Enhanced Expression Of Protease By Bacillus Licheniformis And Its Application In Enzymolysis Procedure Of Soybean Peptide

Soybean peptide,a type of low molecular weight peptide produced by the hydrolyzation of soyabean protein,possess multiple biological activities,and its molecular weight was usually lower than 1000 Da.Since its good water solubility,strong moisture retention and low viscosity,soybean peptide was widely used in the food additives,cosmetic,pharmaceuticals and feed industry.Generally,soybean peptide was mainly produced through three methods:chemical hydrolysis,microbial fermentation and enzymolysis procedure.The enzymolysis method was the most effective method among them.Usually,the protease used in enzymolysis procedure was produced by the microbial fermentation,and the low protease yield hindered the industrial production of soybean peptide.In this study,the protease activity was improved by gene modification and fermentation condition optimization in Bacillus licheniformis,and the enzymolysis procedure of soyabean peptide was also optimized at the same time.1.Four protease genes were selected and applied to constructed four protease expression engineering strains with P43 promoter.The protease activities of the respective strains were as follows:BL10/pP43-aprE?18.41 U/mL?>BL10/pP43-bprA?17.36 U/m L?>BL10/pP43-epr?14.02 U/m L?>BL10/pP43-vpr?10.26 U/m L?.This result implied that AprE possess the highest protease activity,and aprE gene was selected as the target gene for the further research.2.After the optimized protease gene was selected,we also studied the effect of host strains on the protease activity.Four host strains,B.licheniformis DW2,DW2?bacA,WX-02 and BL10 were selected as the host strains,and our results indicated that the highest protease activity?21.00 U/mL?was achieved when DW2?bacA was served as the host strain.3.Promoter could significantly affect protein expression.In this study,bacitracin gene cluster promoter P_{bac A},AprE promoter P_aprE and P_RBS6?modified from promoter P43 in our previous research?were selected.Then,these three recombinant strains with different promoters were constructed,and the corresponding protease activities were measured.Based on our results,the protease activity produced by the recombinant strain with P_RBS6 as promoter was reached 69.40 U/mL,increased by 2.65-fold compared with that of bacA/pP_RBS6-aprE.4.Signal peptide was another important factor affects protein expression.The signal peptide of serine proteolytic Vpr SP_Vpr,mannase SP_SacB,SP_SacC,AprE and SP_aprE were screened,then constructed the recombinant strains.The results were as follows:the protease activity of recombinant strain with SP_SacB,SP_SacC,AprE and SP_aprE as signal peptide were reached 47.40 U/m L,32.80 U/mL,37.90 U/m L and 77.93 U/m L,respectively.Obviously,the recombinant strain with SP_aprE as signal peptide produced the highest protease activity.In conclusion,the engineered strain B.licheniformis?bacA/pP_RBS6SP_aprE-aprE produced the highest protease activity.5.To further increase the protease activity,the fermentation processes were optimization was conducted.Through single factor experiment,the optimum concentration of carbon?corn starch 40 g/L?and organic nitrogen?60 g/L?,inorganic nitrogen??NH₄?₂SO₄ 3 g/L?was confirmed.Followed with orthogonal experiment,the optimum culture medium was determined.In addition,other factors such as inoculum age,inoculation quantity and liquid volume were optimized,and these results were as follows:corn starch 40 g/L,soya bean meal 70 g/L,?NH₄?₂SO₄ 2 g/L,light calcium 1g/L,K₂HPO₄ 3 g/L,inoculum age 6 h-8 h,inoculation quantity?v/v?4%,liquid volume25 mL,and the protease activity reached 747.40 U/mL.6.The application of soybean peptide which produced by the enzymolysis of soybean meal:the enzymolysis process of soyabean peptide was analyzed,the reaction time,protease volume and water ratio:material were optimized,and SDS-PAGE was applied to evaluate the protein expression and removal rate of anti-nutrients in the soybean peptide.Finally,the optimum enzymolysis process conditions were as follows:reaction time 8 h,protease volume?v/v?3%,the ratio of water:material was 10:5,and the protein production rate and removal rate of anti-nutrients were vintage under the optimal conditions.