High-Efficiency Expression Of Key Hydrolysis Enzymes Of Feathers From Bacillus Licheniformis CP-16

The contradiction between supply and demand of protein feed resources is prominent in our country.Meanwhile there are lots of keratin protein resources,which are not fully utilized,such as feathers,fur and hoof from pigs,cattle and sheep.Feathers contain have a large number of keratin of disulfide bonds,hydrogen bonds and hydrophobic groups,which is difficult to be digested by animals because of stable structures.The treating of feathers with chemical and physical methods has some flaws,such as high energy consumption,amino acid destruction,environmental pollution and so on.Efficient biodegradation is more in line with future development needs.Opening the disulfide bonds to destroy keratious hydrophobic structure is the key to biohydrolysis of keratin.The efficient expression of key enzymes that open these chemical bonds is essential for the industrialization of keratin bioprocessing.This study aims to obtain positive mutants strain with high efficiency of keratin degradation through breeding and screening of Bacillus licheniformis CP-16 mutagenesis(physical and chemical),which lay a foundation for efficient hydrolysis of keratin.Meanwhile,the disulfide reductase gene(Trx)and keratinase gene(Ker)were amplified from CP-16,to construct two strains of recombinant Bacillus subtilis WB600 strain(B.subtilis WB600/ pSTOP 1622-pTrx,B.subtilis WB600/ pSTOP 1622-pKer),which secrete key hydrolysis enzymes of feathers.The effects of different signal peptides on the secretion of the target gene(Trx,Ker)were analyzed,and then the fermentation conditions of the recombinant strain were optimized to achieve the mass production of key hydrolysis enzymes of feathers,which was applied in the enzymatic hydrolysis of keratin.Multi-round physical and chemical mutagenesis of original bacteria by UV and diethyl sulfate to obtained the positive mutants with high yield of the key hydrolysis enzymes of feathers by primary screening and rescreening.After mutagenesis of UV,the activities of disulfide reductase and keratinase of mutated bacteria V-8 were 0.15 U/mL and 15.4 U/mL,respectively,which was 83.1% and 83.3% higher than that of the original bacteria.After the mutagenesis of diethyl sulfate,the activities of disulfide reductase of mutated bacteria D-5 was 0.14 U/mL,which was 67.5% higher than that of the original bacteria.The activities of keratinase of mutated bacteria D-2 was 13.9 U/mL,which was 65.5% higher than that of the original bacteria.Two expression plasmids(pSTOP1622-Trx,pSTOP1622-Ker)were constructed and electroporated into WB600 then expressed successfully.On this basis,a series of screening vectors contained different signal peptides were constructed by seamless cloning then expressed in WB600 The results showed that the activity of extracellular secretory keratinase was highest when keratinase was loaded with its own signal peptide.However,when the disulfide reductase carried different signal peptides,it was still expressed intracellularly,and its activity was decreased to some extent.By optimizing the fermentation culture formula of recombinant bacteria,the recombinant bacteria B.subtilis WB600/pSTOP 1622-pTrx could significantly promote enzyme production in corn flour(5.1g/L),glucose(5.9g/L),molasses(11.9g/L),glycerol(3.0g/L),peptone(20g/L),Nacl(10g/L),when fermentation broth OD600=1,add 0.5% xylose to induce fermentation for 48 h,which was 80.00% higher than the initial culture condition of B.subtilis WB600/pSTOP 1622-pTrx,440.00% higher than the optimal positive mutant bacteria V-8,and 912.50% higher than that of CP-16.By optimizing the fermentation culture formula of recombinant bacteria,the recombinant bacteria B.subtilis WB600/pSTOP 1622-pKer could significantly promote enzyme production in the corn flour(5.1g/L),glucose(5.9g/L),molasses(5.9g/L),glycerol(3.0g/L),peptone(20g/L),Nacl(10g/L),when fermentation broth OD600=1,add 0.5% xylose to induce fermentation for 48 h,which was 49.74% higher than the initial culture condition of B.subtilis WB600/pSTOP 1622-pKer,269.48% higher than the optimal positive mutant bacteria V-8,and 577.38% higher than that of CP-16.In conclusion,mutation breeding and the allopathic expression of key hydrolysis enzymes of feathers in Bacillus subtilis both can significantly improve the activity of enzymes,and the allopathic genes expression in Bacillus subtilis is better than mutation breeding as a method that high-efficiency expression of key hydrolysis enzymes of feathers from Bacillus licheniformis CP-16.