The Engineering Transformation And Fermentation Conditions Screening Of Subtilisin

Subtilisin(also known as subtilases)is an extracellular serine endopeptidase,which is found in archaea,fungi,eukaryotes and viruses.Subtilisin is classified as members of the S8 A subfamily within the second largest family of serine peptidases(family S8),characterized by the presence of conservative Asp,His,and Ser catalytic triad.Subtilisin,which has a wide range of substrate specificity and high activity and stability in alkaline environment,is used as a biological additive in washing,tanning,food and other industries.Subtilisin as a detergent additive has been the focus of enzyme development research for the past several decades.In this study,subtilisin Bapb92 gene of Bacillus alcalophilus PB92 was chemically synthesized and actively expressed by the Escherichia coli–Bacillus subtilis shuttle expression vector p BE2 R in the protease-deficient strain Bacillus subtilis WB600.The purified alkaline protease was obtained from the supernatant of recombinant Bacillus subtilis WB600 / p BE2R-Bapb92 by 70% ammonium sulfate precipitation,Sephadex G-25 desalting and Sephadex75 chromatography,with purification fold 6.8,specific activity 2200 U / mg and yield 39%.The target protein BAPB92 with molecular weight 26.7 k Da was identified and calculated by 12% SDS-PAGE and mass spectrometry.Four mutants,BAPB92(A188P),BAPB92(V262I),BAPB92(Q239R)and BAPB92(A188P / V262I),were constructed by site-directed mutagenesis of BAPB92.Compared with the wild type,the enzyme activity of BAPB92(A188P),BAPB92(V262I),BAPB92(Q239R)and BAPB92(A188P / V262I)increased 2.6 times,1.2 times,2.5 times and 3.3 times respectively.Kinetic constants were determined using casein as a substrate,compared with the wild type,the catalytic efficiency of BAPB92(A188P),BAPB92(Q239R)and BAPB92(A188P / V262I)were increased 1.5 times,1.3 times and 4.3 times respectively,but the catalytic efficiency of BAPB92(V262I)was decreased to 0.4 times of the wild type.The results showed that the residual enzyme activity of BAPB92 was 29%,that of BAPB92(A188P)was 41%,that of BAPB92(V262I)was 48%,that of BAPB92(Q239R)was 35%,and that of BAPB92(A188P / V262I)was 51%,compared with wild enzyme incubation at 60 °C for 1 hour.The results of p H stability revealed that the mutants of single mutant A188 P,Q239R and combination mutant A188 P / V262 I were more tolerant to alkaline environment,the single mutant V262 I has the same alkali resistance as the wild type BAPB92.Sodium triphosphate(STPP)and methylglycine diacetic acid(MGDA)are commonly used detergent chelating agents.BAPB92 and its mutants were incubated at 25 °C for 1 h with standard detergent containing STPP and MGDA respectively.The results showed that both BAPB92 and its mutants could exist stably in STPP and MGDA system and maintain high activity.Through comparing the common fermentation medium of recombinant Bacillus subtilis WB600 / p BE2R-Bapb92(A188P)producing alkaline protease,the medium containing dextrin and solublestarch was selected as the initial fermentation medium.On this basis,the optimum fermentation conditions were determined by single factor experiment and orthogonal experiment as follows: dextrin 5%,peanut meal 3%,yeast extract 1%,Mg SO4 0.28%,Na Cl 0.5%,initial p H 7.0,broth content 12%,inoculum size 4%.Under this condition,the enzyme activity of BAPB92 increased 4.9 times compared with the initial culture conditions,that of BAPB92(V262I)increased 3.1 times,that of BAPB92(Q239R)increased 8.4 times,that of BAPB92(A188P)increased 10.6 times,BAPB92(A188P / V262I)enzyme activity reached 3111.94±37.25 U / m L and increased 11.2 times at 37?,200 rpm for 60 h,the enzyme activity was improved significantly.These results can be used for reference in the production and application of subtilisin.