Fermentation Conditions And Characteristics Of Neutral Protease From Bacillus N19

The neutral protease is a protease which suitable pH is 6.0～7.5.It can catalyze the cleavage of bonds in proteins.Since the neutral protease have many features and advantages in industry such as high speed and wide range of conditions of catalyzed reaction,without pollution.This research took a strain of Bacillus N19 which preserved in the enzyme engineering laboratory as the fermentation strain.The fermentation conditions of Bacillus N19,including carbon source,nitrogen source,inorganic salt,iron and so on,were investigated in shake flask level.The orthogonal test of the four factors showed that the optimized culture medium was:beef-extract 11 g/L,yeast-extract 12 g/L,peptone 20 g/L,NaCl 4 g/L.The optimal producing protease conditions were illustrated below:the initial pH 4.5,amount of inoculation 2%(V/V),incubating temperature 42℃,shaking cultivation 250 r/min,liquid volume 40 mL/300 mL,fermenting time 44h.By adopting filtration,ammonium sulfate fractionation,sephadex G-25 gel filtration, DEAE ion-change chromatography,Sephacryl^TMS-200 gel filtration,the neutral protease which came from Bacillus N19 fermentation liquor was purified.The result of PAGE verified that a single protein band was obtained.The specific activity of neutral protease had reached 6214.7U/mg which was 20.1 folds purified with yield of 12.87%.Characteristics of the purified neutral protease from Bacillus N19 which took Casein as substrate were as follows:the optimum temperature of the neutral protease was 70℃,When was below 60℃or above 80℃,relative enzyme clearly dropped.The neutral protease kept in 50℃for 2 h the residual enzyme activity was 26.35%,60℃for 2 h the residual enzyme activity was 3.6%,70℃for 20 minute the residual enzyme activity was 14.3%;the optimum pH of the neutral protease was 6.8,it has a higher enzyme activity in the pH 6.0～8.0;it had a good pH tolerance.Fe²⁺had a significant activation on the neutral protease,the enzyme activity could be increased 394.65%,EDTA could inhibit enzyme activity;for Casein the Km was 3.1×10^-3g/mL.which tested by Bi-reciprocal graph,SDS-PAGE indicated that the molecular weight of the neutral protease was 37.7 KDa.This paper had done some research on the Bacillus N19 including the optimization of Bacillus N19 cultivation conditions,isolation and purification of neutral protease,the enzyme properties which wound be a foundation for its industrial production.