Enzymolysis And Purification Of A Keratinase From Keratin-degrading Bacterium Stenotrophomonas Maltophilia DHHJ

As encrusted protein, keratin has the characteristics of no-dissolve, anti-decomposition and anti-pain, and it also cannot be dissolved in normal conditions. So keratin is very difficult to be delt with. And at the same time a lot of keratin materials, as wastes, are released in China, such as feathers from poultry farms, leather heels, and so on, but they have not been used properly. With the rapid development of stockbreeding and amino acid industry, the demand of protein resource is increasing recent years. So it is very necessary to research effective methods to make waste feather convert to protein or mixing amino acid and to make it to be used directly or to be the cheap resources for the purification of amino acid. To protect our environment and make full use of the resources, we will turn the wastes to treasures with biotechnology method. In this work, a novel kerationlytic bacterium, Stenotrophomonas maltophilia DHHJ , which isolated and preserved in our laboratory, were studied for the range of enzymolysis of the crude enzyme from the bacterium. And then the crude enzyme was purified and obtained a monomeric enzyme.Firstly, we chose several common keratin wastes and measured enzyme activity on those keratin substrates by crude enzyme. The results showed that fowl and pigeon feather belonging to 6-keratins were more active for degradation compared with other keratin sources. 6-keratin materials had better degradation by the enzyme from Stenotrophomonas maltophilia DHHJ. In addition, this bacterial stain could also degraded a-keratins which include hard keratins (cattle horn,caprine horn,human finger nail,Bauhinia variegata) and soft keratins (wool,human hair,cattle hair). From the results of hydrolysis of different keratin-containing substrates by the keratinase of Stenotrophomonas maltophilia DHHJ, it was concluded that the enzyme could be suitable for development and application in industry, since it had a broad range of hydrolysis.Secondly, the actions of color in feather degrading by the enzyme were studied. Feathers with three different colors and similar length and weight from the same flock in a poultry farm were chosen. Given their physical similarity, we assumed that the different color feathers in our samples had similar surface areas exposed to enzymatic action. Whole feather and feather powder were empolyed as study objects separately. The results displayed that feathers' color would affect keratinase activities; however, it was only for entire feather, not for ground feather. The deeper color of the feather, the lower the active of degradation became. This indicated that melanin maybe only increase superiority of feathers' structure. If feathers were crushed to powder and destroyed feathers' structure, the melanin would have no influences on feather-degrading. So it is important to use suitable treatment in order to reduce negative effects of self structure before feather fermentation.Thirdly, according to our laboratory conditions, we determined ammonium sulphate precipitation followed by gel chromatography and CHT chromatography to purify the keratinase from crude enzyme. For ammonium sulphate precipitation, ammonium sulphate with different concentrations was added to culture filtrate. Considering relative activity and yield, the precipitate resulting from ammonium sulphate fraction 10% concentration (w/v) was suitable for the next purification. Gel chromatography was performed on a Sephadex G-100 column equilibrated with 0.2M phosphate buffer, pH 7.5 at a flow rate of 0.5mL/min. Elution was performed with the same buffer at a flow rate of 0.4mL/min. A portion (0.5mL) from ammonium sulphate fraction 10% concentration was applied to the Sephadex G-100 gel bed. Protein was collected with 30 fractions (1-mL). The fractions possessing highest specific activity were pooled and used for the next purification. For CHT chromatography, the CHT column was equilibrated with 5mM phosphate buffer (buffer A), pH 7.5 at a flow rate of 0.5mL/min. Elution was performed with 400mM phosphate buffer (buffer B) and buffer A at a flow rate of 1mL/min. Both buffer A and B contained 30mM NaCl to strengthen the effect of purification. The portion (1.4mL) obtained from gel filtration on Sephadex G-100 was applied to the CHT chromatography colomn. Elution was performed with buffer A for 2min, followed by 0%-100% gradient buffer B for 4min, and then buffer B for 19min. Protein were collected with 25 fractions (1-mL). After those three steps in purification of crude enzyme, the fraction obtained from CHT chromatography showing 17.24-fold purification.Finally , the purified enzyme was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). It was carried out with a 5% stacking gel and a 12% separating gel. The results of SDS-PAGE at native and non-native electrophoretic conditions indicated that the purified keratinase is a monomeric enzyme with a molecular mass of 35.2kDa.