Secretion And Expression Optimization Of Keratinase From Bacillus Licheniformis BBE11-1 In Bacillus Subtilis

Keratinase is a group of enzymes that can specifically degrade keratin.It has broad application prospects in the fields of waste recycling,leather textile and feed addition.However,low extracellular expression remains a major problem in the development of recombinant keratinase.Therefore,how to increase the extracellular expression of recombinant keratinase has become one of the current research hotspots.In this paper,the recombinant Bacillus subtilis WB600-p43NMK-Ker was used as the research object,and the extracellular expression of keratinase was improved through the screening and rational design of signal peptides,semi-rational design of ribosome binding sites,and modification of Bacillus subtilis secretion system.The main research contents are as follows:（1）Based on signal peptide database search,prediction of secretion pathway and subcellular localization,a signal peptide library containing 20 endogenous signal peptides of B.subtilis strain 168 was established,and the original signal peptide was replaced to verify the effect on the extracellular expression of keratinase.After fermentation verification,10 recombinant strains with high extracellular secretion ability were obtained,among which the extracellular enzyme activity and specific enzyme activity of the recombinant strain containing SP_{dac B} reaches 84.3KU?m L^-1 and 182.0 U?μg^-1,which is 8.1-fold and 5.0-fold of the starting strain,respectively.（2）The signal peptide was rationally designed based on the analysis of the structure,sequence and amino acid properties of the signal peptide,and the extracellular secretion ability of the signal peptide was improved by the method of site-directed mutagenesis and fragment fusion of the signal peptide.Two strains with improved extracellular secretion ability were obtained,among which the extracellular enzyme activity and specific enzyme activity of WB600-p43NMK-Ker-SP_bsn-T2K reaches 76.7 KU?m L^-1 and 167.9 U?μg^-1,which is 48%and37%higher than that of WB600-p43NMK-Ker-SP_bsn,respectively.（3）Based on the high-throughput screening method,the ribosome binding site is optimized through semi-rational design of mutation sites and saturation mutation strategy to improve the translation initiation rate of m RNA.Two recombinant strains with enhanced keratinase extracellular enzyme activity were obtained.Among them,the extracellular enzyme activity and specific enzyme activity of WB600-R16D12 reaches 109.1 KU?m L^-1 and 221.2 U?μg^-1,which is10.5-fold and 6.4-fold of the starting strain,respectively.（4）The secretion ability of Bacillus subtilis was improved by modifying the secretion system.Additional copy of genes encoding related molecular chaperones,including transmembrane accessory proteins Sec A,Sec DF,and postfolding accessory protein Prs A,were inserted into the genome,and their expression strength was regulated by promoter optimization.In addition,a recombinant strain knocked out the repressor expression gene hrc A that represses the expression of the CIRCE regulator was constructed.The optimized recombinant plasmids were respectively introduced into the recombinant strains above.The extracellular enzyme activity and specific enzyme activity of WB600-Prs A-P8-Ker is 157.6 KU?m L^-1 and 233.6 U?μg^-1,which is 15.2-fold and 6.8-fold of the starting strain,respectively.