Efficient Preparation And Substrate Specificity Engineering Studies Of Keratinase From Pseudomonas Aeruginosa 4-3

Keratins are a class of naturally occurring,difficult-to-degrade polymers rich in various essential amino acids,possessing extensive value for resource utilization.However,due to the compact structure and stable nature of keratins,they are resistant to degradation by ordinary proteases and are often carelessly discarded,resulting in severe environmental pollution and wastage of protein resources.Keratinases are hydrolytic enzymes capable of degrading keratins,caseins,collagens,and other soluble or insoluble proteins,holding significant application prospects in the food,feed,and leather industries.Nevertheless,existing keratinases generally suffer from low yields,poor activity,insufficient thermal stability,and inadequate substrate specificity,failing to meet industrial demands and hindering their commercialization.This research screened and obtained a novel keratinase with commercial application potential.Through the establishment of a heterologous expression and fermentation system,efficient production of the keratinase was achieved.Protein engineering techniques were employed to modify critical performance indicators such as catalytic efficiency,substrate specificity,and thermal stability.By screening,engineering,and fusion expression of substrate-binding domains,a novel keratinase with targeted adsorption and specific degradation of feathers was obtained.The specific research content and achievements are as follows:(1)A novel keratinase exhibiting outstanding thermal stability and organic solvent tolerance was obtained.Initially,a strain of Pseudomonas aeruginosa4-3 capable of efficiently degrading feathers was screened.Under conditions of40°C and an initial p H8.0,this strain achieved almost complete feather degradation(95.31%)after 48 h of fermentation,with the fermentation broth containing 2568.3 μg/m L of soluble protein and a keratinase yield of 295.2U/m L.Through chromatographic purification,the A2 protein(4-3Ker)with keratinase activity and the A1 protein with aminopeptidase activity were obtained.Both are metalloproteinases that exhibit negligible activity loss after incubation for 1 h at their respective optimal temperatures and demonstrate excellent tolerance towards organic solvents such as methanol,acetone,hexane,and heptane.Further investigation revealed that the keratinase is the primary enzyme contributing to feather degradation by P.aeruginosa 4-3,while the aminopeptidase assists in promoting the conversion of soluble proteins into free amino acids.(2)A heterologous expression and fermentation system was established to achieve efficient production of the keratinase.Through genome sequencing of P.aeruginosa 4-3,the gene encoding the 4-3Ker was successfully identified,and the recombinant expression strains Escherichia coli Trans B(DE3)/p ET22b-4-3Ker and Bacillus subtilis/p MA5-4-3Ker were constructed.By optimizing the fermentation conditions and media components,highly efficient expression of 4-3Ker was achieved in E.coli and B.subtilis,with a maximum enzyme activity reaching 6642.38 U/m L.Under shake flask fermentation conditions,the yield of recombinant 4-3Ker reached 1.36 g/L.Furthermore,the recombinant 4-3Ker exhibited consistent properties with the wild-type enzyme in terms of optimal temperature,optimal p H,thermal stability,and organic solvent tolerance,laying the foundation for subsequent protein engineering modifications.(3)Through protein engineering,a keratinase mutant(M128R/A138V/V142 I,4-3Ker-MAV)exhibiting significantly enhanced catalytic activity,substrate affinity,and thermal stability was obtained.By employing bioinformatics analysis,structural alignment,and conservation analysis,the key amino acid residues in the S1’ substrate-binding pocket were identified.Leveraging rational design,single-site saturation mutagenesis,and combinatorial mutagenesis strategies,the optimal triple mutant 4-3Ker-MAV was obtained.Compared to the wild-type enzyme,this mutant demonstrated a1.21-fold increase in catalytic activity towards keratin substrates,with the catalytic activity ratio for keratin to casein rising from 0.48 to 1.01.Additionally,the half-inactivation temperature was elevated by 3.13°C to69.08°C.Moreover,its catalytic efficiency towards natural feathers was enhanced by 32.85%,achieving a degradation rate of 70%.Molecular dynamics simulations revealed that the improved properties of the mutant originated from changes in the flexibility and hydrophobicity of its substratebinding region,as well as enhanced flexibility of the substrate entry and exit channels.(4)A substrate-binding domain engineering strategy was employed to construct a keratinase capable of targeted adsorption and specific degradation of feathers.To overcome the challenges of low substrate specificity towards insoluble substrates like feathers and the inefficient catalysis resulting from non-specific binding between the free enzyme and substrate,relying on free diffusion,this study screened and obtained the E423-PPC1 substrate-binding domain,which specifically adsorbs to feather keratins.This substrate-binding domain primarily utilizes hydrogen bonding and hydrophobic interactions as driving forces for temperature-and time-dependent targeted adsorption to feather keratins.By fusing and expressing it with 4-3Ker-MAV,followed by optimization,a novel keratinase(4-3Ker-MAV-PPC)capable of targeted degradation of feather keratins was successfully obtained.Compared to 4-3Ker-MAV,the fusion enzyme did not exhibit a significant change in affinity towards keratin substrates.However,its catalytic efficiency increased by 0.97-fold,achieving a degradation rate of up to 80% for natural feathers within 6 h,providing a new strategy to enhance the catalytic efficiency and substrate specificity of keratinases.In summary,this study has produced an engineered novel keratinase that combines high yield,high activity,high efficiency catalysis,high substrate specificity,and high thermostability,which holds immense potential for industrial applications.Simultaneously,this research provides a new technical route for precise modulation of the substrate specificity of keratinases.