Preliminary Study Of Keratin Degradation Mechanism Of Bacillus Amyloliquefaciens

Keratin belongs to the hard protein,mainly exist in the epidermal tissue of animals,such as hair,scales,feather,hoof,horn and some other structures.Keratin has the property of resistance the chemical reagent and hydrolysis enzyme attacks.Keratin is often been discarded as industrial by-products because it is difficult to degrade,resulting in a great waste.Keratin-Degrading microbial are a kind of microorganisms that can directly use keratin as carbon and nitrogen source.Keratin-Degrading microbial can secrete a variety of enzymes which have the relative activity of keratin degradation such as keratinase,protease and disulfide reductase.These enzymes and bacteria growth produce the cellular redox system together which promotes the denaturation of keratin and degrade it.In this paper,the keratin degradation ability and extracellular protein secretion of 10 strains of Bacillus Amyloliquefaciens were compared,and find that Bacillus Amyloliquefaciens ACCC 19735 can degrade the feathers completely within 24 h.Thirteen keratin hydrolysis-related enzymes have been identified with the technology of SDS-PAGE and LC-ESI-MS/MS,including nine proteolytic enzymes: four peptidase belong to the S8 proteinase family,two peptidase belong to the M28 peptidase family,a peptidase in M42 family,one peptidase of M17 family and one carboxypeptidase of M32 family,a leucyl aminopeptidase;three NADH metabolic related enzymes: two dihydrolipoyl dehydrogenases and a NAD(P)H dependent oxidoreductase;a enzyme associated with an amino acid metabolism: urocanate hydratase.Complete genome sequence of Bacillus Amyloliquefaciens ACCC 19735 has been determined and the information of the whole genome sequence has been annotated by bioinformatics analysis.Combining with the technologies of SDS-PAGE and LC-ESI-MS/MS,15 gene sequences have been obtained from the genome sequence,including the keratinase gene kerk and the gene of aminopeptidase pam,pam1 and pam2,the minor extracellular protease named as mep,bacillopeptidase1 to 3 named as bap1,bap2 and bap3;the serine protease named as serp,the carboxypeptidase named as cap and the leucyl aminopeptidase named as lap,a NAD(P)H-dependent oxidoreductase named as nado,and two dihydrolipoyl dehydrogenase named as did1 and did2,the urocanate hydratase named as uhy.The Kerk has been proven to be the key enzyme in the degradaion of keratin.The study of the degradation of keratin has not correlated with many other genes.In this study,we use the Escherichia coli induced expression vector pET28 a to construct two expressive vector pET28a-pam,pET28a-pam2,expressing two aminopeptidase named as PAM and PAM2,and we have succeeded to characterize these two aminopeptidase and got physicochemical properties of these enzymes.Purified recombinant PAM showed optimal activity at pH 7.5 and 45?.Purified recombinant PAM2 showed optimal activity at pH 8.0 and 50?.The specific activity of these two enzymes are 745U/mg and 896U/mg respectively when the Leu-pNA was substrate.The catalyze efficiency of the supernatant of feather fermentation culture of B.Amyloliquefaciens 19735 to feather powder have been enhanced about 28% and 46% when it synergize with these two aminopeptidase while the final concentration of the aminopeptidase are about 5 u M.Replace the feather powder with the keratin azure,the catalyze efficiency have been enhanced about 30% and 35%.These date indicate that these two aminopeptidase play important roles during the process of feather degradation.