【作者】 王秀娟；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 经济微生物， 2011， 博士

【摘要】 纤维素酶自1906年Seilliere在蜗牛的消化液中发现以来一直是研究的热点,那是因为纤维素废料是地球上最丰富的可再生资源,利用纤维素酶对其降解既无污染又可以得到各种有用的产物,是一条变废为宝的好途径;再者纤维素酶已经被应用在纺织、造纸、饲料、食品加工以及洗涤剂生产等工业领域;目前利用纤维素酶降解木质纤维素生产燃料乙醇又成为了研究热点。因此,能获得一个高产纤维素酶菌株,并且其酶活力、热稳定性和pH稳定性等酶学指标都能满足工业生产的需要,这是每个纤维素酶研究者梦寐以求的。本实验室一直从事嗜热真菌产酶的研究,分离到了嗜热子囊菌光孢变种（Thermoascus aurantiacus var.levisporus）和嗜热毛壳菌（Chaetomium thermophilum CT2）产酶菌株,其生长上限温度较高,产生的纤维素酶的活性及耐热性都很高,具有极大的研究和应用价值。本研究先构建来源于嗜热毛壳菌（Chaetomium thermophilum CT2）的纤维二糖水解酶Ⅱ和纤维二糖水解酶Ⅰ的工程菌WTCBHⅡ、WTCBHⅠ,然后利用定向进化的方法对其进行改造。采用易错PCR方法建立突变体库后,以高活力作为筛选压力,先后以小量发酵法和摇瓶发酵法筛选突变体库。对工程菌WTCBHⅡ进行定向进化得到了两株酶活力是出发菌株所产酶活力的3倍的突变菌株:CBHⅡX16和CBHⅡX305,测得的序列与野生基因比较,发现CBHⅡX16中有5个氨基酸发生突变,它们是R1S、A29T、L203Y,、Q204K和E252G; CBHⅡX305中有6个氨基酸发生突变,它们是A29T,、T115I,、I195V、L203Y,、Q204K和E252G。对工程菌WTCBHⅠ进行定向进化得到了两株酶活力是出发菌株所产酶活力的2倍的突变菌株:CBHⅠX88和CBHⅠX26,测得的序列与野生基因比较,发现CBHⅠX88有10个突变位点它们是C13Y、S15P、S84P、N86D、N179D、D212E、C225R、M348K、D383G和S412G; CBHⅠX260有7个突变位点它们是C13Y、S15P、S101Y、M208T、D212E、N290T和Q473R。通过硫酸铵沉淀、DEAE-Sepharose阴离子交换柱层析等步骤纯化了突变蛋白,酶学性质与野生酶进行了比较,突变酶在最适反应温度、最适反应pH值和热稳定性方面都有提高。用同源建模的方法对6个工程菌所产酶的三维结构进行预测,对突变酶性质改变的可能机制从空间结构上进行了探讨,发现在突变酶CBHⅡX16和CBHⅡX305的L203 Y、CBM1中A29T,可能与酶活提高有关; K204Q、E252G、突变位点29和位点37、突变位点203和位点227之间氢键的增加都有利于突变酶的稳定性, T115I有利于热稳定性的提高, Q204K、E252G与突变酶最适反应pH值提高可能有关;在突变酶CBHⅠX88中的C225R和突变酶CBHⅠX260中的M208T,都是位于劈开纤维素的活性位点附近,CBHⅠX260中的Q473R是位于结合结构域（CBM）,它们的改变可能与提高酶活有关;两个突变酶的突变位点C13Y、S15P、S84P、C225R,这些改变的氨基酸的侧链都比原来的变大而复杂了,加强了包埋效应,提高了热稳定性,C225R、M348K和Q473R,R和K都是碱性氨基酸,可能是突变酶反应pH值升高的原因。利用定点突变的方法对本实验室已经构建成功的来自嗜热子囊菌光孢变种（Thermoascus aurantiacus var.levisporus）的内切葡聚糖酶eg1进行分子改造,先对比了多个纤维素酶第5家族（Cel5）内切葡聚糖酶序列,排除共同保守的氨基酸,选择其附近的非保守氨基酸作为突变点。选择了5个突变位点:N41D、L52M、Y129H、W165Y和H193A。通过硫酸铵沉淀、DEAE-Sepharose阴离子交换柱层析等步骤纯化了点突变突工程菌L52M EG1、Y129H EG1和W165Y EG1所产的内切葡聚糖酶,酶学性质与野生酶进行了比较,突变酶在最适反应温度、最适反应pH值和热稳定性方面都有改变。并用同源建模的方法对4个工程菌所产酶的三维结构进行预测,对突变酶性质改变的可能机制从空间结构上进行了探讨。His193是糖苷水解酶第5家族（GH5）的His¹⁹⁸保守氨基酸,致使H193A点突变的工程菌H193AEG1所产酶活力大幅度降低,N41D点突变的工程菌N41DEG1所产酶的活力也大幅度降低,可能与该位点静电力的改变有关。更多还原

【Abstract】 Many researches have been focused on cellulases since Seilliere found the enzyme in the digestive juice of snails in 1906. It has been shown that cellulases unpollutedly degrade cellulose waste, the most abundant renewable resources on earth, into a variety of useful products. Cellulases can also be used in industries including textile, paper pulp, food, feed and detergent. Recently, lignocellulose is degradated by cellulases to produce the fuel ethanol. Therefore, many researchers have begun to find a stain that can produce cellulases with the higher activity, the enhanced thermal stability and the increased pH stability to meet the needs of industrial production.In our laboratory, thermostable cellulases have been isolated from the thermophilic fungi and thermophilic fungi Thermoascus aurantiacus var. Levisporus and Chaetomium thermophilum CT2. In this study, the cellobiohydrolase genes cbh2 and cbh1 were isolated from C. thermophilum CT2 and were expressed in Pichia pastoris. Then, two expression strains