【作者】 白洪志；

【导师】 杨谦；

【作者基本信息】 哈尔滨工业大学 ， 环境科学与工程， 2008， 博士

【摘要】 利用纤维素的最佳方法是微生物降解转化,鉴于目前纤维素降解菌株酶活力低、纤维素酶诱导产酶及纤维素降解机理不完全清楚等限制纤维素资源有效利用的情况下,为了有效利用纤维素这种可再生能源,筛选高效降解菌,研究菌株产酶机制以及纤维素降解机理仍然具有重要的理论和实践意义。本文从57份土样中,通过分离纯化得到97株菌株。对其进行刚果红鉴别培养基和滤纸条液体培养法初筛,并结合复筛得到2株纤维素酶活较高的菌株。经形态学鉴定H-11为简青霉（Penicillium simplicissimum）,C-08为绿色木霉（Trichoderma viride）。研究了几种影响二菌株发酵产酶的因素,液体发酵结果表明:麸皮和稻草为适合碳源,两者比例H-11为4:3,C-08为5:2;最适有机氮源为豆饼粉,而无机氮源分别是KNO3和（NH4）2SO4。H-11产酶最适为豆饼粉,而C-08产酶的（NH4）2SO4:豆饼粉比例为1:5。最适产酶碳源和氮源比例,H-11为7:1,C-08为5:1;最适百分含量,都为3.6%;最适产酶pH,H-11为3.6,C-08为3.2;最适温度都为30℃;最适产酶时间H-11为144h,C-08为96h。固态发酵结果表明:秸秆粉和麸皮为2∶4最适合产酶;尿素和氨态氮适合H-11产酶。氨态氮适合C-08产CMCase,NO3-利于产Xylanase,其中NH4NO3最好;料水比1∶1比较适合H-11产CMCase,1∶1.5适合产Xylanase。料水比1∶2适合C-08产CMCase,1∶2.5适合产Xylanase;H-11随底物粒度的降低CMCase增加,在60¹00目时产Xylanase最低。稻草粉粒度40¹00目时,适合C-08产CMCase,此范围外适合产Xylanase;初始pH为3和4时则适合H-11菌株产CMCase和Xylanase。pH值为4适合C-08产酶;孢子接种量为7⁸×107孢子/mL时适合两株菌产酶;28℃适合H-11产酶,在低于28℃时适合C-08产酶;发酵时间H-11为60h,C-08为72h。固定化研究表明,制备小球的最适海藻酸钠溶液浓度为5%。麸皮汁较适合菌株产酶,H-11比C-08产酶能力强。菌株间亲和性不强。H-11则在pH为3和4时,更适合产CMCase和Xylanase,C-08产酶适宜pH为7。固定化细胞产酶能力强于游离细胞。Tween-80有利于提高菌株的产酶活力。利用液体培养和洗涤菌丝诱导培养法研究了碳源对菌株的诱导特性。结果发现D-甘露糖对H-11有较好的诱导作用,葡萄糖对C-08有较好的产酶作用。葡萄糖促使H-11、C-08产生FPase、CMCase、C1和β-Gase,各种酶产量在菌株间及菌株内差别很大。制备细胞外、细胞壁膜、细胞内纤维素酶,并用其分别转化纤维二糖,对转化产物高效液相色谱定性分析,从而探讨碳源的诱导本质。结果显示:H-11胞内酶和C-08的胞外酶的转化产物,除产生葡萄糖外,亦产生一种未知的物质A。胞壁膜酶无转化作用。H-11的胞外酶和C-08的胞内酶液对纤维二糖除水解外亦有转化作用。从而证明纤维二糖不是菌株的诱导物。将H-11液体发酵粗酶液经硫酸铵分级沉淀、柱层析后得到电泳纯的β-GaseⅠ、β-GaseⅡ和CMCase组分。对纯组分的理化性质研究表明:三组分的分子量分别为126.0KDa、77.8KDa和33.2kDa;最适温度都为60℃,分别在40℃、40℃和50℃以下稳定;最适pH值为pH4.4⁵.2、3.6⁴.0和2.8,在pH值4.4⁶.8、2.8⁶.8和2.8⁶.8范围内稳定。Mn²⁺、Ca²⁺、Sn²⁺和Li⁺对β-GaseⅠ有促进作用,Mg²⁺、Zn²⁺、Cu²⁺、Co²⁺、Fe²⁺和Fe3+对β-GaseⅠ有抑制作用;Mn²⁺、Sn²⁺和Fe²⁺对β-GaseⅡ有促进作用,Zn²⁺、Cu²⁺、Co²⁺和Fe3+对β-GaseⅡ有抑制作用;Sn²⁺对CMCase酶活力有明显的增强作用,Mn²⁺和Cu²⁺对酶有显著的抑制作用。β-GaseⅠ水解水杨素的Km和Vmax值分别622.306mg·mL^-1和15.480mg·mL^-1·min^-1,β-GaseⅡ水解水杨素的Km和Vmax值分别26.689mg·mL^-1和0.508mg·mL^-1·min^-1,CMCase作用于CMC-Na的动力学参数Km和Vmax值分别1.744mg·mL^-1和14.124mg·mL^-1·min^-1。三种酶的底物特异性较差,β-GaseⅠ、β-GaseⅡ不以CMC-Na为底物,CMCase不作用于水杨素,除此之外,三组分对MCC、滤纸、几丁质、pNPC和pNPG都有作用。通过对三组分的红外图谱特征频率区进行分析,结果表明其蛋白质二级结构都主要以α-螺旋形式存在。H-11的粗酶液中C1、CMCase对MCC都具有吸附-解吸附作用,而β-Gase对MCC不具有吸附-解吸附作用,三种纯组分和C-08粗酶液中的C1、CMCase、β-Gase对MCC都具有吸附-解吸附作用。β-GaseⅠ和β-GaseⅡ对CMCase降解纤维素有很强的协同降解作用,三种纯组分对C-08粗酶液降解纤维素没有协同作用。H-11的固态、液态发酵和C-08的固态发酵都存在氧化降解机制。更多还原

【Abstract】 Microbe degradation is a most potential and ideal way to the application of cellulose. Because of low degradation activity of microbe and ambiguous inducement and biodegradation mechanism, farther application of cellulose is relatively deferrable. In order to utilize cellulose efficiently as a tremendous green resource, to screen microbes with high degradation activity and research inducement and biodegradation mechanism would appear important theory and practice value.Ninety-seven strains were achieved by enrichment and isolation from 