinivibrio sp.MK070915,Idiomarina sp.MK070916 and Salinivibrio sp.MK070917 were screened from brine in Changyi,Weifang,Shandong,China.The physiological and biochemical characteristics,growth curve,the effects of environmental factors and carbon and nitrogen sources on enzyme production,enzymatic properties of the enzyme produced by the strain and optimization of fermentation conditions of the strain were explored.The main research contents are as follows:(1)Halophilic bacteria producing protease were screened and their physiological and biochemical characteristics were explored.The results showed that 161 halophilic strains were screened out,and three strains MK070915,MK070916 and MK070917 with the highest protease-producing ability were screened out.The stability of protease-producing of the three strains was verified by rescreening.All the three strains were Gram-negative bacteria,which had high hydrolysis ability to gelatin and strong metabolic ability to other enzymes.The three strains were identified by molecular biology.They are named Salinivibrio sp.MK070915,Idiomarina sp.MK070916 and Salinivibrio sp.MK070917,respectively.(2)The growth curves of three halophilic strains were determined by turbidimetry,and the environmental factors and carbon and nitrogen sources affecting protease production of three halophilic strains were explored.The results showed that the optimal enzyme production conditions of Salinivibrio sp.MK070915 were 5%NaCl,pH 8.0,culture temperature 20?,the best carbon source was maltose,the best nitrogen source was peptone,the enzyme activity was 34.29 U;The results showed that the optimal enzyme production conditions of 10%NaCl,pH 8.0,culture temperature 35?,the best carbon source was yeast extract,the best nitrogen source was skim milk,the enzyme activity was 23.91U;Salinivibrio sp.MK070917 optimal enzyme production condition was 10%NaCl,pH 7.0,culture temperature 30?,the best carbon source is dextrin,the best nitrogen source is peptone,the enzyme activity was 20.69 U.(3)The enzymatic properties of protease produced by three halophilic strains were studied used Folin.For strain Salinivibrio sp.MK070915,the optimum reaction temperature was 55?,the optimum pH was 7.0,and and the addition of Cu2+promoted the enzyme activity.For strain Idiomarina sp.MK070916,the optimum reaction temperature was 50?,the optimum pH was 8.0,and the metal ions such as Fe3+,Cu2+ promoted the enzyme activity.For strain Salinivibrio sp.MK070917,the optimum reaction temperature was 55?,the optimum pH was 7.0,and the addition of Fe3+,Co2+,Cu2+and Ca2+ promoted the enzyme activity.The proteases produced by the three strains have the same characteristics:high concentration of salt inhibited the enzyme activity.(4)Three halophilic strains producing protease were optimized for protease fermentation by response surface methodology.The optimum culture conditions were obtained by using Gibbons medium as the base.For the strain Salinivibrio sp.MK070915,the amount of maltose added was 1.85%,the amount of peptone added was 1.17%,the amount of KCl added was 1‰ and the amount of sodium citrate added was 5‰,and the predicted maximum enzyme yield was 44.09 U,and the experimental results were 44.22 U,with a difference of 0.299%compared with the theoretical values.For strain Idiomarina sp.MK070916,the addition of skimmed milk was 1.5%,the amount of MgSO4·7H2O added was 3%,the final concentration of Fe3+ was 3.13 mM,the inoculation was 4%,the maximum enzyme yield was predicted to be 30.56 U,and the validation experiment result was 30.19 U,the difference was 1.2%compared with the theoretical value.For strain Salinivibrio sp.MK070917,the addition of dextrin was 0.66%,the addition of peptone was 1.11%,the addition of KC1 was 1.16‰ and the addition of sodium citrate was 2.10‰,and the maximum enzyme yield was predicted to be 30.192 U.The validation experiment result was 30.18 U,the difference was 0.5%compared with the theoretical value.