Characterization And Evaluation Hydrolysis Ability On Feed Protein Of Bacillus Subtilis H6-3 Neutral Protease

This study conducted on pig source Bacillus subtilis H6-3 (H6-3) which preserved by animal medical microbiology laboratory of Sichuan Agricultural University. The nutritional needs and culture conditions of H6-3 were investigated in shake flask level. Physical and chemical factors effects on neutral protease activity were tested. Proteolytic activity on soybean meal, rapeseed meal and corn were estimated. In addition, neutral protease gene of H6-3 was amplificated.The orthogonal test of five factors L16 (45) showed that the optimal producing protaese medium was:glucose 15g/L, oligochitosan 5g/L,peptone 15g/L,yeast extract 5g/L and potassium dihydrogen phosphate 5g/L.The optimal culture producing protaese conditions illustrated were:the initial pH 7.5,amount of inoculation 1%(V/V),incubating temperature 39℃,liquid volume 40 mL/100 mL, optimal culture time 61 hour.Characterization of the purified bacillus subtilis H6-3 neutral protease which took casein as substrate were follows:the optimum temperature of the neutral protease was 60℃. When the temperature below 60℃or above 80℃the relative enzyme activity dropped dramatically. The neutral protease kept in 60℃for 2 hours the residual enzyme activity was 40%,60℃for 120 minute the residual enzyme activity was 7.5%. The optimum pH of the neutral protease was 7.0. It has a higher enzyme activity in the pH 6.0～8.0, incubation at pH 6.0～8.0 for 1 hour the residual enzyme activity remain above 80%. Mg2+ could promote enzyme activity, Hg+,Fe2+, EDTA, OP and DMSO could inhibit enzyme activity.When hydrolysis soybean meal, rapeseed meal and corn, Bacillus subtilis H6-3 neutral protease increased protein degradation significantly. with add dose was 50IU/g hydrolysis degree increased 34.74%,34.70% and 55.54% respectively, nitrogen dissolve index and trimethoxysilane acetic nitrogen dissolve index increased fairly, average length of peptide chain droped dramatically. Purified by filtration, ammonium sulfate fractionation and separated by Macro-Econo-Pac Prep High Q ion exchange chromatography, the specific activity has been improved 8.56 fold. SDS-PAGE indicated that only a single band was obtained. The molecular weight of H6-3 neutral protease was about 40.92 KD.Bacillus subtilis H6-3 neutral protease gene was cloned successfully and the homology to reported Bacillus sp.RH219 nprE reached 100%.This paper had done some research on H6-3 neutral protease including the optimization of the cultivation conditions, purification and characterization of neutral protease. The protease properties which woud be good potential to be applicated in feed industry.