【作者】 燕红；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 环境工程， 2007， 博士

【摘要】 纤维素是地球上数量最大的可再生资源,微生物对它的降解、转化是碳素循环的主要环节。它的有效利用对于解决目前面临的环境和能源危机具有重大的现实意义。本文采用刚果红染色平板和液体摇瓶发酵培养,从土壤样品和实验室保藏的菌种中筛选出两株高产纤维素酶的细菌菌株。其中X18为我实验室购买的菌种,名为地衣芽胞杆菌（Bacillus licheniformis）;而X10-1-2经鉴定为蜡样芽胞杆菌（Bacillus cereus）。这两株菌均产生胞外纤维素酶,且它们所产纤维素酶对结晶型纤维素的吸附能力都较弱,认为它们的纤维素酶缺乏纤维素结合区。通过对筛选得到的两株高产纤维素酶的芽胞杆菌X18和X10-1-2的发酵条件进行优化,得出以下结论:麸皮作为天然纤维素可较大幅度地提高纤维素酶的产量,两株菌的最佳麸皮浓度为2.5%和3%。两株菌的最佳氮源均为豆饼,最佳豆饼浓度分别为4%和1%。产酶的最佳温度均为30℃。X10-1-2的最佳产酶pH为5,而X18的Cx酶在pH6⁸范围内产酶最高,本试验采用pH8。X18和X10-1-2的最佳产酶时间分别为20h和60h。通过测定各种纤维素酶活性研究了不同碳源对两株芽胞杆菌产纤维素酶的诱导作用。研究发现,这两株芽胞杆菌不能由纯的纤维素材料作单一碳源诱导产纤维素酶,而仅由带有还原性基团的单糖或双糖诱导产酶,且纤维素酶系各组分的合成表达是协同发生的。同时,利用高效液相色谱对可诱导菌体产纤维素酶的物质经菌体胞壁（膜）酶和胞内酶的转化产物进行定性分析后,发现,两株芽胞杆菌的胞壁（膜）酶和胞内酶均未对这些可诱导菌体产纤维素酶的糖起到任何转化作用。利用这两株芽胞杆菌分别对麦麸和稻草进行降解。对发酵过程中上清液的还原糖浓度、可溶性总糖浓度、纤维素酶和半纤维素酶活的变化进行了分析,同时测定底物残渣的失重、结晶度、红外光谱和表面结构的变化。结果表明,菌体产酶的过程也是木质纤维素的降解糖化过程。经过5d的培养,麦麸和稻草中的纤维素、半纤维素和木质素有了不同程度的降解。经过降解,麦麸和稻草的表面结构发生了明显的变化。将X10-1-2和X18分别培养60h和20h的发酵液采用3000rpm离心15min,上清液经（NH4）2SO4分级沉淀、Sephadex G-75凝胶过滤层析分离分别得到一个电泳纯的内切型-β-葡聚糖酶（CMCase）组分。经SDS-PAGE电泳检测均为单一条带,来自X10-1-2和X18的CMCase的分子量分别为51.3kDa和37.2kDa。来自X10-1-2和X18的CMCase的最适酶促反应温度分别为55℃和50℃,X10-1-2的CMCase在55℃以下具有良好的热稳定性,而X18的CMCase的热稳定性较差。两株菌的CMCase最适pH值分别为8.0和9.0,且在pH 7.0⁹.0的中、碱性范围内都保持着较好的稳定性。Mn²⁺、Fe²⁺和EDTA对X10-1-2的CMCase有一定的激活作用;而Ba²⁺、Cu²⁺、Mg²⁺和C2O42-对CMCase有不同程度的抑制作用。对于X18的CMCase,Fe²⁺、Mn²⁺、Mg²⁺、Cu²⁺、Co²⁺和K+对纤维素酶在酶促反应中有一定的激活作用;Zn²⁺、脲、SDS、Ba²⁺和Na+对纤维素酶在酶促反应中均有不同程度的抑制作用。对不同纤维素底物的专一性实验结果表明,这两株菌的CMCase对羧甲基纤维素钠的水解活性较强,但对Whatman滤纸和木聚糖也显示出微弱的降解酶活性。以不同浓度的CMC为底物,根据米氏方程求出X10-1-2和X18的CMCase的动力学参数分别为Km=2.12mg/mL,Vmax=5.37μg/mL·min和Km=9.84mg/mL,Vmax= 11.19μg/mL·min。通过对红外图谱特征频率区的分析,说明这两个CMCase都具有纤维素酶在红外图谱中的特征吸收频率,其蛋白质的二级结构都主要以α-螺旋的结构形式存在。通过CMCase的核磁共振一维氢谱可知,在两株菌的CMCase一级结构中,芳香族氨基酸含量均较少,主要由脂肪族氨基酸组成。更多还原

【Abstract】 Cellulose is the most abundant renewable polysaccharide in nature. Its biodegradation by microorganisms is one of the major steps of the carbon cycle on Earth. The efficient utilization of this process could make a significant contribution to the solving of present environmental and ecological problems.Two strains of bacteria producing higer cellulase activities were selected by screening plate stained with Congo red and liquid culture from soil samples and preserved strains by our labrotory. X18 was purched by our labrotory, and named as Bacillus licheniformis. X10-1-2 was identified as Bacillus cereus according to classification basis. Cellulases produced by two Bacillus sp. were exocellular enzyme. Adsorption ability of produced cellulases by two Bacillus sp. on crystalline cellulose was very poor. It could be concluded, these cellulases were lack of cellulose-binding domains.Optimal culture conditions for production of cellulases using Bacillus sp. X18 and Bacillus sp. X10-1-2 were investigated. The highest level of cellulase activities were induced in the medium containing wheat bran. Optimal mass concentration for Bacillus sp. X18 and Bacillus sp. X10-1-2 were 2.5% and 3%, respectively. Bean cake was the best nitrogen source. Optimal mass concentration for Bacillus sp. X18 and Bacillus sp. X10-1-2 were 4% and 1%, respectively. Optimal culture temperature of cellulase production of Bacillus sp. X18 and Bacillus sp. X10-1-2 was 30℃. Optimal culture pH of cellulase production of