Study On The Mechanism Of Stenotrophomonas Maltophilia DHHJ Strain Using Keratin Monomer

With the continuous development of productive forces,a large number of animal feathers and fur as waste generated every day,containing extremely rich keratin,this kind of protein has excellent biocompatibility and environmental biodegradability,But the by-products of these production have been completely discarded,not only polluting the environment but also seriously wasting resources.Therefore,the recovery of keratin gets more and more people's attention.Conditions of microbial degradation are mild,not only to avoid contamination,but also to convert it into available peptide and amino acids.Today,many keratin-degrading bacteria have been isolated and studied.It has been found that the keratinase activity isolated from these microorganisms is very high.Looking for keratinase having high activity will be an important part of developing and utilizing animal feathers and hair.Sustainable development was emphasized in today's society,the development of keratinase,and the recycling of keratin waste are very importance for the protection of the environment.Therefore,analyzing the mechanism of using keratin by keratin-degrading bacteria has far-reaching influence on the development of keratinase and the recycle of keratin.In our previous study,keratin monomers and Stenotrophomonas maltophilia DHHJ were cocultured to resolve the role of keratin monomers in bacterial keratinase.The study found that when the strain degrades keratin,it will bind with keratin monomers,and bacteria accumulate in large quantities and produce keratinase activity to degrade feathers.On the basis of these previous studies,this dissertation studied the mechanism of using keratin monomers by the strain by using fluorescent-labeled protein technology and RNA-seq.Using FITC to label the keratin monomer,the FWP and S.maltophilia DHHJ strains were cocultured,adding different concentrations of BSA,DNA,ATP and metal ions,the results found that BSA competes with keratin monomer for binding sites on bacterial surfaces;DNA is inaccessible to bacteria and therefore does not affect bacterial binding to keratin;addition of ATP indicates that this process of binding is a process consuming energy;Mg2+,Ca2+,Mn2+ are conducive to the combination of keratin and bacteria,and the promotion effect of Ca2+ is very obvious;after the enzymatic hydrolysis of keratin solution,the amount of bacterial combination decreased significantly.The determination of keratinase activity was also conducted in each experimental group,the results are consistent with the previous conclusion that these factors affect the binding process of bacteria and keratin,and this process directly determines the activity of keratinase.Therefore,we think keratin monomer is the key factor to stimulate the production of bacterial enzyme.We used RNA-seq to analyse the transcriptome of the strain,which can degrade keratin monomers.Two strains were prepared,one of which was sample K induced by the keratin monomer to produce keratinase and the other one was sample S cultured in a non-induced source medium.The samples were subjected to RNA-seq to understand the expression of global genes.Making the genes with substantial deference for GO enrichment analysis.The aspects whose number of the genes expressing differently are cellular processes,metabolic processes,single biological processes,catalytic activities,binding and transporter activity.Genes relating to the transmembrane transport activity,ABC transport activity and amino acid transmembrane transport activity enrich significantly.This may be due to that after the combination of keratin monomers and bacterial surface binding sites,bacteria need to use the transport system to uptake the keratin monomer,thus contributing to the large number of these genes expressed.The genes with obvious difference were enriched and analyzed by KEGG.Among them,metabolic pathway,biosynthesis of secondary metabolism,amino acid biosynthesis,bacterial secretion system and ABC transport system all had very active up-regulated expression.We believe that when bacteria and keratin were co-culture,keratin is taken into the cell through the protein transport system as a nutrient source,thereby stimulating bacteria to begin the secretion of keratinase,seven candidate genes were selected and the relative expression of these genes in each group was analyzed by q RT-PCR.The results showed that these genes were up-regulated in K group and they all were related to protein binding and transport,which is also consistent with the previous conclusion Consistent.