| Keratin wastes have gained much more attention because of its high protein content in recent years. A solution on how to make the keratin degradation effectively has become the focal point. Among the present methods, microbial degradation of keratin attracts more attention with its advantages like mild conditions, low energy, wide application, simple to purification and non-pollution. In addition, the mechanism of microbial degradation of keratin is still a challenge in the world. In this work, a kerationlytic bacterium, Stenotrophomonas maltophilia DHHJ, that isolated and preserved in our laboratory was studied for the mechanism of microbial degradation of keratin, including the change of the activity of keratinase and cells surfaces grow on the different media, purification methods to purify the crude enzyme and protein analysis to get the amino acid sequence to lay a foundation for the further study.S. maltophilia DHHJ grew on the LB medium and feather medium. Then get the enzyme activity diagram of intracellular enzyme and extracellular enzyme. The charts show that the keratinases secreted by S. maltophilia DHHJ are inducible enzyme. But they have their basic expression, fermentation liquid contain a small amount of secretion of keratinases when the cells grow on the LB medium. This also indicates that keratinases that secreted by S. maltophilia DHHJ have widely substrate specificity.At the same time, we observe the change of the cells surfaces through SEM, some dents are observed on the cells surfaces not only grow on the feather medium but also grow on the LB medium, but dents are deeper and larger when cells grow on the feather medium. The results show that the feather have been partial digested by the basic expressed extracellular keratinase, and then keratin particles enter into bacteria cell and had been further degraded by intracellular enzyme. In the same time, it promote the expression of extracellular enzyme.To primary purify the extracellular enzyme which secreted by S. maltophilia DHHJ, ammonium sulfate fractionation and column chromatography were used. When extracellular enzyme got through ammonium sulfate fractionation. the concentration between0%-20%of ammonium sulfate are suitable to get the primary purified enzyme according to the salting out curve. Then we chose CHT and DEAE as the medium of column chromatography, the results suggest that DEAE chromatographic column shows a better separation ability than CHT. After that, we also use the G-50sephadex to make the enzyme for further purification. But G-50sephadex can not isolate keratinases from other proteins. Then we decide that the steps of keratinases purification contain ammonium sulfate fractionation and DEAE column chromatography.The protein analysis are used to get the sequence of the purified protein. The report shows that the keratinases that secreted by S. maltophilia DHHJ may contain a functional domain which exists in the most hydrolytic enzyme-α/β hydrolase fold and the α/β hydrolase fold may take an important role in the degradation process of keratin. The molecular mass of α/β hydrolase fold is about31kDa. So there’s a wedge there between α/β hydrolase fold and the protein banding we get. A concept can explain this phenomenon is that a self degradation of the keratinases are happened during the process of purification. The protein dispersion zone below20kDa can support this concept. Some protein also take a role in the degradadtion process of keratin such as, phosphate binding protein, outer membrane protein H1, outer membrane protein, extracellular solute binding protein, periplasm amino acid transporters, ompF family protein, ABC transfer substrate binding protein etc. |
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