Study On Microbial And Enzymatic Degradation Of Feathers And Its Degradation Characteristics

The vigorous development of the poultry meat processing industry has resulted in the production of millions of tons feather waste worldwide every year,and the disposal of the waste has become an urgent problem to be solved.Compared with the disadvantages of high energy consumption and high pollution associated with physical and chemical methods,biodegradation of feathers has the advantages of environmental friendliness,low energy consumption and high nutritional value of hydrolysates.Keratinase can specifically degrade keratin,but it needs to synergize with reducing agents to achieve efficient degradation of feathers,but reducing agents such as DTT、β-mercaptoethanol and sulfite are usually toxic or cause the hydrolyzate to be too salty.Natural feather-degrading strains due to their own redox metabolism system and keratinase-producing ability,but low degradation efficiency is an obstacle for industrial application.This study took the highly active Ker Z1 and B.licheniformis BBE11-1 as research objects,and optimized the combined system to promote feather degradation rate.In addition,the disulfide reductase was heterologously expressed and optimized in B.subtilis WB600,and the feather degradation ability was enhanced through the synergistic of Ker Z1 and disulfide reductase.The key findings include:（1）Construction of a combined system for degrading feathers.Based on the analysis of Ker Z1 and B.licheniformis BBE11-1 for degrading feathers alone,a system for combined keratinase and wild-producing enzyme strains to degrade feathers was developed.The B.licheniformis BBE11-1 culture system and the complex conditions of Ker Z1 were optimized by single factor and orthogonal experiments.The degradation rate of 100 g·L^-1duck feather at 40℃,p H 10.0,keratinase activity of 50 k U·m L^-1,and B.licheniformis BBE11-1 inoculum of 10%,in36 h reached 74%,an increase of 18%and 15%to Ker Z1 and B.licheniformis BBE11-1 degrade alone;at 40℃,p H 10.0,keratinase activity 30 k U·m L^-1,B.licheniformis BBE11-1 inoculum amount 8%,the degradation rate of 100 g·L^-1chicken feather was 64%in 32 h,29%and 28.5%higher than Ker Z1 and B.licheniformis BBE11-1 alone.（2）Recombinant expression of disulfide reductase.Four disulfide reductases derived from B.subtilis:alkyl peroxide reductase（Ahp C）,dihydrolipoic acid dehydrogenase（DLD）,peptide methionine sulfoxide reductase（Msr A）and thioredoxin reductase（Trx R）was successfully extracellular expressed in B.subtilis WB600,and the specific enzyme activities were 0.53 U·mg^-1,1.69 U·mg^-1,16.7U·mg^-1and 2.06 U·mg^-1,respectively.The combination of disulfide reductases and Ker Z1 can promote the degradation of feathers by keratinase.Among them,the soluble protein content of Msr A+Ker Z in feathers hydrolyzate is 3 times than that of Ker Z1 alone.（3）Signal peptide screening to enhance disulfide reductase activity.12endogenous signal peptides of B.subtilis were selected for screening,among which Pel C,Npr E,Pho D,SPker,Ywb N,Yxk H increased the Msr A extracellular disulfide reductase by 1.4,1.8,2.0,2.2,2.6 and 4.2 times,respectively.And Yxk H significantly increased its extracellular secretion.（4）Fermentation optimization of recombinant strains expressing disulfide reductase.Fermentation optimization was performed on the Msr A strain carrying Yxk H(WB600-p P43NMK-mrs A_{SPyxk H}).The optimized fermentation medium was1.5%soluble starch,4%corn steep liquor,2%NH₄Cl,0.2%Na₂HPO₄12H₂O,0.1%KH₂PO₄,0.08%Ca²⁺,and the conditions were initial p H 9.0,temperature 37℃,inoculum size 5%,the disulfide reductase activity is 4.9 U·m L^-1,increased by 5 times.The degradation rate of feathers is increased by 11%combined with Ker Z1.（5）Compositional analysis of feather hydrolysate.The molecular weight distribution of soluble protein and peptide in the hydrolyzate were determined by HPLC.Its contents in duck and chicken feather hydrolyzate were 5.6 g·L^-1and 3.2g·L^-1,respectively.The amino acid in feather hydrolyzate was analyzed by an amino acid analyzer,and its content was 7472.33 mg·L^-1and 5589.67 mg·L^-1,respectively.Adding 10%(v·v^-1)feather hydrolyzate based on LB,the cell density of WB600-p P43NMK-ker was increased by 2-2.4 times compared with the control,and the keratinase activity was increased by 1.5 times;10%feather hydrolyzate completely replaced the carbon and nitrogen source of LB,the cell density and enzyme activity had no significant difference with the control.Using 10%(v·v^-1)feather hydrolyzate to irrigate corn seedlings,its fresh weight increased by 1.4-1.7times compared with the control after three weeks.