Efficient Expression And Modification Of Keratinase In Bacillus Subtilis For Functional Keratin Preparation

Keratin is a kind of natural biological material,which exhibits favorable biocompatibility and biodegradability nature,thus showing application potential in the field of tissue engineering materials.Keratinase is of great significance in keratin materials biodegradation and functional keratin preparation due to its unique substrate specificity in decomposing keratins.However,the industrial production of keratinase is limited by its low yield and poor stability,which seriously hinders its commercial application.In this study,the efficient expression of keratinase was achieved based on the strategy of pro-peptide engineering and promoter engineering modification.The thermal stability of keratinase was improved by combining the computer-aided design and molecular evolution.Furthermore,using the keratinase as a catalyst,the biological preparation process of wool active keratin was explored.The main results were described as follows:(1)The keratinase activity was improved using pro-peptide engineering modification.Pro-peptide plays an important role in guiding the correct folding of mature enzyme.In this study,the full-length pro-peptide area of recombinant Bacillus subtilis keratase Ker Bp was proved crucial in the activation of mature enzyme by truncated mutation.By replacing the amino acid at the pro-peptide cleavage site(P1),phenylalanine,an aromatic hydrophobic amino acid,was found to be beneficial to the cleavage of the pro-peptide,with the mutant Y(P1)F increased keratinase activity by 1.7-fold.Furthermore,site-directed mutagenesis in the pro-peptide region was performe based on homologous sequence analysis,and six mutants with improved activity were obtained.Among them,I(62)K mutants significantly increased the activity of keratinase by about 2.2-fold.It was suggested that the mutation of the appropriate site of the pro-peptide could change the folding rate of the protein and thus increase the expression level of the enzyme.A saturated mutant library was further constructed for these six sites and 15 mutants with activity over 800 U·m L^-1 were obtained after three rounds of effective screening,among which the enzyme activity of mutant M7 reached 1114 U·m L^-1,which was 6times higher than that of the wild type.Sequencing analysis showed that M7 contained mutations in 5 sites which were found mostly occurred in or near irregular coils.This kind of mutation lead to decreased rigidity and increased flexibility of the pro-peptide region,which was conducive to guiding the folding of mature enzymes.(2)The growth and enzyme production process of host cells were regulated using promoter engineering strategy.It was found that with the increase of keratinase activity and yield,keratinase showed the ability to degrade the endogenous proteins of the host B.subtilis,which affected their normal growth and metabolism process,leading to the decrease of biomass and enzyme production.In this study,the promoter engineering was employed to regulate the growth and enzyme production of host cells.It was found that by replacing the constitutive promoter with the density-dependent promoter,the cell density was increased from 8 to 10 and the maximum keratinase activity reached 2300 U·m L^-1,twice of the constitutive promoter.By further optimizing the copy number of the promoter,the two-tandem promoter P_{apr E}-P_{apr E} with the highest activity 3080 U·m L^-1 was obtained.Furthermore,the-10 and-35 regions of promoter P_{apr E}-P_{apr E} were mutated into the conserved sequences for?^A recognition,and the enzyme activity was increased to 3500 U·m L^-1.By optimizing the carbon sources of the broth,it was found that adding 2%glucose could prolong the growth time of the strain,thus promoting their growth and accumulation.Therefore,the maximum cell density reached 15.6 and keratinase activity increased to 4200 U·m L^-1 after 84 h of culture.(3)The thermal stability of keratinase was improved by modification of critical Loop region based on computer aided strategy.The thermal stability of keratinase is a prerequisite for efficient degradation of keratin resources such as wool fiber at high temperature.In this study,the thermal stability of keratinase Ker Bp was improved by combining computer aided design with protein engineering modification.Three highly flexible loops of keratinase Ker Bp and one Loop associated with Ca²⁺binding were predicted by computer aided calculation and analysis,and the amino acid residues contributing less to stability were located.In addition,the amino acid residues with lower frequency in Ker Bp were replaced with the"high frequency amino acids"based on homologous sequence alignment.A total of 65 single-point mutants were constructed,and 6mutants with improved thermal stability while maintaining enzyme activity were obtained by combining virtual and experimental screening.Finally,the synergistic modification increased the half-life at 60?from 17.3 min to 66.1 min,and the optimal reaction temperature was increased from 40?to 60?.(4)Macromolecular functional keratin was prepared by controlled degradation of wool fibers by keratinase.Keratin is a kind of biomaterial with excellent performance and great application potentials.However,the natural keratin is difficult to utilized as biomaterial for its compact structure and poor solubility.The treatment of keratin by chemical method often destroys the active group on the molecule,so it is of great significance to explore the enzymatic preparation of macromolecule and soluble keratin.In this study,soluble wool keratin with molecular weight about 45 k Da and 28 k Da was prepared by using the developed keratinase in previous work.The wool dissolution rate was 68.1%,and the extraction rate of keratin was 33.7%.The process of enzymatic degradation of wool was explored,which showed that keratinase was degraded layer by layer from the scale layer of wool fiber,and finally the complex structure of wool was destroyed and dissolved.The structure analysis of the keratin extracts by FT-IR,NMR,XRD,and CD spectrum showed that the enzymatic hydrolysis disrupted the disulfide bonds and hydrogen bond between the macromolecular chain of keratin protein and molecular internal.The amount of?-structure was decreased and the tight structure of wool fibers became loose and stretched.The results of in vitro evaluation showed that the keratin extracts had good biocompatibility,showing obvious effects of promoting cell proliferation and migration,and had strong antioxidant activity.To explore the application potential of keratin extracts in the field of tissue engineering,hydrogels were prepared based on their self-assembly characteristics.In vitro culture experiments of mouse fibroblasts showed that the keratin gel had excellent biocompatibility and could be used as scaffold for supporting cell growth.In vivo study on promoting wound healing in mice showed that the keratin hydrogel could promote wound repair by promoting the growth and migration of fibroblasts and accelerating the formation of new blood vessels and collagen deposition,which proved that the keratin hydrogel was an excellent wound dressing.