嗜热革节孢第一家族和第六家族糖苷水解酶的分子改造

发布时间：2018-03-20 14:29

本文选题：纤维素　切入点：外切葡聚糖酶　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：纤维素在自然界中广泛存在,而且含量丰富,是一种可再生资源,对解决能源短缺和资源枯竭具有重要意义。但是,我国的纤维素利用率很低,造成大量的浪费。在纤维素的分解过程中,纤维素酶发挥非常大的作用,将纤维素最终分解为葡萄糖,使得纤维素能够得到充分的利用。嗜热革节孢是一种嗜热真菌,能够产生热稳定性酶,是纤维素的分解者之一。本研究对来自嗜热革节孢的外切葡聚糖酶基因cbhk和β-葡萄糖苷酶基因bgk进行克隆,并在毕赤酵母中进行表达,得到CBHK和BGK重组酶。对重组酶进行分离纯化,SDS-PAGE电泳检测蛋白分子量,外切葡聚糖酶CBHK的表观分子量为72kDa,大于理论分子量50kDa,可能是糖基化造成的;β-葡萄糖苷酶BGK的分子量大小为55kDa与理论的分子量大小基本一致。改善酶活性对提高纤维素的降解具有十分重要的作用。基于嗜热革节孢β-葡萄糖苷酶Hi BG的结构,本研究对嗜热革节孢β-葡萄糖苷酶活性位点进口端的12个氨基酸残基进行定点突变。通过同源建模,对来源于嗜热革节孢的外切葡聚糖酶基因进行四个位点的定点突变。并将它们在毕赤酵母中进行了突变基因的高效表达。与野生型外切葡聚糖酶相比,突变体的酶活性都有所降低,其中W163H酶活性降低最多,降低了50%;E429D和Q459T分别降低了40%和35%;突变后W397H活性完全丧失。与野生型β-葡萄糖苷酶相比,所有突变体的酶活力都降低了,其中A260N、F348G、Y179F和F180H酶活性降低较少,分别降低了20%、20%、30%和30%;D237S、L173Q酶活性分别降低了55%和60%;而突变W168H、N335F和W349G的酶活性完全丧失。在纤维素的分解过程中,β-葡萄糖苷酶起着关键作用,酶的催化存在产物抑制现象,大多数酶都存在产物抑制现象,但是一些酶对葡萄糖具有耐受性。嗜热革节孢糖苷水解酶第一家族β-葡萄糖苷酶对葡萄糖具有耐受性,但是具体的作用机制尚不清楚。本研究,通过对嗜热革节孢糖苷水解酶第一家族β-葡萄糖苷酶的12个进口端位点进行定点突变,研究这些位点对于嗜热革节孢第一家族β-葡萄糖苷酶酶活性及葡萄糖耐受性的影响。突变L173Q丧失葡萄糖耐受性,突变Y179F在高浓度葡萄糖时丧失葡萄糖耐受性。初步证明进口端位点对于酶活性及葡萄糖耐受性都具有一定的影响,催化活性通道的结构特异性可能是葡萄糖耐受机制。
[Abstract]:Cellulose exists widely in nature and is rich in content. It is a kind of renewable resource, which is of great significance to solve the energy shortage and resource depletion. However, the utilization rate of cellulose in China is very low. In the process of cellulose decomposition, cellulase plays a very important role in the final decomposition of cellulose into glucose, so that cellulose can be fully utilized. In this study, the exon glucanase gene cbhk and 尾-glucosidase gene bgk from the thermophilic Arthrospora were cloned and expressed in Pichia pastoris. CBHK and BGK recombinant enzymes were obtained. The protein molecular weight was determined by SDS-PAGE electrophoresis. The apparent molecular weight of the exoglucanase CBHK is 72 kDa, which is larger than the theoretical molecular weight of 50 kDa, which may be caused by glycosylation, and the molecular weight of 尾-glucosidase BGK is 55 kDa, which is consistent with the theoretical molecular weight. The degradation of vitamin plays a very important role. Based on the structure of thermophilic actinomycetes 尾 -glucosidase Hi BG, In this study, 12 amino acid residues at the import end of 尾-glucosidase activity site were mutated by homology modeling. Four site-directed mutations of exopolydextranase genes from P. thermophilus were carried out. They were highly expressed in Pichia pastoris and compared with wild type exoglucanase. The enzyme activity of mutant W163H was decreased by 40% and 35T, respectively, and the activity of W397H was completely lost after mutation. Compared with wild-type 尾 -glucosidase, the enzyme activity of all mutants decreased. The enzyme activities of A260NX, F348GN, Y179F and F180H decreased by 20% and 30%, respectively, and the activities of D237SN L173Q decreased by 55% and 60g, respectively, while the enzyme activities of mutant W168HN335F and W349G were completely lost. 尾 -glucosidase played a key role in the decomposition of cellulose, and 尾 -glucosidase played a key role in the decomposition of cellulose. The enzyme catalyzes the existence of product inhibition phenomenon, most of the enzymes have product inhibition phenomenon, but some enzymes have tolerance to glucose, the first family of thermophilic gamma-glycoside hydrolase 尾 -glucosidase has tolerance to glucose. However, the specific mechanism is still unclear. In this study, a site-directed mutation was carried out on 12 entry sites of the first family of thermophilic gamma-glycoside hydrolases, 尾 -glucosidase. To study the effects of these loci on 尾-glucosidase activity and glucose tolerance in the first family of Arthrospora thermophilus. The mutant L173Q lost glucose tolerance. The mutation Y179F lost glucose tolerance at high glucose concentration. It was preliminarily proved that the inlet site had certain influence on enzyme activity and glucose tolerance. The structure specificity of catalytic active channel may be the mechanism of glucose tolerance.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936

【参考文献】