Analysis Of Key Steps In Degradation Of Feather Keratin By Bacillus And Efficient Expression Of Keratinase

Keratinase is a protease that specifically hydrolyzes keratin and has important application prospects in the biological recovery of keratin waste.However,it is inefficient in catalyzing natural keratin substrates.This research focuses on analyzing the key step of Bacillus degrading feather keratin and the high-efficiency expression of keratinase to alleviate the low efficiency of feather keratin waste biotransformation based on keratinase.The main research results are as follows:?1?Constructing artificial flora for efficient degradation of feather keratin based on co-culture mode.Microbial flora can distribute the metabolic burden and play a synergistic effect through the division of labor between strains,and has the ability to effectively transform complex substrates and can be modularized.In this study,by analyzing the characteristics of the feather keratin degradation system of the two strains,it was found that the keratinase activity secreted by B.licheniformis BBE11-1 was lower than that of S.maltophilia BBE11-1,but the extracellular enzyme system was more abundant.Based on the theoretical basis of the multi-factor synergistic effect of keratinase as the main factor in the process of microbial degradation of keratin,a co-culture system was developed and optimized for feather keratin degradation.Combined with the temperature conversion strategy,the degradation rate of 50 g×L^-1feather reached 81.8%after only 48 h in the 3-L fermenter,which greatly improved the efficiency of feather degradation by the keratinase secreting strain.?2?Cysteine cycle metabolism is a key step for Bacillus to degrade keratin.Cysteine is a characteristic product of keratin degradation.In this study,it was found that cysteine can produce sulfite through a two-step metabolic reaction in Bacillus cells,and the synergistic effect of sulfite on the hydrolysis of feather keratin by keratinase was confirmed in vitro.To this end,it is proposed that the cysteine cycle metabolism steps mediate the ability of keratinase-secreting Bacillus to degrade feather keratin.Subsequently,by knocking out key genes in the cysteine metabolism and sulfite transport pathways,it was found that the mutant strains?yde D,?Cdo1,?Ast1,?SSU1 and?Cdo1?SSU1 lost the ability to convert L-Cys into sulfite and the ability to degrade feathers.Furthermore,the synergistic system of keratinase and sulfite was implanted into B.subtilis WB600 and a metabolic model was established.By enhancing the expression intensity of Cdo1and Ast1,the ability of strains BSKer-43-CA and BSKer-566-CA to metabolize L-Cys to produce sulfite increased by 69.63%and 37.06%,respectively,and the soluble protein content in feather hydrolysates also increased correspondingly 35.82%and 26.87%.Therefore,cysteine-mediated cyclic metabolic steps mediate the ability of keratinase-secreting Bacillus to degrade feather keratin.?3?Constructed and optimized heterologous expression system of keratinase.Highly active keratinase corresponds to better feather keratin degradation ability.In this study,the most active extracellular keratinase Ker Z1 was obtained by using the constitutive promoter p43.The optimum pH and temperature of Ker Z1 are 10 and 60?,respectively.Using RBS calculator v2.0 to predict the translation level of keratinase,it is found that as the RBS sequence moves closer to the translation region,its impact on translation initiation efficiency decreases.Saturation mutation was performed in the region from-13 to-18,and the best mutant RBS3 had an extracellular keratinase activity of 22600.1 U×m L^-1.The extracellular keratinase activity of RBS3 in the optimized medium reached 94389.2 U×m L^-1.The extracellular keratinase activity and productivity of RBS3 in the 15-L fermentor were 426600.3 U×m L^-1 and15235.7 U×m L^-1×h^-1.Keratinase and sulfite synergistically catalyze 100 g×L^-1 feathers for 12 h,and the total amino acid content in the hydrolysate reached 56.6 g×L^-1.?4?Engineering pro-peptide enhances keratinase activity.Pro-peptide is essential to keratinase for active expression.In this study,it was confirmed that the pro-peptide determines the extracellular activity of keratinase by truncating the secondary structure of the leader peptide one by one and replacing the amino acid at the cleavage site P1 of the leader peptide.Through sequence alignment and site-directed mutagenesis,the keratinase activity of 7 mutants was increased by 16%to 66%.Simultaneously modified six amino acids were based on three codon saturation mutations.By constructing a smaller mutant library,the best mutant 2-D12 keratinase activity in the shake flask could reach227300.6 U×m L^-1.Using industrialized culture medium,through real-time control and combined with the feeding strategy,the keratinase activity of mutant 2-D12 in the 15-L fermenter reached 610800.9 U×m L^-1,which is the highest level currently reported.The hydrolysis rate of 2-D12 synergistic sulfite on 100 g×L^-1feathers reached 90.67%in 12 h.The total amino acid content in the feather hydrolysate was 65.8 g×L^-1,and it also contained short biologically active peptides with a molecular weight of about 1 k Da or less.