Isolation, Screening, Molecular Ecology And Functional Use Of Keratin-degrading Bacteria

Safety production of fruits is one of the major challenges for fruit industry in China.The“Reduced Application of Chemical Fertilizers&Pesticides”policy inaugurated by the Ministry of Agriculture has further highlighted the demand.Therefore,increasing the application of organic fertilizers(including amino acid foliar fertilizer)has become an inevitable choice for fruit cultivation.With the rapid development of livestock and poultry industry in China,the feather wastes have accumulated year by year.In this context,producing amino acid fertilizer with feather wastes not only solves the problem of environmental pollution but also promotes the safety production of fruit,exhibiting a clear value of application and popularization.Consequently,with the focus on the feather keratin-degrading bacteria,the study in this dissertation conducts the isolation and screening of degrading bacteria,reveals their molecular ecological characteristics,performs the preliminary application of fermentation products and its optimization,and further carries out the cloning and expression of key enzymes.These works will provide theoretical basis for better use of feather keratin-degrading bacteria.The main results of this dissertation are as follows:1)Isolation was conducted with the conventional soils induced with feather addition and the soils from feather-damping sites in chicken farm,and 40 feather keratin-degrading bacteria were isolated.Nine strains were from the conventional soils with feather enrichment,and 31 strains were from the soils in chicken farm.Therefore,keratin-degrading bacteria can be successfully isolated from conventional soils with feather induction or from keratin-waste-rich sites.Phylogenetic tree based on 16S rDNA sequences showed that the 40 strains belong to four phylums,including Firmicutes,Actinobacteria,Proteobacteria and Bacteroides.The former two are gram-positive bacteria while the latter two are gram-negative bacteria,and Bacillus spp.belonging to Firmicutes dominate in them.2)In these feather-degrading bacteria,strain 205 was identified as Sporosarcina sp.according to the morphology observation and 16S rRNA gene sequencing,and was the first report of Sporosarcina sp.capable of degrading feathers.Strain 205 was confirmed to be a novel species with further identification,providing a new microbial resource for the degradation of poultry feather keratin.Strain 205 was named as Sporosarcina guangzhouensis GIMN1.015~T,preserved in China Center for Type Culture Collection(CCTCC),with accession number CCTCC M2011367.3)In the screening test,four efficient feather-degrading strains used the feather as the sole carbon and nitrogen source with the feather degradation rate of>90%,almost degrading the intact feather including rachis.Amino analysis after feather fermentation indicated that 17types of amino acids were present and the total content of amino acid was up to 3.84mg/m L.Additionally,several different plant hormones were detectable.Pot experiments with citrus and lettuce as test plants were conducted,and the results showed that,after foliar application of medium concentration amino acid fertilizer,the growth of citrus shoot and leaf was increased.The aboveground biomassof lettuce(fresh weight and dry weight)was increased with different amino acid fertilizer treatments,and the fresh weight of lettuce was increased by 20.8~32.7%.The contents of soluble protein and VC in leaves of lettuce were increased by spraying higher concentration amino acid fertilizer(35.3%and 13.3%higher than that of the control,respectively).In addition,the content of nitrite nitrogen in lettuce leaves was significantly decreased by foliar spraying of amino acid fertilizer compared with urea treatment.4)The degenerate primers were designed after searching for keratinase gene from CODEHOP database,and the 40 isolated feather-degrading bacteria and 10 non feather-degrading bacteria were used to verify the effectiveness of the primers.PCR-DGGE analysis and fluorescence quantitative PCR with the specific primer showed that exogenous addition of feathers had tremendous impact on the expression of keratinase-encoding gene in soils,with greater influence on the strains in conventional soils than those in feather dampingsoils.Featheradditionsignificantlypromotedtheexpressionof keratinase-encoding gene and species diversity.The sequencing results showed that the detected bands mainly represented actinomycetes and myxobacteria.These taxa have been seldom reported as keratin-degrader,indicating that these are new keratinase gene resources and deserve further study.5)Genome walking amplication of the full-length ORF of keratinase-encoding gene was performed with TAIL-PCR,and the full-length ORF sequences of serine protease-encoding gene were obtained from two stains(strain 205 and strain 6-16).Bioinformatics analysis of putative amino acid sequences showed that the keratinases from strains 6-16 and 205belonged to the subtilisin protease family group,containing the catalytic triad of serine proteases(Asp,His and Ser active sites).This is in accordance with the most keratinases reported previously.The coding regions of the two keratinases were cloned into the prokaryotic expression vector pET28a-EGFP,and transformed into E.coli BL21(DE3).The recombinant bacteria were screened and used to produce fusion protein.SDS-PAGE analysis showed that the two encoded keratinases were 45kDa and 35kDa after protein purification with His-tag.6)In order to optimize the fermentation conditions of high efficient strains and obtain the highest yield of kertainase,the culture conditions of high efficient strains(strains 3-2 and6-16)were optimized with response surface method(RSM).With OVAT test of single factor,the keratinase production conditions were optimized with RSM in terms of feather content,temperature and initial pH.The errors between the test values and predicted values of the fitting quadratic model of RSM were 7.19%and 3.76%,respectively,laying a foundation for the industrial application of these high efficient strains.