were gotten through screening and were named as WTCBHⅡand WTCBHⅠ. After that, methods of direct evolution and high throughput screening for higher activity in P. pastoris were used to enhance the activity and stability of the cellobiohydrolase from C. thermophilum CT2. As a result, CBHⅡof two transformants showed 2 fold higher activities than that expressed from a wide-type cbh2 gene. The mutant CBHⅡin the two selected transformants were designed CBHⅡX16 and CBHⅡX305, respectively. CBHⅡX16 had five mutant amino acids: R1S, A29T, L203Y, Q204K and E252G. Meantime, A29T, T115I, I195V, L203Y, Q204K and E252G were exhibited in CBHⅡX305. In contrast, CBHⅠof two transformants exihibited 1 fold higher activities than that expressed from a wide-type cbh1 gene. The mutant CBHⅠin the two selected transformants were designed CBHⅠX88 and CBHⅠX260. We found 10 mutant amino acids (C13Y, S15P, S84P, N86D, N179D, D212E, C225R, M348K, D383G and S412G) in CBHⅠX88 and 7 mutant amino acids (C13Y, S15P, S101Y, M208T, D212E, N290T and Q473R) in CBHⅠX260. After the mutant and wild-type cellobiohydrolases were purified by using methods of the fractional ammonium sulphate precipitation and ion exchange chromatography on DEAE-Sepharose, their natures were compared.To understand how mutant amino acids affect CBH property, 3-dimentional structures of the wide-type and mutant CBH proteins from C. thermophilum in the study were predicted by the method of homology modeling. In CBHⅡX16 or CBHⅡX305, L203Y and A29T suggested that the activity was improved, and K204Q, E252G and T115I indicated the increasing of the thermal stability, while Q204K and E252G might be the reasons that the optimum reaction pH was enhanced. In CBHⅠX88 or CBHⅠX260, C225R, M208T and Q473R might lead to the increase in activity. In C13Y, S15P, S84P and C225R, the side chain of Y, P and R could improve the packing efficiency which might cause the thermal stability to be increased. Meantime, C225R, M348K and Q473R might be the reasons that the optimum reaction pH was enhanced.The method of site-directed mutagenesis was used to modify endo-β-glucanases encoded by eg1 gene from T. aurantiacus var. Levisporus. The enzymes with five mutant non-conservative amino acids (N41D, L52M, Y129H, W165Y and H193A) were expressed in P. pastoris. Endo-β-glucanase activities were significantly reduced by two amino acid mutation N41D and H193A. The enzymes with L52M, Y129H and W165Y were separately expressed in P. pastoris, and the constructed engineering stains were named as L52MEG1, Y129HEG1 and W165YEG1, respectively. After the mutant and wild-type endo-β-glucanases were purified by using fractional ammonium sulphate precipitation and ion exchange chromatography on DEAE-Sepharose, their characterizations were compared. The three-dimensional structure of enzymes produced from L52MEG1, Y129HEG1 and W165YEG1 were predicted by using the method of homology modeling, and the possible mechanisms of the changes in the natures of the mutant enzymes were discussed.更多还原