本文選題：纖維素　切入點：外切葡聚糖酶　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：纖維素在自然界中廣泛存在,而且含量豐富,是一種可再生資源,對解決能源短缺和資源枯竭具有重要意義。但是,我國的纖維素利用率很低,造成大量的浪費。在纖維素的分解過程中,纖維素酶發(fā)揮非常大的作用,將纖維素最終分解為葡萄糖,使得纖維素能夠得到充分的利用。嗜熱革節(jié)孢是一種嗜熱真菌,能夠產(chǎn)生熱穩(wěn)定性酶,是纖維素的分解者之一。本研究對來自嗜熱革節(jié)孢的外切葡聚糖酶基因cbhk和β-葡萄糖苷酶基因bgk進行克隆,并在畢赤酵母中進行表達,得到CBHK和BGK重組酶。對重組酶進行分離純化,SDS-PAGE電泳檢測蛋白分子量,外切葡聚糖酶CBHK的表觀分子量為72kDa,大于理論分子量50kDa,可能是糖基化造成的;β-葡萄糖苷酶BGK的分子量大小為55kDa與理論的分子量大小基本一致。改善酶活性對提高纖維素的降解具有十分重要的作用�；谑葻岣锕�(jié)孢β-葡萄糖苷酶Hi BG的結(jié)構(gòu),本研究對嗜熱革節(jié)孢β-葡萄糖苷酶活性位點進口端的12個氨基酸殘基進行定點突變。通過同源建模,對來源于嗜熱革節(jié)孢的外切葡聚糖酶基因進行四個位點的定點突變。并將它們在畢赤酵母中進行了突變基因的高效表達。與野生型外切葡聚糖酶相比,突變體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分別降低了40%和35%;突變后W397H活性完全喪失。與野生型β-葡萄糖苷酶相比,所有突變體的酶活力都降低了,其中A260N、F348G、Y179F和F180H酶活性降低較少,分別降低了20%、20%、30%和30%;D237S、L173Q酶活性分別降低了55%和60%;而突變W168H、N335F和W349G的酶活性完全喪失。在纖維素的分解過程中,β-葡萄糖苷酶起著關(guān)鍵作用,酶的催化存在產(chǎn)物抑制現(xiàn)象,大多數(shù)酶都存在產(chǎn)物抑制現(xiàn)象,但是一些酶對葡萄糖具有耐受性。嗜熱革節(jié)孢糖苷水解酶第一家族β-葡萄糖苷酶對葡萄糖具有耐受性,但是具體的作用機制尚不清楚。本研究,通過對嗜熱革節(jié)孢糖苷水解酶第一家族β-葡萄糖苷酶的12個進口端位點進行定點突變,研究這些位點對于嗜熱革節(jié)孢第一家族β-葡萄糖苷酶酶活性及葡萄糖耐受性的影響。突變L173Q喪失葡萄糖耐受性,突變Y179F在高濃度葡萄糖時喪失葡萄糖耐受性。初步證明進口端位點對于酶活性及葡萄糖耐受性都具有一定的影響,催化活性通道的結(jié)構(gòu)特異性可能是葡萄糖耐受機制。
[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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q936

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