本文選題：B.cellulosilyticus + DSM�。� 參考：《江蘇大學》2017年碩士論文

【摘要】：β-葡萄糖苷酶和木聚糖酶是兩類重要的碳水化合物水解酶,廣泛應用在食品、生物質(zhì)能源和飼料等領域。溫度特性是評價酶的一個重要參數(shù),目前相關研究多集中在中溫和高溫酶上,而這些酶難以滿足某些工業(yè)需要低溫酶的特殊性。適冷酶是指具有較低的最適反應溫度,在低溫條件下與底物結(jié)合能力及催化活性都很強的酶。適冷β-葡萄糖苷酶和木聚酶與中、高溫β-葡萄糖苷酶和木聚糖酶相比,其能提高底物利用率,降低能耗,節(jié)約成本,在工業(yè)應用中存在巨大的潛力。目前適冷β-葡萄糖苷酶和木聚糖酶可獲得資源相對較少(主要來源冰川和海洋),并且已被研究的為數(shù)不多的酶活性不高,離實際的應用還存在一定的差距,因此產(chǎn)業(yè)化應用受到了限制。所以,獲得具有性質(zhì)優(yōu)良的適冷β-葡萄糖苷酶和木聚糖酶具有重要的意義。從極端環(huán)境中挖掘基因資源是獲得具有新性質(zhì)酶的重要途徑,解纖維芽孢桿菌(Bacillus cellulosilyticus DSM 2522)因其來源于耐鹽堿的土壤中,生長于低溫環(huán)境和纖維素降解能力而被基因測序。測序結(jié)果表明,其含有大量的糖苷水解酶,可稱之為碳水化合物酶庫。本課題通過數(shù)據(jù)比對和分析,選擇其中一個葡萄糖苷酶基因(Bcell_0282)和一個木聚糖苷酶基因(Bcell_0547)進行異源表達和性質(zhì)表征研究,以期能獲得具有冷適應性的酶。本論文的主要研究成果歸納如下:1.本研究中發(fā)現(xiàn),β-葡萄糖苷酶(BcBGlA)和木聚糖酶(Xyn10A)最顯著的特點是冷適應性和耐鹽性。重組BcBGlA屬于GH1家族,BcBGlA的最適pH是7.0,在pH 5.0-8.0具有較好的穩(wěn)定性。最適溫度是40℃,在30℃-45℃孵育24h后,仍剩余50%以上酶活,當在50℃中,孵育1h后幾乎失活,而4℃的低溫下,仍保持27.5%的相對酶活;重組Xyn10A屬于GH10家族,Xyn10A的最適pH是8.0,在p H 4.0-11.0具有較好的穩(wěn)定性。最適溫度40℃,在4℃和20℃的低溫仍然可以保持38.3%和55.7%的相對活性。重組BcBGlA當以纖維二糖作為底物時,Kcat/Km值為45.56/s/mM,在已報道的β-葡萄糖苷酶中較高。2.重組BcBGlA在研究時,表現(xiàn)出的對于葡萄糖的耐受量具有一定的優(yōu)勢,可以緩解生物轉(zhuǎn)化過程中由纖維二糖積累而引起的內(nèi)切葡聚糖酶和纖維二糖水解酶的分解代謝作用;重組Xyn10A水解木聚糖的產(chǎn)物分析中,主要產(chǎn)物為木二糖、木三糖和木四糖,但不產(chǎn)生木糖。3.當在反應液中添加0.2M NaCl,此時BcBGlA的相對酶活最高,當鹽濃度達到1.0M時,其殘留的酶活量仍保留在60%以上。在反應液中加入0.5M NaCl,此時Xyn10A的相對酶活最高,同時加入0.5M NaCl還可以提高溫度的穩(wěn)定性;當鹽濃度達到3.0M時,其殘留的酶活量仍為66.5%。本研究中利用基因克隆表達技術(shù)實現(xiàn)了葡萄糖苷酶和木聚糖酶的異源表達,獲得了在低溫下具有較高催化活性且穩(wěn)定的重組酶,其展現(xiàn)出了在食品工業(yè)和生物質(zhì)能源領域的產(chǎn)業(yè)化應用潛力。
[Abstract]:尾-glucosidase and xylanase are two important carbohydrate hydrolases, which are widely used in food, biomass energy and feed. The temperature characteristic is an important parameter to evaluate the enzymes. At present, most of the related studies focus on the moderate and high temperature enzymes, but these enzymes are difficult to meet the special needs of some industries. Chilling enzyme is an enzyme with low reaction temperature and strong binding ability and catalytic activity with substrate at low temperature. Compared with moderate, high temperature 尾 -glucosidase and xylanase, cold 尾 -glucosidase and xylanase can improve substrate utilization, reduce energy consumption, save cost, and have great potential in industrial application. At present, the available resources of 尾 -glucosidase and xylanase are relatively few (mainly from glaciers and oceans), and the few enzyme activities that have been studied are not high, which is still far from the actual application. Therefore, the application of industrialization is limited. Therefore, it is of great significance to obtain 尾-glucosidase and xylanase with excellent properties. Mining gene resources from extreme environments is an important way to obtain new enzymes. Bacillus cellulosilyticus DSM 2522 was sequenced because it originated from saline-alkali tolerant soil, grew in low temperature environment and degraded cellulose. Sequencing results show that it contains a lot of glycoside hydrolase, which can be called carbohydrate enzyme library. In this study, one glucosidase gene (Bcell0282) and a wood-polyglucosidase gene (Bcell0547) were selected for heterologous expression and characterization by data comparison and analysis, in order to obtain cold-adaptive enzyme. The main research results of this thesis are summarized as follows: 1. In this study, it was found that the most significant characteristics of 尾 -glucosidase (BcBGlA) and xylanase (Xyn10A) were cold adaptability and salt tolerance. The optimal pH of recombinant BcBGlA belongs to GH1 family BcBGlA is 7.0, and it has good stability at pH 5.0-8.0. The optimum temperature was 40 鈩，

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