Optimal Carbon Source For Keratinase Production From Pichia Pastoris And Characteristics Of Recombinant Keratinase And Its Efficacy For Pigs

The present study was conducted to optimize the carbon source of fermentation medium for keratinase production by recombinant Pichia pastoris pPICZaA-kerAoptil developed in our laboratory. The enzymatic properties of the recombinant keratinase and the hydrolysis products of feather in vitro were both evaluated. Furthermore, the keratinase was added to a feather meal-containing diet to test the effect on growth performance, nutrient digestibility, intestinal microflora, serum thyroid hormones level, and urea nitrogen of growing pigs.These results would be reference and evidence for the development and application of keratinase in animal nutrition and feed industry.Experiment 1:Optimization of carbon source of fermentation medium for keratinase production by small-scale fermentation and assay of functional characteristicsThe small-scale fermentation was performed in a 30L fermenter with two kinds of carbon sources, glycerol and glucose. The highest expression level was 568 U/mL, and was achieved after 72 h fermentation in glucose culture medium. The enzyme was characterized to have a molecular weight of 39 kDa, an optimal pH of 7.5 and an optimal temperature of 50℃. In addition, this enzyme was stable at 50℃, retained almost 100% of the highest activity at this temperature for 30 min. Analysis of the degradation products showed that keratinase treatment promoted the release of sulfur-containing amino acids from azure keratin in vitro.Experiment 2:In vitro digestibility and the hydrolysis products of feather by keratinaseCompared to blank group and pepsin-trypsin group, the digestibility of feather powder was significantly increased by keratinase in vitro. The content of most essential amino acids in supernatant fluid also increased significantly. In addition, when feather was treated with keratinase before hydrolyzed by pepsin-trypsin, the digestibility of feather powder was 16.56%, the content of soluble protein, most essential amino acids and total free amino acids were significantly increased.Experiment 3:The application efficacy of recombinant keratinase in growing pigsThe keratinase was added to a feather meal-containing diet to test the effect on growth performance, nutrient digestibility, intestinal microflora, serum thyroid hormones level, and urea nitrogen in growing pigs. Sixty DLY growing pigs (58.1 ±1.1 kg of initial body weight) were allotted to five groups. The dietary groups included:(1) basal diet (CD); (2-5) 5% of feather meal with keratinase supplementation at level of 0 U/kg、3000 U/kg、6000 U/kg,9000 U/kg. The experiment was conducted for 14 days. Results showed that feather meal supplementation significantly declined and keratinase supplementation improved pig performance.ADG was increased by 21.3% when pigs received a diet supplemented with keratinase at 9,000 U/kg. In addition, keratinase supplementation increased the apparent digestibility of amino acids, energy, dry matter, phosphorus, and organic matter (p<0.05). Analysis of hindgut microflora indicated that keratinase supplementation increased the copies of lactobacilli, but decreased the number of E. coli (p<0.05). The potential benefits of keratinase for growing pigs should make it an attractive applicant for industrial enzyme production.