| Feather, one of the natural protein resources, is composed of almost pure keratin. As by-products of the poultry farming, a large amount of feathers is released every year in our country. Most of feathers are waste due to low utilization and slow degradation, which pollutes the environment. So it is very necessary to find effective methods to degrade feather and to make feather waste to be the cheap resource used for production of protein, development of green material, and so on. The work is helpful to protect the environment and achieve waste recycling, and has the remarkable social and economic benefits. In this paper, Stenotrophomonas maltophilia DHHJ, which was screened and saved by our research group, was mutagenized by UV light and the mutant strain L2was gotten. This research contents included the bacterial culture conditions, the properties of keratinase, the kinetics of enzyme-catalyzed reaction, the kinetics models of batch fermentation process, the mechanism of feather degradation, and the application of degradation products. Drawing the following conclusions:1. Original strain (S. maltophilia DHHJ) was irradiated under UV light for90seconds, then the mutant strain L2with good performance and genetic stability was achieved. The effects of feather content, initial pH, culture temperature, carbon and nitrogen source, metal ion, chelating agent and surfactant on feathers biodegradation were respectively studied. The results showed that the growth of L2, feather degradation and enzyme production were good on the conditions of feather content2%～4%, initial pH7.0-8.0, culture temperature35℃～45℃. Adding2%glucose or0.2%casein in the medium was good for feather degradation. EDTA could completely inhibit bacterial growth and make the enzyme deactivation. Metal ions Na+promoted the enzyme production. The effects of surfactant on bacterial growth and enzyme production were related to concentration and dose. The4g/L Tween80had positive effects on bacterial growth, enzyme production and feather degradation.2. Response surface method was used for optimizing culture conditions. Through the analysis of experimental data, the initial pH was what affected bacterial growth and enzyme production the most among different factors. The next factors were temperature, time and content of feathers. The third one was the content of glucose and the last one was the content of casein. The study showed that the optimal culture conditions were incubation time103h, incubation temperature39℃, initial pH7.6, glucose content2%, casein content0.2%, and feather content2.6%. The enzyme activity under the optimized conditions was1.8times as much as that under the non-optimized conditions. Meanwhile, cell concentration in the fermentation system also increased significantly.3. The study showed that keratinase produced by the mutant L2could degrade a variety of protein substrates. The soluble protein was more easily degraded than insoluble protein, and β-keratin was more likely to be hydrolyzed than a-keratin. The optimal pH and temperature was7.8and50℃for this keratinase, and it was stable at pH6.5-8.0, temperature30～60℃. Ca2+could promote the enzyme activity, Zn2+inhibited enzyme activity at different levels, and heavy metal ions (Hg2+, Pb2+and Cd2+) significantly inhibited enzyme activity. PMSF, Iodoacetamide, and EDTA could deactivate enzyme, which indicated that the enzyme could be a kind of serine protease, the metal ion played an important role on keeping the enzyme activity, and-SH could exists in enzyme molecule. Surfactants had no significant effects on enzyme activity. The kinetics of enzyme-catalyzed reaction was studied, and the related parameters were obtained. Vmax was0.0774U/mg·min and Km was32.5161mg/mL.4. The batch fermentation kinetics models of feather degradation were studied. The kinetics models of bacterial growth and product formation were established based on the5L fermentation experiments. On the basis of shake flask tests, the dynamics of feather consumption was preliminary discussed. It was proved that the growth rate of bacteria had a greater effect on abilities of enzyme production and degrading feather than the bacterial concentration. So strains should be kept in the exponential growth phase during the fermentation process, which was good for feather degradation and enzyme production.5. Mechanism of feather degradation was studied. In the pretreatment process of feathers, the high pressure steam could damage surface of feathers and change the microstructure of feathers, which contributed to feathers degradation. SEM revealed that bacterial cells grew closely adhered to barbules of feathers, which caused the mechanical damage to feathers. Biochemical studies indicated that intracellular fluid contained disulfide reductase-like enzyme which could break the disulfide bonds and the extracellular fluid contained protein hydrolysis enzyme which could convert protein into polypeptide and amino acids. The amount and type of amino acid in the fermented liquid increased significantly along with the fermentation, which demonstrated that feather keratin degradation did occur.6. Application of fermentation products of feathers were discussed. The fermentation liquor could be used as additive of hair care products because it had good effects on hair care and finalize the design. Feathers residue had good adsorption to the acid and neutral dyes, so it could be used as an excellent adsorption material for dye wastewater treatment. |
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