Efficient Degradation And Transformation Mechanisms For Protein Biomass Waste By Bacillus Based On Multi-omics Method

All along,the scarcity of natural resources and environmental pollution have posed potential risks to human survival and sustainable development.Protein-rich biomass generated during the production processes of industrial and agricultural sectors is an important biomass resource.Therefore,the efficient degradation and transformation of protein-rich biomass waste has become a hot topic of research in current biotechnology field.Feather waste,which is primarily generated by poultry farming and processing industries,represents a significant biomass waste stream.Due to its high content of keratin and other unique chemical components,effective treatment of feather waste has been a challenge in terms of environmental protection.In recent years,keratinases have been found to have the potential to catalyze the hydrolysis of keratins,including feathers from poultry,and are considered to be ideal catalysts for the conversion of keratin waste.However,the currently reported keratinases have limitations in terms of catalytic efficiency and time consumption,making them unsuitable for large-scale industrial applications.Therefore,the comprehensive and systematic screening,exploration,and study of novel keratinase-producing microorganisms,as well as the continued research on keratinase resources produced by these strains,remain the focus of research in this field.Focusing on this problem,a series of studies have been carried out in this study with the screened Bacillus with high activity of keratinase as the main research object.The main research results obtained in this paper are as follows:1.A strain of Bacillus with high proteolytic activity was screened from the natural chicken manure compost habitat,and the protease diversity of this bacterium degradome was revealed based on genomic analysis.Six Bacillus strains were obtained after the chicken manure compost samples underwent screening,isolation,purification,and identification.Among them,Bacillus sp.CN2,which exhibited the highest proteolytic activity,was selected for whole-genome sequencing.The genome size was approximately 3.9 Mb with 4049 protein-coding genes.A total of 149 proteases or protease-homolog encoding-genes among 58 families were identified.All the proteases derived from strain CN2 are grouped into five catalytic classes.Serine proteases and metalloproteases were the most abundant groups in strain CN2,followed by cysteine proteases,and the distribution of protease families also exhibits a similar pattern.This suggests that these three types of proteases play an important role in the degradation of proteinaceous biomass in composting habitats.Among these extracellular proteases,serine proteases accounted for 52%,followed by metalloproteases(28%),and cysteine proteases(16%).GO functional classification results indicated that most proteases were primarily involved in metabolic and catalytic processes.2.The substrate degradation preference of Bacillus sp.CN2 was analyzed using functional proteomics,and a degradation model for the efficient protease degradation system of this strain was proposed.The metabolic utilization capabilities of Bacillus sp.CN2 for four different nitrogen sources and its characteristics for inducing proteases secretion were analyzed using functional proteomics.The results indicated a preference for utilizing wheat bran,corn steep liquor,and maize protein powder,and the fermentation broth had a high specific activity of protease.At least 8 extracellular protease bands were detected by active zymography,and inhibitor experiments confirmed that these proteases were mainly serine proteases.More importantly,the analysis of the strain CN2 secreome revealed the existence of an efficient protein degradation system.Specifically,a variety of proteases were secreted into the medium after being induced by soluble proteins,among which,three S8 serine endopeptidases were responsible for decomposing proteins into polypeptides.Then,a small amount of M4 metalloendopeptidases and exopeptidases degraded them into small peptide fragments,which were transferred to cells via transporters.In the cell,polypeptides or small peptide fragments were further hydrolyzed by one S8 serine endopeptidase and a large number of aminopeptidases to produce oligopeptides or free amino acids for growth.3.Using feather keratin as substrate,electrochemical experiments and proteomics analysis demonstrated that Bacillus sp.CN2 utilizes a T3 y-glutamyltransferase to provide reducing power to open disulfide bonds of the keratin.Using feather waste from livestock and poultry breeding as substrate,the functional enzyme system of Bacillus sp.CN2 and the degradation mode of recalcitrant proteins were explored.Bacillus sp.CN2 showed strong feather keratin degradation activities,which could degrade native feathers efficiently,resulting in 86.70%weight loss in 24 h,along with the production of 195.05 ± 6.65 U/mL keratinases at 48 h.The extracellular protease consortium had wide substrate specificity and exhibited excellent biodegradability toward soluble and insoluble proteins.Importantly,analysis of the extracellular proteome revealed the presence of a highly efficient keratin degradation system.Firstly,T3 y-glutamyltransferase provides a reductive force to break the dense disulfide bond structure of keratin.Then S8B serine endopeptidases first hydrolyze keratin to expose more cleavage sites.Finally,keratin is degraded into small peptides under the synergistic action of proteases such as M4,S8C,and S8A.Consistent with this,high-performance liquid chromatography(HPLC)and amino acid analysis showed that the feather keratin hydrolysate contained a large number of soluble peptides and essential amino acids.This insight increases our understanding of keratinous substrate degradation and may inspire the design of optimal enzyme cocktails for more efficient exploration of protein resources in industrial applications.4.The ProS8A keratinase derived from Bacillus sp.CN2 was cloned and heterologically expressed,and biodegradation experiments showed that single ProS8A keratinase could completely degrade keratin in the presence of reducing power.Bioinformatics analysis revealed that three extracellularly highly expressed proteases in Bacillus sp..CN2 belong to the S8A subfamily,but their sequences and domain compositions are different from each other.In order to explore the function of the three proteases,a total of six recombinant expression vectors of three serine proteases with and without a signal peptide were constructed.Using the Bacillus subtilis WB800N heterologous expression system,the active recombinant enzyme ProS8A was successfully obtained and its enzymatic properties were characterized.ProS8A exhibited degradation activity against casein,BSA,soy protein isolate,and gelatin,with a broad substrate specificity.Biochemical results revealed that ProS8 A had the optimal reaction temperature and pH of 40℃ and pH 8.0,respectively,and displaying good thermal stability and maintaining stable in the range of pH 7.0-10.0,indicating that Pros8A belonged to mesophilic alkaline protease.In addition,ProS8A demonstrated excellent tolerance to non-ionic detergents,indicating that it has certain stress resistance.The reducing agents DDT and β-ME can promote its enzyme activity.ProS8A was able to degrade intact feathers within 6 h in the presence of reducing agents.A large number of essential amino acids and soluble peptides were detected in the feather hydrolysate,implying that ProS8A plays an important role in the reuse of keratinaceous waste.