【作者】 张亚波；

【导师】 李多川；

【作者基本信息】 山东农业大学 ， 环境生物学， 2009， 博士

【摘要】 纤维素是地球上分布最广、蕴藏量最丰富的生物质,也是最廉价的可再生资源,但是其中有大部分不能直接利用,成为纤维素废物。我国几乎所有的纤维素废物,如作物秸秆、废纸、废木料等,白白烧掉或直接丢弃。这种处理方式不但造成数十亿直接经济损失,而且焚烧产生的大量浓烟及排放的很多有害气体对环境和生态都造成了严重的影响。纤维素废料之所以难于利用是由于纤维素难于降解分子结构,而纤维素酶可以将纤维素降解为工业上易于利用的葡萄糖,纤维素酶还可用于纺织、造纸、洗涤、饲料食品加工以及洗涤剂生产等工业领域。该酶主要来自于真菌、细菌和原生动物,是降解纤维素生成葡萄糖的一组酶的总称,它不是单成分酶,而是由多个起协同作用的酶构成的多酶体系。尽管纤维素酶已经在工业上有所应用,但是其酶活力、热稳定性和pH稳定性等酶学指标仍需要提高才能满足工业生产的需要。嗜热微生物是高热稳定纤维素酶的主要来源,嗜热真菌产生的纤维素酶在高温条件下具有高活力和稳定性,并且与中温真菌一样,嗜热真菌产生的各类纤维素酶普遍含有多种类型的组分。嗜热子囊菌光孢变种(Thermoascus aurantiacus var.levisporus)和嗜热毛壳菌(Chaetomium thermophilum CT2)是广泛分布的,生长上限温度较高的嗜热真菌,从这两种真菌中已分离了多种嗜热酶,并且证明了其纤维素酶存在多个组分,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。本研究中从嗜热子囊菌光孢变种(T.aurantiacus var.levisporus)中分离到一种eg1基因(GenBank登录号为AY 847014),并在毕赤酵母中高效表达,筛选到一株工程菌命名为GpN24,该重组毕赤酵母工程菌株所表达的内切葡聚糖酶Ⅰ具有良好的热稳定性。工程菌GpN24产生的酶蛋白分子量为33kDa,最适温度和pH分别为55℃和5.0,在90℃的条件下保温30min后仍具有50%的酶活力,在pH3.0-5.0的条件下酶活力保持稳定。为了提高酶活力和稳定性,本研究以内切葡聚糖酶eg1作为出发基因,采用易错PCR方法建立突变体库后,以高活力作为筛选压力,先后以双层平板法和小量发酵法筛选突变体库。经过突变和筛选,获得了一个酶活力提高3.5倍的突变体,并且最适pH由原来的5.0变为了4.0。测得的序列与野生基因比较,发现第41位氨基酸N突变为D;第129位氨基酸Y突变为H;第235位氨基酸G突变为E。其中第129和第235个氨基酸均属于保守性的氨基酸。通过硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子层析、Phenyl-Sepharose疏水层析等步骤纯化了该蛋白,酶学性质与野生酶进行了比较,并初步探讨酶活力提高的可能机制。利用融合PCR技术在eg1的5’末端连接了来自于嗜热毛壳菌(C.thermophilum CT2)的cbh1基因,经测序验证读码框正确后,构建表达载体转化毕赤酵母GS115中,筛选到一株表达量较高的融合基因eg1cbh1的酵母工程菌株,命名为GPNI3。该菌株表达的融合酶的分子量为88kDa,最适温度和pH分别为50℃和4.0,对不同底物的酶活力均高于内切葡聚糖酶eg1的活力。大多数纤维素水解酶至少由催化区和结合区组成,催化区含有催化水解纤维素β-1,4糖昔键的活性位点;纤维素吸附区(CBD),主要功能是结合纤维素,对纤维素的超分子结构具有特异性,能够提高酶对纤维素的吸附,从而增加纤维素底物的水解。CBD在纤维素酶对不溶性纤维素底物的水解中起着关键作用,关于CBD作用的多数信息,是通过去除该结构域、改变结构域或进行定点诱变研究而得到,本实验室已经从嗜热毛壳菌中分离到了cbh1和cbh3基因,cbh1具有完整的结构即:催化区、结合区以及连接两个区域的连接桥;而cbh3缺少结合区,本研究在无结合区的嗜热毛壳菌cbh3酶蛋白的C端通过融合PCR技术连接了cbh1基因的纤维素结合区CBD,经测序验证读码框正确后,构建表达载体转化毕赤酵母中,筛选到一株高表达量的融合基因cbh3-1的酵母工程菌株,命名为GPB103。该菌株表达的融合酶蛋白分子量为51kDa,最适温度和pH分别为60℃和4.0,滤纸酶活较cbh3提高了1.2倍,DEAE-Sepharose Fast Flow阴离子层析纯化了该蛋白,并对酶学性质进行了研究。更多还原

