Heterogenous Expression And Feather Degradation Of Keratinases From The Genus Of Deinococcus

Keratin is a nonnutritious hard protein widely distributed in feather,wool,animal hoof,horn,toenail and so on.There are a large number of hydrogen bonds and cysteine disulfide bonds in its structure.The disulfide bond interacts to form a dense structure of keratin,which is difficult to be degraded and utilized.Keratinase is a kind of enzymes that can specifically degrade keratin.It can destroy the dense structure of keratin to achieve the purpose of degradation.keratinase is used to degrade feather keratin in mild conditions and has a good application prospect.In order to better develop the use of keratin and keratinase genes in this study,three keratinase genes were identified from(Deinococcus gobiensis)I-0 and(Deinococcus radiodurans)R1.Kerdg1(DGO_RS02895)and Kerdg2(DGO_RS03965),Kerdr(A2G07_RS01950)was expressed and purified in Escherichia coli and its enzymatic properties were analyzed.The results are as follows:1.In order to study these three genes,It was found that Kerdg1 was composed of 1239 bases to encode 412 amino acid residues and the molecular weight was 41.1k Da.Kerdg2 was composed of 1599 bases to encode 532 amino acid residues,the molecular weight was53.78 k Da.Kerdr size was 1599 bp encoding 532 amino acid residues,the molecular weight was 53.39 k Da.Sequence comparison revealed that Ker DG1,Ker DG2,Ker DR protein contained transmembrane structure and leading peptide.Signal peptide,N-terminal propeptide and mature protein region,C-terminal propeptide were similar to peptidases_s8_s53 family proteins.In addition,there are three major amino acid residues(Asp,His,Ser)in mature protein region consistent with the known keratinase(Ker A)of Bacillus spp.,and the three genes are confirmed to be keratin genes.2.The three keratin genes were ligated into pET22 b vector and transformed into the expression host BL21 for prokaryotic expression.The degradation ability of the recombinant strain was analyzed,and the results showed that the degradation of feather was obvious by the recombinant strain.The results showed that the degradation products mainly produced 21 amino acids,such as arginine,tyrosine,proline,cystine and so on.3.The recombinant strain was induced to express,and SDS-PAGE electrophoresis showed that the target protein was obtained in the supernatant of the bacteria.The optimum p H and temperature of Ker DG1 were 5.0 and 60?,and the enzymatic activity of 3.5 U/m L was obtained.The optimum p H and temperature of Ker DG2 were 8.0 and 40?,and enzymatic activity is 3.0 U/m L.The optimum p H and temperature of Ker DR were 7.0 and 40?,and the enzyme activity was 3.0 U/m L.The tolerance test showed that keratinase could tolerate certain temperature and p H,and it is also found that the enzyme has certain resistance to metal ions and chemical reagents.