Gene Cloning,Expression And The Characterization Of The Keratinase Produced By Bacillus Sp.CDJYB

Keratin is mainly present in animal hair,scales,feathers,hooves,horns,etc.Because of its rich disulfide bonds and hydrophobic amino acids,keratin is often difficult to degrade,and keratinase can efficiently degrade angles under mild conditions.Protein,which allows keratin to be used environmentally.The role of keratinase is broad,and it has application value in the fields of food,medicine,cleaning and fur processing.In this paper,In this paper,the keratinase gene?Ker?of a strain of Bacillus sp.CDJYB was screened by multiple sequence alignment and molecular cloning,and the recombinant plasmid pET-28a-Ker was constructed.The keratinase was obtained by induction and purification.Its enzymatic properties and its application in the degradation of feather meal and feathers.The specific research contents and results are as follows:1.Using multiple sequence alignment,design primers,clone the keratinase gene?Ker?of Bacillus sp.CDJYB,construct recombinant plasmid pET-28a-Ker,and recombinantly express keratinase in Escherichia coli BL21 strain.At the optimal induction temperature of 20?,the keratinase activity in the crude enzyme solution was389.7 U/mL.2.The optimum reaction pH of recombinant keratinase was determined by single factor method to be 10,and the optimum reaction temperature was 50?.The enzyme was poor in thermal stability,and the enzyme activity loss exceeded 80%after incubation at 50?and above for one hour.Metal ions such as Ca²⁺,Mg²⁺,Fe²⁺,Co²⁺,Ba²⁺,Cu²⁺,Zn²⁺and Fe³⁺promote the keratinase,and Cu²⁺and Co²⁺promote the recombinase significantly.When soluble keratin is used as a substrate,the reducing agents DTT and GSH can partially inhibit the activity of keratinase,and the surfactant can also inhibit the recombinant enzyme.The inhibitory effect of SDS is over 70%.but it is strongly inhibited by low concentration of PMSF?0.01%,w/v,mg/mL?,indicating that the enzyme is a typical serine protease.The kinetic parameters V_max and K_m of keratinase for sulfonated keratin were 62.5?g/?mL·min?and 19.56 mg/mL,respectively.3.The application of recombinant keratinase to degrade feather powder and feathers found that under the action of keratinase,the feather powder?partial disulfide bond and other chemical bonds were destroyed?,the release of tyrosine was 9.74 mg/g feather powder after 6 h,which reached 39.4%of the acid hydrolysis effect;DTT plays an important role in the degradation of feathers,when the concentration of DTT reaches0.25%?w/v?,the promotion effect on feather degradation is maximized(the OD₂₈₀ of product is 0.37),and the degradation effect is 19.5 times that of the DTT-free group(the OD₂₈₀ of product is 0.019).Secondly,after using lipase and cooking pre-treatment feathers,the speed of keratin degradation feathers can be accelerated,after the cooking process The effect is remarkable,but the effect of the lipase and cooking process is not better than the single cooking method.Electron microscopy scans show that the surface of the feathers has become rougher after pre-treatment with lipase and cooking,but the surface of the feathers after cooking is more dry and fluffy.