【作者】 吴小倩；

【导师】 周美华；

【作者基本信息】 东华大学 ， 环境科学与工程， 2013， 博士

【摘要】 羽毛富含角蛋白,是天然的蛋白质资源。作为家禽养殖业的副产物,我国每年产生大量羽毛。因为利用率低,降解缓慢,大部分羽毛作为废弃物存在,造成环境污染。因此研究开发有效的处理方法降解羽毛角蛋白,使羽毛废弃物成为廉价易得的原料,用于蛋白质生产、绿色材料研制等领域,既可保护环境又实现废物的资源化利用,具有显著的社会效益和经济效益。本论文采用课题组筛选的嗜麦芽寡养单孢菌Stenotrophomonas maltophilia DHHJ诱变菌降解羽毛角蛋白,分别对发酵条件、酶学性质、分批发酵动力学和羽毛降解机理进行研究,为该菌降解羽毛角蛋白提供了相应的科学依据；研究降解产物的应用,实现全部产物包括发酵液和羽毛残渣的再利用,给下游产品开发提供了技术支撑。论文得到以下研究结论：1、原始菌株(S.maltophilia DHHJ在紫外光下照射90秒,筛选得到性能良好,遗传性稳定的变异菌株L2。采用单因素实验方法考察各因素对S.maltophilia DHHJ L2降解羽毛的影响,得到：在羽毛含量2%～4%,初始pH值7.0～8.0,培养温度35℃～45℃条件下,菌株L2的生长情况良好,羽毛降解能力和产酶能力较高；外加2%的葡萄糖、0.2%的干酪素可促进菌株对羽毛的降解；螯合剂EDTA完全抑制细菌生长,使酶失活；Na+可促进产酶；4g/L Tween80对细菌生长、产酶、羽毛降解有一定促进作用。2、采用响应面法优化发酵条件,结果表明：对细菌生长和产酶的影响最大的因素是初始pH,其次是培养温度、时间和羽毛含量,再次是葡萄糖含量,最后是干酪素含量。最佳发酵条件为：培养时间103h,培养温度39℃,初始pH值7.6,葡萄糖含量2%,干酪素含量0.2%,羽毛含量2.6%,该条件下所得酶活力是未优化条件下的1.8倍,同时菌体浓度也明显提高。3、对角蛋白酶的理化性质及反应动力学研究表明：变异菌株L2所产角蛋白酶对可溶蛋白的降解能力优于不可溶的角蛋白,对p-角蛋白的降解能力优于α-角蛋白；该酶最适pH值和温度是7.8和50℃,并在pH7.0～8.0,温度20℃-50℃下能保持稳定；金属离子Ca2+可促进角蛋白酶的活性,Zn2+不同程度的抑制酶活,重金属离子Hg2+、Pb2+、Cd2+明显抑制酶活；丝氨酸蛋白酶抑制剂PMSF,巯基抑制剂Iodoacetamide,金属蛋白酶抑制剂EDTA均会使酶失去活性,说明该酶是一种丝氨酸蛋白酶,在酶的活性部位存在着对酶活起重要作用的金属离子,同时酶分子中还可能有-SH存在；表面活性剂对酶活没有明显影响。由L-B作图法求得该酶最大反应速度Vmax为2.15U/(mg.h),米氏常数Km为29.65mg。4、分批发酵动力学研究结果表明：菌株降解羽毛的能力和产酶能力受菌体生长速率的影响远大于菌体浓度的影响,使发酵体系中的菌株处于指数生长期对羽毛降解和产酶有利。5、羽毛降解机理的研究结果表明：高压蒸煮预处理破坏了羽毛表面的某些物质,改变了羽毛的微观结构,使羽毛更易被微生物降解；SEM检测表明细菌依附在羽枝上生长,产生的机械破坏作用有利于羽毛降解；细菌胞内液中含有二硫键还原酶类似物,可使羽毛角蛋白硫解变性,胞外液中的蛋白质水解酶可使变性蛋白水解为多肽和氨基酸；随着发酵的进行,发酵液中氨基酸的种类和总量明显增加,说明羽毛角蛋白降解确有发生。6、探讨了羽毛发酵产物——发酵液和羽毛残渣的利用,实验结果表明：发酵液在护发、修复、定型三方面有较好的效果,具有作为护发品添加剂的潜在应用价值；羽毛残渣对酸性、中性、碱性三种类型的染料表现出不同程度的吸附能力,对酸性、中性染料吸附效果良好,可作为优良的吸附材料用于染料废水的处理。更多还原

【Abstract】 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.更多还原