Bacillus sp. X18 and Bacillus sp. X10-1-2 were 6⁸ and 5, respectively. Optimal time of cellulase production of Bacillus sp. X18 and Bacillus sp. X10-1-2 were 20h and 60h, respectively.The induction of cellulase production in two Bacillus sp. was studied, by means of measuring cellulase activities under the condition of different carbon sources. The results indicated that cellulases could not be induced by pure cellulose material as a sole carbon source. Instead, they could be induced by monosaccharide or disaccharide with reducing group. Moreover, the expression of cellulase components was synergistic. When cell wall/ envelope enzyme and endoenzyme from two Bacillus sp. acted on these inducers, analysis of reaction products by HPLC revealed that cell wall/envelope enzyme and endoenzyme from two Bacillus sp. were inactive on these inducers.Wheat bran and straw were employed for degradation by Bacillus sp. X18 and Bacillus sp. X10-1-2. During the research of fermentation, concentration of reducing sugar and soluble total sugar, activities of cellulase and hemicellulase were assayed, while weight loss, crystallinity, FT-IR and surface structure of residue were analyzed. The results showed that the process of biodegradation of lignocellulose was actually simultaneous saccharification and fermentation. Cellulose, hemicellulose and lignin in wheat bran and straw were biodegraded to diffirent tent after 5d cultivation. Surface structure of wheat bran and straw had changed evidently after biodegradation.CMCase produced by Bacillus sp. X10-1-2 and Bacillus sp. X18 were purified to homogeneity from culture supernatant. The enzyme from Bacillus sp. X10-1-2 and Bacillus sp. X18 had molecular mass of 51.3kDa and 37.2kDa, respectively.The optimum temperature of CMCase from Bacillus sp. X10-1-2 and Bacillus sp. X18 was 55℃and 50℃, respectively. The CMCase from Bacillus sp. X10-1-2 was stable below 55℃, but the CMCase from Bacillus sp. X18 had bad thermostability. The cellulases from Bacillus sp. X10-1-2 and Bacillus sp. X18 were optimally active in the pH8 and pH9, respectively, and they were stable at 50℃in a pH range 7.0⁹.0. The activity of the enzyme from Bacillus sp. X10-1-2 was increased by Mn²⁺, Fe²⁺, and EDTA. Ba²⁺, Cu²⁺, Mg²⁺, and C2O42- caused loss in enzyme activity to a lower extent. In contrast, the activity of the enzyme from Bacillus sp. X18 was increased by Fe²⁺, Mn²⁺, Mg²⁺, Cu²⁺, Co²⁺, and K+. Zn²⁺, urea, SDS, Ba²⁺, and Na+ caused loss in enzyme activity.The cellulases from Bacillus sp. X10-1-2 and Bacillus sp. X18 showed highest activity with carboxymethylcellulose. Slight activities were also observed with filter paper and xylan as substrate. For carboxymethycellulose the CMCase from Bacillus sp. X10-1-2 had a Km of 2.12mg/mL and Vmax of 5.37μg/mL·min, while CMCase from Bacillus sp. X18 had a Km of 9.84mg/mL and Vmax of 11.19μg/mL·min. The FT-IR spectrum of two CMCases showed characteristic cellulase peaks. Infrared spectrum of the amide I and II bands of CMCase showed that secondary structure of two CMCases mainly consist ofα-helix structures in solid phase. According to the proton magnetic resonance spectrum of two CMCases, we concluded that primary structure of two CMCase contain less aromatic amino acid and more alkalinity amino acid.更多还原