【作者】 薛栋升；

【导师】 姚善泾；

【作者基本信息】 浙江大学 ， 生物化工， 2012， 博士

【摘要】 纤维素酶是将纤维素降解为葡萄糖的酶,由内切酶、外切酶和p-葡糖苷酶组成。p-葡糖苷酶将纤维二糖降解为葡萄糖,是酶解纤维素过程中的关键步骤之一。耐盐纤维素酶可以在高盐环境下高效降解纤维素,造纸和纤维素预处理废水的治理,海洋垃圾、海藻和海草中纤维素的高效利用,盐碱地的修复等需要耐盐的纤维素酶。本论文从海洋中筛选产耐盐纤维素酶的菌株,通过对具有良好耐盐性能的纤维素酶和p-葡糖苷酶的发酵工艺、酶学性质、普遍的耐盐机制等的研究,为高盐环境中纤维素的降解打下基础。论文研究包括以下内容：首先,从海洋中筛选得到产耐盐纤维素酶的真菌,根据该真菌的形态,ITS序列和5%NaCl (w/v)最适生长盐度,鉴定该真菌为嗜盐海洋黑曲霉。海洋黑曲霉产生的纤维素酶最适温度和pH为58℃和4.5,在12%NaCl (w/v)溶液中的酶活最大,是在无NaCl溶液中酶活的1.33倍。该纤维素酶在黑液废水中降解纤维素,降解12h产生的葡萄糖比在自来水中降解纤维素产生的葡萄糖多了11.5%。研究结果表明,海洋黑曲霉产生的纤维素酶是一种具有良好耐盐耐热性能的纤维素酶。其次,研究了纤维素酶的固体发酵工艺,实现了纤维素酶的环境友好型生产。固体发酵的天然培养基最优组成为：76.9%水葫芦(w/w),8.9%玉米芯(w/w),3.5%稻草粉(w/w),10.7%麸皮(w/w)和干固体基质2.33倍的天然海水。天然海水中的主要无机盐都能增加纤维素酶的产量。发酵144h,纤维素酶的酶活达到17.80U/g干固体基质。第三,研究了液体发酵时海洋黑曲霉纤维素酶酶活和胞外蛋白表达对不同碳源的响应。海洋黑曲霉纤维素酶的酶活和胞外蛋白对不同碳源的响应有较大差异。在以玉米芯或稻草秆作为碳源时,液体发酵产生的内切酶、β-葡糖苷酶和纤维素酶的酶活分别为2.00U/mL和1.70U/mL、3.25U/mL和2.62U/mL、0.13和0.08U/mL,比活力分别为1.39U/mg蛋白和1.31U/mg蛋白,3.25U/mg蛋白和2.62U/mg蛋白,0.181U/mg蛋白和0.165U/mg蛋白。二维电泳分析表明,以玉米芯或稻草秆作为碳源时,海洋黑曲霉产生的胞外蛋白的种类差异达到15种。第四,用丝瓜囊作为固定化载体固定海洋黑曲霉,连续批次发酵生产p-葡糖苷酶。丝瓜囊固定的菌丝量达到3.1g/L,固定效率达到95%以上。固定化发酵缩短了发酵周期,达到最大产量的时间由游离发酵的7d降为固定化发酵的4.5d。固定化发酵的第3、4批次的产量高于游离发酵的产量,固定化连续5批次发酵的p-葡糖苷酶产量均高于110U/mL。第五,研究了p-葡糖苷酶在不同盐度下的酶学性质和热失活动力学。粗p-葡糖苷酶在6%NaCl(w/v)溶液中的酶活最高。在0%和6%NaCl(w/v)溶液中的最适温度和pH相同,都为66‰和5.0。在6%NaCl(w/v)溶液中的酶活是在无NaCl溶液中的1.46倍。在62℃,65℃,67℃和70℃,p-葡糖苷酶在6%NaCl (w/v)溶液中的半衰期均高于在无NaCl溶液中半衰期的2倍以上。p-葡糖苷酶在6%NaCl (w/v)溶液中的热失活自由能比在无NaCl溶液中的热失活自由能和熔点温度分别高了约2kJ/mol,熔点温度也略微提高。纯化后测得p-葡糖苷酶的分子量约为110kDao在高盐度环境下,纯p-葡糖苷酶的酶活和半衰期的增长与粗p-葡糖苷酶酶活和半衰期的增长相似。研究结果表明该海洋黑曲霉产生的p-葡糖苷酶是耐盐耐热型p-葡糖苷酶。第六,构建了p-葡糖苷酶的分子模型,探讨了该类酶的耐盐机制。建立了该类酶的分子模型,大量的谷氨酸和天冬氨酸残基分布在分子模型表面。在分子表面的大量酸性氨基酸残基赋予p-葡糖苷酶高负离子型表面,高负离子型表面和分子内核的作用可能增强了p-葡糖苷酶在高盐环境下的热稳定性。p-葡糖苷酶在高盐度下活性增加的原因可能是高盐的环境使p-葡糖苷酶异构化,异构体的催化位点更容易与底物纤维素二糖作用。本论文对耐盐纤维素酶和β-葡糖苷酶的高效生产,高盐环境下纤维素酶和p-葡糖苷酶性质、p-葡糖苷酶耐盐机理等的研究,对高盐环境下的纤维素的高效降解具有重要的意义。更多还原

