【作者】 刘小杰；

【导师】 何国庆；

【作者基本信息】 浙江大学 ， 食品科学， 2003， 博士

【摘要】 纤维素是自然界中存在最广泛的一类碳水化合物，同时它也是地球上数量最大的再生资源。目前，自然界中纤维素只有一小部分得到了利用，绝大多数纤维素不仅被白白浪费，而且还会造成环境污染。利用微生物生产的纤维素酶将其转化为人类急需的能源、食物和化工原料，对于人类社会解决环境污染、食物短缺和能源危机具有重大的现实意义。 本论文主要工作包括：纤维素酶高产菌株的选育及产酶条件的研究；康氏木霉（Trichoderma koningii）和黑曲霉（Aspergillus niger）纤维素酶发酵培养基的优化研究；β-葡萄糖苷酶的纯化及酶学性质研究；康氏木霉和黑曲霉混菌发酵产纤维素酶的研究；纤维素酶降解稻草粉的实验研究和利用稻草粉产乙醇的初步研究。经过大量实验工作，得到了如下结果： 1)以稻草为主要碳源时，在所实验的菌种中，康氏木霉ZJ4产CMC酶和滤纸酶的活性均为最高。康氏木霉ZJ4经紫外线和硫酸二乙酯复合诱变，得到的突变株ZJ5产纤维素酶的水平明显提高。研究了其纤维素酶的产生条件：以稻草粉为碳源、（NH₄）₂SO₄为氮源、稻草与麦麸比为2：1时，康氏木霉ZJ5产酶最高。该菌株产酶最适培养温度为28℃，最适起始pH 5.0，装液量30mL，转速200r／min，当培养时间为144h时，CMC酶、滤纸酶活和β-葡萄糖苷酶活均达到最高值，分别为582.6U／mL、72.56U／mL和34.62U／mL。 2)研究了CMC酶和滤纸酶粗酶液的酶学性质，结果如下：CMC酶的最适作用温度为50℃，在40℃和50℃时热稳定性较好；滤纸酶的最适作用温度为50℃，在40℃时热稳定性较好；CMC酶的最适反应pH为4.5，在pH 2.5～pH 6.0之间较稳定；滤纸酶的最适反应pH为5.0，在pH 3.5～pH 6.0之间较稳定；Zn²⁺、Al³⁺、Mg²⁺和Mn²⁺对CMC酶酶促反应均有一定的促进作用，Mg²⁺和Mn²⁺对滤纸酶酶促反应有一定的促进作用。 3)稻草先粉碎到80目，再经15％醋酸预处理可以显著提高康氏木霉ZJ5发酵产纤维素酶的水平。首次利用预处理的稻草粉为主要碳源，经过正交实验，得到了康氏木霉ZJS发酵产纤维素酶的最佳培养基组成（留L）:稻草粉，33;麦鼓，20;大麦粉，23;困H4)2504，8。 4)首次用响应面方法对康氏木霉ZJS产纤维素酶的培养基进行了优化。获得的优化培养基组成为（g/L）:稻草粉，33;麦数，15;大麦粉，15;（N H4）250;，4。在优化培养基中发酵144h，CMC酶活、滤纸酶活和卜葡萄糖苔酶活分别达到了765.8U/mL、155.3U/mL和39.54U/mL。 5)本研究首次将响应面分析法用于黑曲霉ZJI产p一葡萄糖普酶培养基组成的优化，取得了较好的效果。在培养基优化前p一葡萄糖营酶活为1875U/mL，经优化后，该酶活性达到了403 .7U/mL，显著大于优化前的水平。经响应面分析获得的优化培养基组成为（g/L）:稻草粉，6.963:麦鼓，20;大麦粉，16.5;困H4)2504，2 .44;KHZPO4，0.5:MgSO4·7HZO，0.5。 6)利用Sephadexo一00柱和oEAE一sephadexA一50柱分离纯化了D一葡萄糖营酶，并研究了其酶学性质和酶促反应动力学。p一葡萄糖营酶的最适作用温度为50℃，在40℃时热稳定性较好;日一葡萄糖营酶的最适反应pH为5.5，在pH 30一pH 8.0之间较稳定;zn2+、川3+、ca2+和MnZ十对卜葡萄糖普酶酶促反应均有一定的促进mg ZJZg作用。确定了以水杨素为底物时的动力学参数:式行4.530mg/mL，蛛二0.06974（mLmin），动力学方程为:VP0 .06974Cs4 .530+Cs7)利用稻草为主要碳源，通过正交实验，得到了康氏木霉ZJS和黑曲霉固体混菌发酵产纤维素酶的最佳培养基组成:稻草粉，6g;麦鼓，49;（N H4）2504，0.89;KHZPO4，0.059:MgSO4·7HZO，0.059;加水30mL，大麦粉，起始pHS.o在优化培养基中发酵96h，CMC酶活、滤纸酶活和p一葡萄糖首酶活分别达到了2 135U/mL、738.2U/mL不{1 1 125U/mL。 8)利用单因子实验得到了稻草粉酶解糖化的最佳条件:稻草粉先经1.0%NaOH+1 .0%HZo:预处理，底物浓度为2.5%，糖化温度50OC，反应体系pH4.8，滤纸酶:p一葡萄糖昔酶二1:1。运用二次通用旋转组合设计，考察了底物浓度（X，）、酶解pH（xZ）和酶解温度（X3）对稻草粉糖化率（Y）的影响，得到了下列回归方程: Y=0 .5744一o.o5214x一0.07一29x2+o.o443sx3一o.07929x22一。一27x32一0 .0 1 785XIX2+0.00708lXIX3一0.05478X2X3 9)本文研究发现利用纤维素酶降解稻草粉时，稻草粉的酶解率与处理时间和酶的浓度都有关。通过对实验数据的分析，得到了纤维素的残留量（St）和酶解量（Y）与处理时间（t）之间的经验公式: [st]/[s。]==e一kI;[Y]/[S。]=（l一e一“） 10)本文首次利用稻草研究了糖化发酵生产乙醇的几种工艺，在糖化、发酵二步工艺中，采用正交实验，得出了台湾酵母（5扮ccharo梢lyce:cerevisia。）利用稻草粉水解液发酵产乙醇的最佳培养条件:起始pH为5.0、接种量巧%、培养时间48h、培养温度为30℃;在此条件下，乙醇浓度可以达到3.105%（v/v）。在边糖化边发酵工艺中，利用正交实验，得到了酒精酵母利用稻草粉产乙醇的最佳培养条件:起始pH为5.0、滤纸酶:p一更多还原