【Abstract】 Cellulose is the most abundant organic compound on Earth,widely spreading as one of the cheapest renewal resources, but most of it can’t be used directly and becomes cellulose waste.Almost all of the cellulose waste, such as crop residual, used paper, forest waste and so on, is merely burned or deserted without treatment in China, which causes many environmental, ecological problems and several billion yuan economic losses in one year.The cellulose waste is difficult to be used because it is a tough molecule to break down, but it can be converted into sugars, which can be easily used in industry, by cellulases.Cellulases can also used in textile industry, paper and pulp industry, food industry, feed industry, detergent industry and so on.The cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and protozoans that catalyzes the hydrolysis of cellulose by Hydrolyzing 1,4-beta-D-glycosidic linkages in cellulose.Although the cellulase has been widely used as an important industrial enzyme, it should be improved in enzyme activity, thermostability, pH stability to meet the industry need.Thermophiles are main sources of cellulases with high thermostability.Cellulases from the thermophilic fungi have been reported to be stable and highly active at high temperature and, like mescophilic fungi, thermophilic fungi can produce multiple forms of the cellulase components, mostly.Thermoascus aurantiacus var.levisporus and chaetomium thermophilum CT2 are both widespread thermophilic fungi.They can thrive at temperature between 45℃and 50℃, while most fungi would die above 40℃.Several thermostable enzymes have been isolated from the thermophilic fungi.Current study shows that both the thermophilic fungi’s cellulases have multiple components and have high activity and thermostability, which makes the thermophilic fungi’s cellulases have great research and industry value.In this study, an endo-β-glucanase encoding gene, eg1 (GenBank accession number: AY 847014), has been isolated from T.aurantiacus var.levisporus and expressed in pichia pastoris.A high expression efficiency strain named GpN24 was gotten through screening, and this strain can express recombinant endo-β-glucanase with excellent thermostability.The molecular of the enzyme was 33kDa.The optimum pH and temperature of the recombinant enzyme activity were 5.0 and 55℃respectively.The recombinant enzyme remained 50% of its original activity after 30 min at 90℃and the activity of the recombinant enzyme can remain stable in pH between 3.0 and 5.0.Direct evolution based on eg1’s error-prone PCR and high throughput screening for higher activity in pichia pastoris was used to enhance activity and stability of the endo--β-glucanase from T.aurantiacus var.levisporus and the expression efficiency of its encoding gene eg1.A mutant library of eg1 was constructed by error-prone PCR.In this research, small amount ferment in 1.5ml centrifuge tube after two-layer plate was used to screen for higher enzyme activity.Through mutagenesis and screening, a mutant showed 3.5 fold enzyme activity and optimum pH changing from 5.0 to 4.0.The result of alignment of the mutant’s and the wild type amino acid sequences showed that the wild type‘s 44 amino acid N was replaced with D,and the wild type’s 129 amino acid Y changed to H, and wild type’s 235 amino acid G mutate to E.The mutant site 229 and 235 belong to conserved sites.Both the mutant endo-β-glucanase and wild type endo-β-glucanase were purified by fractional ammonium sulphate precipitation, ion exchange chromatography on DEAE-Sepharose and Phenyl-Sepharose hydrophobic interaction chromatography.The preliminary mechanism was studied by comparing the two purified endo-β-glucanases.Cbh1 of C.thermophilum was linked to 5’end of eg1 by fusion PCR.After sequencing to make sure a correct ORF, an expression vector was constructed to transform into pichia pastoris, and then a high expression strain of fusion gene eg1cbh1 was screened, and named GPNI3.The molecular of the enzyme was 88kDa .The fusion protein expressed by this strain exhibited maximal activity at pH 4.0 and 60oC and showed higher enzyme activity on all the different substrates than endo-β-glucanase eg1Most cellulases have catalysis domain (CD) and binding domain (BD) .The CD has active sites of catalyzingβ-1,4 glycosidic bond , while the CBD have specificity to the supramolecular structure of cellulose, and increase the absortion to cellulose, and accelerates the hydrolysis of cellulose.As the CBD has key role in the hydrolysis of cellulose, lots of researchs were done by deletion、change or site-directed mutagenesis to the CBD.Cbh1 gene and cbh3 gene were cloned from C.thermophilum, and cbh1 has CD CBD and a link, while cbh3 don’t have CBD.We linked the CBD of cbh1 to the C- terminal through fusion PCR.The expression plasmid was constructed and transformed into Pichia pastoris.A strain with high expression was selected and named as GPB103.After DEAE-Sepharose chromatography, the fusion enzyme homogeneously by SDS-PAGE was purified and the characterization of the purified enzyme was carried out.The molecular of the fusion protein expressed was 51kDa.The optimum temperature and pH for enzyme activity were 60℃and 4.0 respectively, and the activity increased 1.2 times on filter paper.更多还原