【Abstract】 Cellulase, consisted of endoglucanase, exoglucanase and β-glucosidase, can hydrolyze cellulose into glucose. P-glucosidase can transform cellobiose into glucose. Hydrolyzing cellobiose is a critical step of enzymatic cellulose hydrolysis.Under high salinity condition, salt tolerant cellulase can efficiently hydrolyze cellulose into glucose. Salt tolerant cellulase has important significance for treatment of papermaking and cellulosic materials pretreatment sewage, efficient utilization of cellulose in marine litter, algae and seaweed and reclamation of saline and alkaline lands.A marine fungus with salt tolerant cellulase was selected. The production process, properties and universal salt tolerant mechanism of β-glucosidase was studied. The results provided the foundation for practical application of salt tolerant cellulase. This article was consisted of the following:Firstly, a marine fungus was obtained to produce salt tolerant cellulase. According the morphology, ITS sequence and optimum growth salinity of5%NaCl (w/v) solution salinity, the marine fungus was identified as a halophile Aspergillus niger. Optimum pH, temperature and NaCl concentration of cellulase activity was4.5,58℃and12%(w/v). Cellulase activity in12%NaCl (w/v) solution was1.33times higher than that in NaCl free solution. The glucose production of hydrolyzing cellulose of cellulase in black liquor for12h increased by11.5percent compared with that in tap water. Cellulase from the marine Aspergillus niger was salt tolerant and thermostable.Secondly, a natural medium was designed to environment friendly produce cellulase under solid state fermentation conditions. The optimum natural medium was consisted of76.9%Eichhornia crassipes (w/w),8.9%raw corn cob (w/w),3.5%raw rice straw (w/w),10.7%raw wheat bran (w/w) and natural seawater (2.33times weight of the dry substrates). Incubation for144h, cellulase production was17.80U/g dry substrates.Thirdly, responses of cellulase activity and total extracellular proteins to different carbon sources under submerge fermentation were analyzed. The results showed that responses of cellulase activity and total extracellular proteins to different carbon sources were remarkably different. When corn cob or rice straw was used as carbon sources, endoglucanase, P-glucosidase and cellulase production was2.00and1.70U/mL,3.25and2.62U/mL,0.13and0.08U/mL. Endoglucanase, P-glucosidase and cellulase activity was1.39and1.31U/mg protein,3.25and2.62U/mg protein,0.181and0.165U/mg protein. Two-dimension electrolphoresis showed15different kinds of proteins in total extracellular proteins when corn cob or rice straw was used as carbon sources.Forthly, P-glucosidase was produced by immobilizing the marine Aspergillus niger on loofa sponges. Biomass of mycelia immobilized on loofa sponge carrier could be3.1g/L and the immobilization percentage could be over95%. The immobilization technology reduced the production circle, time to the maximum production by free or immobilized mycelia was7or4.5d. P-glucosidase production of the fourth and fifth bacthes was more than that by free mycelia β-glucosidase activities of five repeat batches were all over110U/mL.Fifthly, properties and thermodynamics of β-glucosidase was analyzed at different salinities. Crude P-glucosidase activity in6%NaCl (w/v) solution was the maximum. In free or6%NaCl (w/v) solution, optimum temperature and pH of crude β-glucosidase activity was same,66℃and5.0. Crude β-glucosidase activity in6%NaCl (w/v) solution was1.46folds higher than that in NaCl free solution. At62℃,65℃,67℃and70℃, half life of crude in6%NaCl (w/v) solution were more than twice of these in NaCl free solution. Gibb’s free energy for denaturation and melt temperature of crude β-glucosidase was about2kJ/mol higher and slightly warmer than that in NaCl free solution. After purification, molecular weight of β-glucosidase was measured to be about110kDa. The increasing of activity and half life of pure P-glucosidase at high salinity was similar with that of crude β-glucosidase. P-glucosidase from the marine haloversatile Aspergillus niger was salt tolerant and thermostable.Sixthly, molecular model of β-glucosidase was constructed and the probable mechanism for salt tolerance was explained. Homology molecular model for the kind of β-glucosidase was constructed. Many glutamic acid and aspartic acid residues, acidic amino acid residues, on the surface of β-glucosidase molecular model, endow β-glucosidase molecular with a high electronegativivty surface. The interaction between high electronegativivty surface and molecular core probably enhances the kind of β-glucosidase thermostability at high salinity. The high concentration of Na+probably isomerizes β-glucosidase and catalytic sites of the isomer were probably more efficient to intreract with the substrate of cellobiose.Efficient producing of cellulase and β-glucosidase, studying properties of cellulase and P-glucosidase at high salinity, the explanation of mechanism for β-glucosidase salt tolerance was valuable for hydrolyzing cellulose at high salinity.更多还原