Identification, Characterization And Application Of A New Kerationlytic Bacteruium That Completely Degrades Feather Keratin

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 are released in China, but they have not been used properly. With the rapid development of stockbreeding and amino acid industry, the demand of protein resource is increasing these years. So it is very necessary to research effective methods to make waste feather convert to protein and mixing amino acid and to make it to be used directly or to be the cheap resources for the purify 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 new Kerationlytic bacteruium were studied for the use of feather wastes.First of all, strain K1, which showed the activity of feather degradation, was isolated from soil. After observing the shape feature and sequencing the 16SrRNA, strain K1 was identified and named as Stenotrophomonas maltophilia DHHJ. Stenotrophomonas maltophilia was found for the first time to be able to degrade feather.At the optimal exposure time, the technique of UV-induced mutation was applied to Stenotrophomonas maltophilia DHHJ, which isolated from soil. The mutant strain L4 with high activity of feather degradation was obtained by a series of screening methods. The mutant strain L4 could make the integrated feather fibres breakdown after incubated for one day. The feather was completely disintegrated by the mutant strain L4 after incubation for six days, and the calamus was degraded partially. The degrading rate reached 80%, which increased by over 20% than the original strain. The maximum of protein contents, enzyme activity and amino acid contents reached 7.05mg/mL, 8U/mL, and 0.19mg/mL, respectively. All of these results indicated that the mutant strain L4 possessed the stronger capacity of degrading feather keratin than that of the original strain. The crude enzyme from original Stenotrophomonas maltophilia DHHJ's optimum conditions of activity were pH 7.5 and temperature 40℃, The enzyme was stable at pH between 6.5～8.0 and temperature is up to 40℃; The crude enzyme from the mutant strain L4's optimum conditions of activity were pH 7.8 and temperature 50℃, The enzyme was stable at pH between 7.0～8.0 and temperature is up to 60℃The crude enzyme was activated by Ca²⁺, Ba²⁺, Cu²⁺, Na⁺, K⁺ and Mg²⁺ ions, on the other hand, the crude enzyme was inhibited by Hg²⁺, Cd²⁺, Pb²⁺, Zn²⁺ and PMSF. Since it was inhibited by PMSF, the keratinas produce by Stenotrophomonas maltophilia DHHJ appears to belong to the serine-protease type. Sodium dodecyl slfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the crude enzyme is composed of two subunits; Their molecular mass were 141KD and 119KD respectively.Keratinolytic Stenotrophomonas maltophilia DHHJ was used to study the mechanism of keratinolysis. Scanning electron microscopy studies revealed that bacterial cells grew closely adhered to barbules of feathers, completely degrading them within 96 h. Biochemical studies indicated that Stenotrophomonas maltophilia DHHJ strain produced an extracellular protease, which had keratinolytic potential. The extracellular keratinolytic activity (5.5U) was synergistically enhanced by the addition of intracellular disulfide reductases (16.3U). Howerver, these enzymes alone (keratinase and disulfide reductase), whithout live bacterial cells, failed to degrade the feather. It's emphasizing that bacterical adhension plays a key role during the degradation process. The bacterical cells probably provide a continuous supply of reductant to break disulfide bridges. In addition, sulfide detected in the extracellular broth during feather degradation indicated that sulfitolysis may also play a role in feather degradation by the bacterium.Immobilized technology is the effective method to promote the biological catalysis utilization ratio and biological treatment velocity as well as realize the high-efficiency continuous production process. The method of chitosan surface adsorption to fix Stenotrophomonas maltophilia DHHJ was studied in the article. As the long utilization life, enough toughness, biologic compatibility, easy to reclaim and the stability of degradation, it's worthy of researching further in the future.A process of feather-biodegradation by microorganisms was put forward; and high-efficiency hydrolytic equipment for feather-keratin was created and studied. The equipment was designed with garbage compost and active bio-filter equipments for reference. Feathers pretreated by autoclaving used for "packing materials" and put in the middle of the equipment on which microorganisms grew. Inorganic culture media were sprayed on the "packing materials" from the top and hydrolyzate with large content of soluble proteins was led off from the bottom. Its operating and efficient parameters were as follows: airflow rate (1200L/d), influent flow rate (0.5L/d), return flow rate (8L/d), conversion percentange (42%), volumetric soluble protein production rate 5.46g/L.Wang Jing (Eovironmental Science)Supervised by Professor Zhou Mei Hua...