| Alkaline proteases?AprEs?can hydrolyze peptide bonds to generate amino acids or peptides.They have good heat resistance and alkali resistance,so they are widely used in detergent,leather,textile,medicine,food,and other fields.Although AprEs have broad application prospects,the current research on AprEs is still focused on detergent additives.Therefore,it is of great significance to explore novel AprEs and broaden their application.The study started with screening highly active AprE-producing strains.The strain with high AprE activity was subjected to genomic data mining,in which the AprE genes were cloned and expressed.The enzymatic properties of recombinant AprEs and application in chiral drug resolution and milk protein desensitization were studiedThe main research results are as follows:?1?Four strains with obvious AprE activity were obtained through plate preliminary screening.After fermentation and measurement of enzyme activity,the strain W3 with high AprE activity of up to 69.88 U·mL-1 was acquired,which had been previously identified and named Bacillus altitudinis W3.?2?According to the genome data of B.altitudinis W3,a total of 51 protease genes were screened,of which 9 genes were initially determined as AprE genes,named baapr1-9.Through bioinformatics analysis and verification by cloning and expression,four active AprEs?BaApr1,BaApr2,BaApr5,and BaApr9?were obtained.When azocasein was used as the substrate,BaApr1 had the highest enzyme activity up to 180.58 U·mL-1.The fermentation growth and enzyme activity curves of the four recombinant strains were measured,and it was found that the growth of the four recombinant strains was inhibited compared to the control strains,especially for B.subtilis WB600-pMA0911?wapA?-baapr1?Bp1?,showing that the growth of recombinant strains was affected by AprEs.Multiple sequence alignments revealed that the four AprEs belong to the S8A subfamily.?3?The enzymatic properties were studied with azocasein as the substrate.The optimal pH of BaApr1,BaApr2,BaApr5,and BaApr9 were 9.5,8.5,10.5,and 10.5,respectively,and the optimal temperatures were 55,50,60,and 45?,respectively.They showed better stability and tolerance under moderate temperature and alkaline conditions.The four AprEs were activated by Ca2+and Mg2+,and inhibited by ethylene diamine tetraacetic acid and phenylmethylsulfonyl fluoride,which belong to typical serine proteases.They were well-tolerated to surfactants,but most organic solvents show inhibition on them except for n-hexane.The four AprEs all showed better activity on casein?or a substrate with casein as the main protein component?and had high specificity.BaApr2 and BaApr5 showed hydrolytic activity on gelatin,while BaApr1 and BaApr9 did not.The kinetic parameters were studied with azocasein,casein,and skimmed milk powder as substrates.For BaApr1,the Km values were 1.16,2.39,and 5.09 mg·mL-1,respectively,and the kcat/Km values were 15926.07,16739.36,and 7582.19 s-1·(mg·mL-1)-1,respectively.For BaApr2,the Km values were 1.07,1.82,and 7.74 mg·mL-1,respectively,and the kcat/Km values were 2091.65,2995.06,and584.93 s-1·(mg·mL-1)-1,respectively.For BaApr5,the Km values were 0.24,0.19,and 0.29mg·mL-1,respectively,and the kcat/Km values were 5532.74,28067.23,and 12207.45s-1·(mg·mL-1)-1,respectively.For BaApr9,the Km values were 1.95,1.31,and 5.75 mg·mL-1,respectively,and the kcat/Km values were 1367.09,10953.37,and 1697.99 s-1·(mg·mL-1)-1,respectively.?4?The effect of BaApr1 on the resolution of chiral drugs was not obvious.BaApr1showed good hydrolysis effect on milk proteins,which could effectively hydrolyze the proteins in the three kinds of milks and release free amino acids or small molecule peptides.The amount of leucine among the free amino acids released in the three milk protein hydrolysates was the highest,which was 38.20,89.41,and 85.02?g·m L-1,respectively,showing great application prospects of BaApr1 in desensitization of milk protein. |
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