解纤维芽孢杆菌来源的适冷木聚糖酶和β-葡萄糖苷酶的异源表达及酶学性质研究

发布时间：2018-07-05 21:01

  本文选题：B.cellulosilyticus + DSM　； 参考：《江苏大学》2017年硕士论文

【摘要】：β-葡萄糖苷酶和木聚糖酶是两类重要的碳水化合物水解酶,广泛应用在食品、生物质能源和饲料等领域。温度特性是评价酶的一个重要参数,目前相关研究多集中在中温和高温酶上,而这些酶难以满足某些工业需要低温酶的特殊性。适冷酶是指具有较低的最适反应温度,在低温条件下与底物结合能力及催化活性都很强的酶。适冷β-葡萄糖苷酶和木聚酶与中、高温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,节约成本,在工业应用中存在巨大的潜力。目前适冷β-葡萄糖苷酶和木聚糖酶可获得资源相对较少(主要来源冰川和海洋),并且已被研究的为数不多的酶活性不高,离实际的应用还存在一定的差距,因此产业化应用受到了限制。所以,获得具有性质优良的适冷β-葡萄糖苷酶和木聚糖酶具有重要的意义。从极端环境中挖掘基因资源是获得具有新性质酶的重要途径,解纤维芽孢杆菌(Bacillus cellulosilyticus DSM 2522)因其来源于耐盐碱的土壤中,生长于低温环境和纤维素降解能力而被基因测序。测序结果表明,其含有大量的糖苷水解酶,可称之为碳水化合物酶库。本课题通过数据比对和分析,选择其中一个葡萄糖苷酶基因(Bcell_0282)和一个木聚糖苷酶基因(Bcell_0547)进行异源表达和性质表征研究,以期能获得具有冷适应性的酶。本论文的主要研究成果归纳如下:1.本研究中发现,β-葡萄糖苷酶(BcBGlA)和木聚糖酶(Xyn10A)最显著的特点是冷适应性和耐盐性。重组BcBGlA属于GH1家族,BcBGlA的最适pH是7.0,在pH 5.0-8.0具有较好的稳定性。最适温度是40℃,在30℃-45℃孵育24h后,仍剩余50%以上酶活,当在50℃中,孵育1h后几乎失活,而4℃的低温下,仍保持27.5%的相对酶活;重组Xyn10A属于GH10家族,Xyn10A的最适pH是8.0,在p H 4.0-11.0具有较好的稳定性。最适温度40℃,在4℃和20℃的低温仍然可以保持38.3%和55.7%的相对活性。重组BcBGlA当以纤维二糖作为底物时,Kcat/Km值为45.56/s/mM,在已报道的β-葡萄糖苷酶中较高。2.重组BcBGlA在研究时,表现出的对于葡萄糖的耐受量具有一定的优势,可以缓解生物转化过程中由纤维二糖积累而引起的内切葡聚糖酶和纤维二糖水解酶的分解代谢作用;重组Xyn10A水解木聚糖的产物分析中,主要产物为木二糖、木三糖和木四糖,但不产生木糖。3.当在反应液中添加0.2M NaCl,此时BcBGlA的相对酶活最高,当盐浓度达到1.0M时,其残留的酶活量仍保留在60%以上。在反应液中加入0.5M NaCl,此时Xyn10A的相对酶活最高,同时加入0.5M NaCl还可以提高温度的稳定性;当盐浓度达到3.0M时,其残留的酶活量仍为66.5%。本研究中利用基因克隆表达技术实现了葡萄糖苷酶和木聚糖酶的异源表达,获得了在低温下具有较高催化活性且稳定的重组酶,其展现出了在食品工业和生物质能源领域的产业化应用潜力。
[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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