Expression Of Keratinase Gene In Escherichia Coli And Application Of Feather Degradation Liquid In Wheat

With the rapid development of our society,poultry breeding and animal husbandry are expanding,and keratin by-products are also increasing.Among them,feather is a well-known potential source of high-quality protein,with a high proportion of keratin and essential amino acids and relatively stable amino acids.Rational use of feathers can not only provide a large number of high-quality protein resources,but also reduce environmental pollution.Aft that feather is degrade by keratinase,the prepared feather degradation liquid can be used as an amino acid fertilize,and the application of the feather degradation liquid can effectively improve the soil environment,improve the photosynthesis of crops,promote the growth of crop roots and shoot parts,and increase the stress resistance of plants,which is in line with the idea of sustainable development of agriculture and has high practical significance.In this paper,Ker N5 gene was selected from the gene bank to remove its own signal peptide.Through codon optimization,the codon adaptation index of Escherichia coli of the optimized gene increased from 0.76 to 0.96,the GC content remained unchanged at 0.5,and the free energy value of the secondary structure increased from-148.03kcal/mol to-155.65kcal/mol.Plasmid p ET-22b(+)was selected,and the restriction sites of Nco Ⅰ and Xho Ⅰ were adopted.Pel B signal peptide and 6×His tag at the C-terminal were introduced into the expression of Ker N5 gene,and a prokaryotic expression system of Kern5 was constructed,which was secreted and expressed in Escherichia coli.The high-yield strain Kc5 was screened by using milk solid medium and the ratio of protein degradation circle to colony as the index.The expression of keratinase Ker N5 was detected by SDS-PAGE gel electrophoresis,and the molecular weight of keratinase was determined to be 40 k Da.The supernatant of fermentation product was directly degraded by feathers,and the activity of keratinase was determined.In this paper,keratinase recombinant Escherichia coli Kc5 was used.By optimizing fermentation time,adding different concentrations of inducer IPTG,adding different concentrations of penetrant glycine and different concentrations of carbon source respectively,and taking the activity of keratinase in the supernatant of fermentation products,the activity of keratinase in recombinant Escherichia coli Kc5 cells and the OD600 value of fermentation broth as indicators,it was determined to add 0.05 mmol/L inducer IPTG,100mmol/L penetrant glycine and 60 g/L carbon.Keratinase was expressed by fermentation under the optimum conditions,and the activity of keratinase in supernatant was 112.56 U/m L.In this study,feather powder was degraded by keratinase to prepare feather degradation liquid,and different concentrations of feather degradation liquid were used to soak seeds to accelerate wheat germination.The results showed that the germination potential of wheat reached 82.7%,which was 7.7% higher than that of the control,and its germination rate was also the highest,reaching 97.3%,which was 6.4% higher than that of the control.Wheat was cultivated by adding feather degradation liquid to hydroponic solution.The results showed that the fresh weight of wheat reached 274.6 mg in hydroponic solution with 2.5% feather degradation liquid and amino acid concentration of 0.205 mg/m L,which was 12% higher than that of control.The maximum dry weight of wheat reached 31.5 mg,2% higher than that of the control;The content of soluble protein in wheat reached the maximum of 27.04 mg/g,which was 58% higher than the control.The root length decreases with the increase of feather degradation liquid,and the root diameter increases with the increase of feather degradation liquid.Wheat was planted in pot and feather degradation solution was applied.The results showed that the fresh weight of wheat was 317.0 mg when the amino acid concentration was2.05 mg/m L,which was 9% higher than that of the control.The maximum dry weight reached32.5mg,which was 4.8% higher than the control.The longest root length is 55.30 cm,which is 82% higher than that of the control.When the amino acid concentration of wheat was 4.1mg/m L,the maximum plant height reached 25.50 cm,which was 7.6% higher than that of the control.The diameter is the largest,reaching 1.37 mm,which is 6% higher than that of the control.