Gene Cloning,Expression,Characteristics And Application Of Keratinase From Bacillus Velezensis

Keratinases are widely used in food,medicine,beauty and leather.Paper from the screening of highly active keratinase producing strains,from which novel keratinase genes were mined by analyzing their genomes,cloned and expressed,and enzymatic properties and degradation of feather powder were investigated.The main research results are as follows:(1)After the detection of enzyme activity of several Bacillus strains,a highly active keratinase producing strain with enzyme activity up to 65.90 U�m L^-1 was screened and previously identified and named B.velezensis SYBC H47.(2)Screening resulted in 23 protease genes by functional annotation of the B.velezensis SYBC H47 known genome-wide;further classification resulted in four keratinase genes:baker1?baker2?baker3?baker4.Bioinformatics analysis of these 4 keratinase genes,and clonal expression validation resulted in 1 active keratinase:BaKer1.When azocasein was used as a substrate,BaKer1 had the highest enzymatic activity,reaching 109.77 U�m L^-1.The fermentation growth curve as well as the enzyme activity curve of this recombinant strain were determined and compared with the empty control strain control,it was found that the growth of the recombinant strain was inhibited to some extent,indicating that keratinase affected the growth of.(3)Upon multiple sequence alignment,it was shown that BaKer1 belongs to the S8A subfamily.Its optimal reaction p H is 9.5;the optimal reaction temperature is 55?,and it has good stability and tolerance in moderate temperature as well as alkaline conditions.Ca²⁺has a significant promoting effect on baker1.Mg²⁺,Zn²⁺,Fe²⁺and Mn²⁺can activate the enzyme.When the concentration of Ca²⁺was 5 mmol�L^-1,the promoting effect of Ca²⁺was weakened.At this concentration,Mn²⁺had obvious inhibitory effect,and other ions had more obvious promoting effect with the increase of concentration.The enzymatic activity of BaKer1 is significantly inhibited by EDTA,which represents the requirement for metal ions for the enzyme to exert activity.PMSF also significantly inhibited the enzymatic activity of BaKer1,indicating that BaKer1 is a typical serine protease.In contrast,DTT did not inhibit BaKer1strongly,indicating the absence of disulfide bonds in BaKer1.Isopropanol,n-hexane,acetone had little effect on the activity of BaKer1 in organic solvents,but dichloromethane and ethanol somewhat inhibited it.The 4 surfactants had little effect on the activity of BaKer1.For BaKer1,the K_m values were1.09 and 1.39 mg�m L^-1,and the k_cat/K_m values were 2536.54and 3733.79 s^-1�(mg�m L^-1)^-1,respectively,when azocasein and casein were used as substrate to study the kinetic parameters.The content of free amino acids in the untreated feather powder was low.The release of lysine and tyrosine was higher after BaKer1 treatment.It indicated that BaKer1 had a good application prospect to improve the nuttional use value of feather powder.The treatment of feather powder with BaKer1 could achieve 33.51%of the effect of acid hydrolysis.Although the effect of enzymatic hydrolysis reached only one-third of that of acid hydrolysis,however,the treatment of feather powder with enzyme was greener and environmentally friendly,and the cost was low,and the obtained products were safer and simple in the subsequent processing,indicating that BaKer1 has a high industrial value for the secondary green processing of feather powder.