Isolation And Identification Of Keratinase-producing Bacterium And Properties And Application Of Keratinase

Keratin is major component of cuticulun, mainly exists in feather, wool, hoof, horn, nails and so on. Due to their intensive cross-linking by hydrogen, hydrophobic and cystine disulfide bonds, keratins presenting high stability and are hardly to be degraded. Keratinases are a group of proteolytic enzymes with the capability to hydrolyze keratin. They have property to attack tough insoluble structural of keratin and then to degrade them. Keratinolytic enzymes have important uses in the biotechnological conversion of keratin-containing wastes from poultry and lether industries in a non-polluting way. Insoluble feather keratins can be converted to feedstuffs, fertilizers, and films after enzymatic hydrolysis. In addition, applications of these enzymes for pharmaceutical and cosmetic purposes have also been reported.We have isolated several feather-degrading bacteria strains from the soil of keratin-enriched environments.We selected a strain which has excellent feather-degradating capacity among these strains for next detailed studies. The strain was identified Bacillus subtilis FJ-3-16 according to 16S rRNA sequencing. B. subtilis FJ-3-16 could product keratinase in FM3 medium. After production optimization, a keratinase named Ker FJ was purified using ammonium sulfate precipitation and ion-exchange column chromatography. Then it’s keratiolytic properities were studied, including the optimal temperature/optimal pH, the thermo-stability,substrate specificity, the influence of inhibitors, reducing agent, oxidizing agent, detergent, metal ion, salt concentration and some commercial laundry detergent products and so on. Finally, some applications researchs toward keratinase Ker FJ were also studied, including its abilities on deharing and as a potential additive for laundry detergents.The results of the study are as follows:(1) We isolated 203 keratin-degrading bacteria strains from the soil of keratin-enriched environments. The strain FJ-3-16 produced keratinolytic enzyme using chicken feather as the sole carbon and nitrogen source and was identified as Bacillus subtilis strain via 16S rRNA sequencing.(2) The culture conditions for enzyme production were optimized, including initial pH, cultivation temperature, time and so on. The optimal conditions for keratinase production were considered as follows:6% logarithmic growth phase cells were invaded into fresh FM3 medium for continuous cultivation at 30℃,200 rpm for 72 h. After fermentation, the supernatant was collected by centrifugation. Then keratinase Ker FJ was purified using ammonium sulfate precipitation and ion-exchange column chromatography.(3)The inhibitor experiments suggested that keratinase Ker FJ was a serine protease. The optimal pH was 9.5 and the optimal temperature was 55℃ when it catalytic keratin as a substrate. The activities of Ker FJ were not decreased rapidly when some kind of oxidant, detergent and metal ions existed, this indicated that the enzyme has excellent tolerance. KerFJ could completely degrade feather and wool in 48 h and it has the deharing abilities toward sheep and black donkey fur within 28 h. It could wash the ditry cotton more cleaner when added to the laundry detergents, compared with the detergents exsist alone. This suggested that the keratinase KerFJ may has some applications in laundry industry.