57 soil samples. Two methods were used in the process. The primary is congo red culture and filter liquid culture, the second is measurement of cellulose activity. Two strains with high cellulase activity were obtained using the methods, identified to be Penicillium simplicissimum H-11 and Trichoderma viride C-08.H-11 and C-08 were fermented in liquid media and fermentation conditions were optimized. The highest level of cellulase activity was induced in the medium containing wheat bran and straw with the ration of 4:3. Soybean powder is the best nitrogen source. The ratio of C to N is 7:1 and the mass concentration for P. simplicissimum H-11 is 3.6%. The culture temperature, pH and time of cellulase production of P. simplicissimum H-11 are 30℃, 3.6 and 114h respectively. The highest level of cellulase activity was induced by the compound containing wheat bran and straw, and their ration is 5:2. Ammonium sulfate and soybean powder are the best nitrogen source, and their ration is 1:5. The ratio of carbon source to nitrogen source is 5:1 and the mass concentration for T. viride C-08 is 3.6%. The culture temperature, pH and time of cellulose production of T. viride C-08 are 30℃, 3.2 and 96h respectively.The ratio of stuff to water is 1:1 for H-11 CMCase production, 1:1.5 for Xylanase production, and the ratio of stuff and water is 1:2 for C-08 CMCase production, 1:2.5 for Xylanase production. The optimal rice straw granularity is 150⁴25μm for C-08 CMCase production. The activity of CMCase of H-11 increase with rice straw granularity,but the activity of Xylanase appear lowest when granularity is 150⁴25μm. Granularity between 150⁴25μm is optimal for C-08 CMCase production, while granularity below 150μm and above 425μm is best for Xylanase production. The optimal pH for C-08 fermentation is 4, and the optimal pH for H-11 CMCase and xylanase production are 3 and 4. The optimal conditions for cellulase and xylanase production as follows: inoculum size is 7⁸×107, incubation temperature is 28℃for H-11, lower 28℃for C-08, fermentation period is 60h for H-11, 72h for C-08.The immobilized cells were prepared through embedment of C-08 and H-11 with sodium alginate. The suitable culture concentration of sodium alginate is 5%. Initial pH value of the substrate is 3-4 for H-11 to product CMCase and Xylanase, while initial pH value is 7 for C-08. Comparing of the immobilized cells and the normal cells, the productivity of immobilized cell is the better. The addition of the surfactant tween-80 is helpful to enzyme production.Inducement characteristics of carbon sources to enzyme production were studied using liquid culture and washed mycelium inducement methods. The results indicated that D-mannose is beneficial in H-11 cellulase production, and glucose has better effect on C-08 cellulase