The Key Hydrolysis Enzymes Of Keratin In B.licheniformis CP-16 And S.maltophilia 10

Feather is a kind of protein with amount of disulfide bond, and they are not able to be hydrolyzed by conventional protease, which limits the utilization rate of protein feed. If the disulfide bond were broken before the function of protease, the low utilization of protein would be solved and the feather could be developed as a new protein feed resource. This research is mainly to discover enzymes with disulfide reductase activity and explore the interaction with keratinase based on B.licheniformis CP-16 and S.maltophilia 10 which have disulfide reductase activity in the process of keratin hydrolysis.The separation and purification of key protease in B.licheniformis CP-16. According to the precious study of our research group, this trial separated and purified a new protease-oligopeptidase using ion exchange chromatography and gel chromatography. And it was found that this enzyme function mainly at the end of the hydrolysis of protein, which facilitates the degree of hydrolysis of keratinase on the casein.The cloning and expression of gene about enzyme with disulfide reductase activity. With the help of bioinformatics analysis technology, this experiment collected 5 protein sequences whose products have disulfide reductase activity in B.licheniformis and S.maltophiliain Uniprot. The primers were designed and the sequences were cloned and expressed in E.Coli BL21, and the expressed proteins were named B-P, S-G, S-T, S-P and S-A respectively.The research on the enzymatic property of recombinant enzymes and the interaction with keratinase. The optimum p H of P-Trx, B-P, S-G, S-T, S-P and S-A are 9.0, 9.0, 6.0, 6.0, 8.0 and 8.0, and the optimum temperature are 40℃, 40℃, 55℃, 40℃, 45℃ and 55℃. The degradation of feather by keratinase could be promoted with any addition of P-Trx, B-P, S-G, S-T and S-P, and the maximum ratio was 47% of P-Trx.P-Trx, B-P and S-P could be degraded by trypsin and chymotrypsin in some degree, and the degradation of P-Trx was the least, but B-P was degraded by chymotrypsin entirely. Although trypsin and chymotrypsin could also be degraded by the recombinant enzymes, the degradation rates were all less than 10%. And the degradation of soybean meal could be accelerated by S-P on 83%, which was the maximum ratio when the soybean meal was degraded by trypsin and chymotrypsin.