Exploration Of The Degradation Mechanism Of Feather Keratin By Bacillus Subtilis 8

A highly feather-degrading strain was obtained in the present study.In feather base medium,the intact feather was completely degraded by the strain in 48 h.The degradation ability was significantly better than that of the reported strains.The species of the strain was identified by biochemical and molecular identification.The degradation process was observed by scanning electron microscope;the varieties and contents of amino acids and the content of three kinds of sulfur compounds in fermentation were determined,too.Purification and identification of enzymes involved in feather-degradation were studied by ion exchange chromatography and mass spectrometry during fermentation.Cloning and expression of the purified enzymes were obtained by one-step cloning technology.The keratin hydrolysis activity of recombinant proteases were confirmed using feather powder as substrate.In this paper,the mechanism of degradation of keratin of the strain was analyzed from the aspects of thiolysis and enzymolysis.It was expected to provide theoretical guidance for the reuse of feather waste resource.The main results are as follows:1.Identification of highly effective feather-degrading strains.Based on the morphological,physiological and biochemical characteristics and sequence analysis of 16S rDNA,BS8 was identified as Bacillus subtilis.The results showed the strain had a different degradation for the hard proteins such as feathers,scales,hair,etc.2.Study on mechanism of degradation of feather keratin by Bacillus subtilis 8.With the development of fermentation,the degradation efficiency of feather was increased gradually when feather used as the sole carbon and nitrogen source in the medium by Bacillus subtilis 8.The maximum of disulfide-bond reductase activity(9.6 U/mL)and keratinase activity(21.6 U/mL)were detected at 24 h and 48 h,respectively,indicating that the enzymatic hydrolysis was involved in the degradation of the feather.No sulfite in prior fermentation period.The sulfite content began to increase slowly after BS8 inoculated,accompanied by the formation of sulfocysteine(CySO3H)and sulfhydryl compounds,which confirmed the presence of thiolysis in the feather degradation process.The release of ammonia and the increase of pH value indicated that the degradation of the feather was accompanied by the transaminase and deamination during the fermentation3.Isolation,purification and identification of extracellular protease from Bacillus subtilis 8 Four prntein bands were obtained by DEAE Sepharose Fast Flow chromatography and gelatin activity staining.Four kinds of proteases which are consistent with the experiment were screened(Serine protease:33.9 kD,EC:3.4.21;Peptidase T:45.6 kD,EC:3.4.11;Membrane bound gamma-glutamyltransferase:64.3 kD,EC:2.3.2.2;Cystathionine gamma-synthase:40.7 kD,EC:4.4.1.1)based on the analysis of the results of LC-MS/MS matching,combined with the molecular weight of protein,GO functional annotation and related literature reports,which named Ser P,PeP T,M-y-Glu and Cys P,respectively.Functional annotation results show that 4 kinds of protease could hydrolyze peptide bond and carbon sulfur bond,and the function of transamination and deamination.4.Cloning and prokaryotic expression of extracellular protease from Bacillus subtilis 8.With the BS8 genome as the template,the full-length sequences of the 4 proteases were cloned(Ser P,960 bp;Cys P,1143 bp;M-y-Glu,1776 bp;PeP T,1233 bp)using the specific primers.The expression vectors were successfully constructed and expressed in E.coli BL21 using One Step Cloning Kit,named P-Ser,P-Cys,P-Glu,P-Pet.The 4 kinds of recombinant protease were found to have a certain degree of keratin hydrolysis activity with feather powder as substrate.The activity of P-Cys was highest(102.4 U/mL),while only P-Ser had the keratinase activity(58.6 U/mL)and the lower disulfide-bond reductase activity(14.1 U/mL),respectively.The key to the degradation of feather keratin is to open the disulfide bond.Therefore,it is speculated that P-Cys and P-Ser may be the two key enzymes of Bacillus subtilis 8 in the degradation of feather.5.P-Ser promoted the natural feather hydrolysis of P-Glu,P-Pet and P-Cys in the combination of different proteases.After 4 kinds of protease were mixed,the activity of keratinase and disulfide-bond reductase reached the maximum and the effect of hydrolysis natural feather was the most obvious.However,the enzymatic hydrolysis of feather was not significant.The results showed that the degradation of feather keratin in BS8 was mainly dominated by thiolysis.