【作者】 王晶；

【导师】 周美华；

【作者基本信息】 东华大学 ， 环境科学， 2007， 硕士

【摘要】 角蛋白作为一种硬性蛋白，具有不溶于水和抗分解的性质，一般条件下不溶解，且不能被一般的蛋白酶水解，这给角蛋白的处理提出了难题。我国的角蛋白资源极其丰富，它们大部分没有被充分利用，有的甚至造成环境污染。而近年来畜牧业及氨基酸工业的大力发展使得蛋白资源极其紧缺，因此，当前应大力研究最有效的处理方法使废弃的羽毛角蛋白直接转变成蛋白质或复合氨基酸，使之能直接得到应用或作为进一步提纯氨基酸的廉价易得的原料，从而变废为宝，既保护环境又资源利用，这将具有十分显著的经济效益和深远的社会效益。本课题针对羽毛角蛋白废弃物的资源化利用，进行了以下的研究。首先对从堆积腐烂羽毛的土壤中分离得到能高效降解羽毛角蛋白的细菌KI进行形态观察和16S rRNA测序。初步鉴定为寡养单胞菌（Stenotrophomonas），与嗜麦芽窄食单胞菌（Stenotrophomonas maltophilia）相似性达98％，命名为嗜麦芽窄食单胞菌DHHJ（Stenotrophomonas maltophilia DHHJ）。迄今为止，国内还未见寡养单胞菌（Stenotrophomonas）降解羽毛角蛋白的相关报道。通过对分离于长期堆积腐烂羽毛土壤中的嗜麦芽寡养单胞菌DHHJ（Stenotrophomonas maltophilia DHHJ）进行紫外诱变，在最佳照射时间90s下，经过一系列筛选方法，得到一株高效的突变菌株L4。该突变菌株接种于含有灭菌处理过的完整羽毛的基础培养基中，发酵1天后即可见羽毛有明显脱落，发酵6天后羽毛已经完全脱落，羽梗也有一定程度的毁解，降解率可达80％，比原始菌株提高了20％。发酵液蛋白质含量最大可达7.05mg／mL，酶活力最大为8U／mL，氨基酸含量最大为0.19mg／mL，这表明突变菌株L4具有较强降解羽毛角蛋白的能力。原始嗜麦芽窄食单胞菌DHHJ（Stenotrophomonas maltophilia DHHJ）产生的粗酶液最适作用pH为7.5，最适作用温度为40℃，在pH6.5～8.0，40℃以下酶活较稳定。突变菌株L4粗酶液的最适作用pH为7.8，最适作用温度为50℃，在pH7.0～8.0，60℃以下酶活较稳定。Ca²⁺，Ba²⁺，Cu²⁺，N^a+，K⁺和Mg²⁺离子对粗酶活力有促进作用，而Hg²⁺，Cd²⁺，Pb²⁺，Zn²⁺和PMSF则对粗酶活力有抑制作用。由于其被PMSF所抑制，可认为嗜麦芽寡养单胞菌DHHJ（Stenotrophomonas maltophilia DHHJ）产生的角蛋白酶应属于丝氨酸蛋白酶。对粗酶液进行了SDS—PAGE电泳，测定该角蛋白酶可能为复合酶，由两个亚基构成，大小分别为141kD和119kD。初步研究了嗜麦芽窄食单胞菌DHHJ（Stenotrophomonas maltophilia DHHJ）降解角蛋白的机理。电镜观察到细菌紧密地生长在羽枝上，在96h左右完全降解羽毛。生物化学研究显示嗜麦芽窄食单胞菌DHHJ（Stenotrophomonas maltophilia DHHJ）产生具有角蛋白降解能力的胞外酶。胞外酶的角蛋白水解活性（5.5U）受到胞内二硫键还原酶的促进（16.3U）。但是，单独的角蛋白酶和二硫键还原酶在没有活的细菌存在下，都不能降解羽毛。这说明细菌附着在降解过程中起了重要的作用。这可能是由于细菌持续提供了一种还原剂破坏二硫键。此外，在羽毛降解过程中，在胞外酶中还检测到亚硫酸盐，说明亚硫酸盐解对羽毛降解也起了一定的作用。固定化技术是提高生物催化利用率、生物反应速率以及实现高效连续生产过程的有效方法。本课题研究了壳聚糖表面吸附法固定嗜麦芽窄食单胞菌DHHJ（Stenotrophomonas maltophilia DHHJ）进行羽毛降解。由于其具有使用寿命长，具有足够的韧性、强度和一定的生物相容性，易回收，降解效果稳定等特点，因此是值得致力研究的方向。提出了羽毛角蛋白微生物水解的工艺，并借鉴垃圾堆肥和活性生物滴滤池技术，设计了羽毛水解装置。将经过加热预处理的羽毛作为“填料”，放置于羽毛反应器的中段，让高效菌株在其中生长代谢，使无机培养液从装置顶部喷淋至“填料”上，富含水溶蛋白的水解液从底部引出。羽毛沥滤床反应器的主要操作和效能参数为：供气量1200L／d，进水量0.5L／d，循环量8L／d，转化率42％，产生可溶蛋白5.46g／L。更多还原

【Abstract】 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更多还原