【Abstract】 Cellulose is the most abundant organic raw material in the world, and it is the only renewable resource that is available in large quantities. Presently, only a few cellulose are utilized, most of cellulose are wasted and pollute environment. So it has great realistic meaning for human being to solve environment pollution, food shortage and energy crisis that using cellulase produced by microorganism transform cellulose to energy , chemical and food.This paper included : 1) study on the screening out of strain with high cellulase activity and the conditions for enzyme production; 2) culture medium optimization for producing cellulase by Trichoderma koningii ZJ5 and Aspergillius niger ZJ1; 3) study on purification and properties of P -glucosidase: 4) study on cellulase production by mixed culture with Trichoderma koningii ZJ5 and Aspergillius niger ZJ1; 5) research on the kinetics of the hydrolysis of rice straw powder by cellulase; 6) study on fermentation of rice straw to ethanol. The main results were as follows:1) This paper compared the ability of producing cellulase of several fungi when rice straw powder as primary carbon source, Trichoderma koningii ZJ4 can produce relatively high cellulase. The productivity of cellulase of Trichoderma koningii ZJ4 mutant Trichoderma koningii ZJ5 was improved greatly after mutated by ultraviolet and diethyl sulfate. The medium with straw powder as carbon source, (NH4)2SO4 as nitrogen source and straw powder/bran=2: 1 was optimal and the maximum cellulase activity was reached at 28C and pH 5.0 when cultivated 144 hours. The maximum of CMCase, filter paper and 3 -glucosidase activity were 582.6U/mL, 72.56U/mL and 34.62U/mL respectively.2) The properties of CMCase and filter paper enzyme were studied and results as follows: The optimal temperature of CMCase was 50C and it was stable under 50C; The optimal temperature of filter paper enzyme was 50 C and it was stable under 40 C; The optimum pH value of CMCase was 4.5 and it was stable between pH 2.5 and pH 6.0, while the optimum pH value of filter paper enzyme was 5.0 and it was stable in pH 3.5~pH 6.0; Zn2+, A13+, Mg2+ and Mn2+ have active effects on CMCase activity , Mg2+and Mn2+ have active effects on filter paper enzyme activity.3) When rice straw was pretreated by 15% acetic acid after it was smashed to 80 mesh, it can increase cellulase production by Trichoderma koningii ZJ5. When pretreated rice straw powder as primary carbon source, the optimal fermentation medium, which was obtained by orthogonal test was as following (g/L): rice straw powder, 33; wheat bran, 20; barley flour, 23; (NH4)2SO4, 8.4) Response surface methodology was used to optimize medium for cellulase production by Trichoderma koningii ZJ5. The composition of fermentation medium was(g/L): rice straw powder, 33; wheat bran, 15; barley flour, 15; (NH4)2SO4 , 4. The maximum of CMCase, filter paper enzyme and P -glucosidase activity were 765.8U/mL, 155.3U/mL and 39.54U/mL, respectively under the optimized culture medium after 144 hours.5) Response surface methodology was also used to optimize a medium for P -glucosidase production by Aspergillius niger ZJ1. P -glucosidase activity was 403.7U/mL after optimization and higher significant than that in fermentation medium before optimized ( 3 -glucosidase activity was 187.5U/mL) .The optimized composition of fermentation medium was (g/L): rice straw powder, 6.963; wheat bran, 20; barley flour, 16.5; (NH4)2SO4, 2.44; KH2PO4, 0.5; MgSO4-7H2O, 0.5.6) & -glucosidase of Aspergillius niger ZJ1 was purified by Sephadex G-100 and DEAE-Sephadex A-50 chromatography, and its properties and kinetics was studied. Its optimum temperature was 50癈, and it was stable under 40"C. Its optimum pH was 5.5 and it was stable in pH 3.0 ~pH 8.0. Metal cation influence enzyme activity, Zn2\ AI3\ Ca2+ and Mn2+ have active effects on enzyme activity. Km and Vm of P -glucosidasefor salicin as substrate was 4.530mg/mL and 0.06974 mg/ (mL-min) respect更多还原