production. Glucose can induce C-08 and H-11 to produce FPase, CMCase, C1 andβ-Gase, but enzyme quantities are different between two strains and within each strain.In order to study the mechanism of cellualase inducement by different carbon sources, the extracellular, plasm-membrane-bound and intracellular cellulases were made to transform cellobiose, and metabolites were analyzed by HPLC. The results showed that, in addition to glucose, the extracellular cellulases of H-11 and the intracellular cellulases of C-08 can also produce an unknown matter A. The plasm-membrane-bound cellulases have no effect on cellobiose. The intracellular cellulases of H-11 and the extracellular cellulases of C-08 can hydrolyze cellobiose, demonstrating that cellobiose is not the inducer of C-08 and H-11.The crude cellulases of H-11 were purified from liquid fermentation media by （NH4）2SO4 precipitation and SephadexG-100. Theβ-Gase and CMCase were purified and physical and chemical properties were studied. The molecular mass ofβ-GaseⅠ,β-GaseⅡand CMCase are 126.0kD, 77.8kD and 33.2kD, respectively. The optimum temperature are all 60℃, the three enzymes are stable when the tempture is below 40℃, 40℃and 50℃. The optimum pH value are 4.4⁵.2, 3.6⁴.0 and 2.8, respectively, and the three enzymes are stable when pH value is 4.4⁶.8, 2.8⁶.8 and 2.8⁶.8. Mn²⁺, Ca²⁺, Sn²⁺ and Li+ are able to enhance the activity ofβ-GaseⅠ, however, Mg²⁺, Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺ and Fe3+ can inhibit its activity. Mn²⁺, Sn²⁺ and Fe²⁺ are able to enhance the activity ofβ-GaseⅡ, whereas, Zn²⁺, Cu ²⁺, Co²⁺ and Fe3+ can inhibit the activity. Sn²⁺ are able to enhance activity of CMCase, however, Mn²⁺ and Cu²⁺ can inhibit the activity. Km and Vmax ofβ-GaseⅠare 622.306 mg·mL^-1 and 15.480 mg·mL^-1·min^-1, respectively. Km and Vmax ofβ-GaseⅡare 26.689 mg·mL^-1 and 0.508 mg·mL^-1·min^-1, respectively. Km and Vmax of CMCase are 1.744mg·mL^-1 and 14.124mg·mL^-1·min^-1, respectively.β-GaseⅠandβ-GaseⅡcan hydrolysis Salicin obviously, and CMCase can hydrolysis CMC-Na obviously.The cellulase peak was showed in the FT-IR spectra ofβ-GaseⅠ,β-GaseⅡand CMCase. The infrared spectrum of the amide I and II bands of them showed that the secondary structure is mainly consist ofα-helix structures in solid phase.C1, CMCase andβ-Gase from the crude enzyme of H-11 can all adsorb and desorb MCC, whereasβ-Gase can’t.β-GaseⅠ,β-GaseⅡ, CMCase from H-11 and C1, CMCase,β-Gase from the crude enzyme of C-08 can also adsorb and desorb MCC.β-GaseⅠandβ-GaseⅡhave strong promoting function for the hydrolyzing of cellulose, whereasβ-Gase,β-GaseⅡand CMCase of H-11 don’t have the function to crude enzyme of C-08.The solid fermentation, liquid fermentation of H-11 and the solid fermentation of C-08 all exist the mechanism of oxidizing hydrolysis.更多还原