嗜冷木聚糖降解酶的基因克

发布时间：2018-05-13 16:42

  本文选题：低温酶 + 基因克隆　； 参考：《天津科技大学》2017年硕士论文

【摘要】：木聚糖是植物细胞壁的主要组成成分,它的完全降解需要多种降解酶的共同作用,其中木聚糖酶和木糖苷酶是木聚糖降解过程中的关键酶。木聚糖降解酶已经广泛的应用饲料、食品、造纸和生物能源等多个工业领域。近年来,人们发现在食品加工等行业中应用嗜冷木聚糖降解酶可以有效减少产品活性成分、风味物质和营养成分的损失,降低生产成本。然而,目前发现的木聚糖酶和木糖苷酶绝大多数都属于中高温酶(http://www.brenda-enzymes.org),已报道有基因序列和酶学性质研究的低温木糖苷酶仍然匮乏,亟待发掘性质优良的嗜冷木聚糖酶和木糖苷酶基因资源,并对其嗜冷机制进行研究,本研究获得了一个序列和性质新颖的嗜冷木聚糖酶Xyn27,并且对嗜冷木糖苷酶AX543的嗜冷机制进行了初步分析和突变研究。具体如下:通过Touch-down PCR和热不对称交错(TAIL) PCR方法,从枝顶孢属菌的基因组DNA中获得了 GH10家族(Xyn27)的新型木聚糖酶,Xyn27包含一个信号肽(20个残基),编码346个氨基酸,与报导的GH10木聚糖酶(XM_003662144)最高一致性为83 %。重组Xyn27在毕赤酵母GS115中成功表达,最适诱导条件为35℃、48h。Xyn27是一种嗜冷木聚糖酶,其中在35℃时的活性最高,在20℃,10℃和0℃下的相对活性仍为60.25%, 38.70%和10.8%。进一步分析表明,与嗜温或嗜热的对应物相比,Xyn27具有较少的精氨酸而有更多的丙氨酸残基。Xyn27的最适pH为7.0,在3.0～9.0的pH范围内反应lh后仍较稳定。此外,Xyn27对于大多数金属离子和有机溶剂有耐受性,优于其他GH10嗜冷木聚糖酶,其中对于重金属离子Ca2+,. Mn2+和Zn2+活性显著增强。性质研究表明,以1%榉木木聚糖为底物时的Km,Vmax,kcat和 kcat . Km-1 分别为 13.42 mg . mL-1, 9.07 umol · min-1 . mg-1, 192.98 min-1和 14.38 mL.min-1.mg,Xyn27可将木聚糖完全降解为木二糖。基于木聚糖酶Xyn27的嗜冷活性和金属离子耐受性,表明其在基础研究和食品等行业有潜在应用。木糖苷酶AX543最适温度为25℃,为目前报道的最适温度较低的酶,通过一系列的序列分析和结构比对,推断loop区结构的差异可能是低温酶嗜冷的主要原因之一,其次还包括氨基酸残基的不同,经分析发现AX543在5个位置的loop区存在显著差异,分别是 loopl (31-46)、loop2 (54-59)、loop3 (164-183)、loop4 (209-224)和 loop5(296-298),于是针对这些位置,设计引物进行突变,成功获得六个质粒,其中前五个突变体成功在毕赤酵母中表达,但由于活性均比较低,未进行下一步研究。
[Abstract]:Xylan is the main component of plant cell wall, and its complete degradation requires the joint action of many kinds of degradation enzymes, among which xylanase and xylosidase are the key enzymes in the process of xylan degradation. Xylanase has been widely used in feed, food, paper making and bioenergy industries. In recent years, it has been found that the application of cold-eosinophilic xylanase in food processing and other industries can effectively reduce the loss of active ingredients, flavor substances and nutrients, and reduce the production cost. However, most of the xylanase and xylosidase found at present belong to medium high temperature enzyme http: / www.brenda-enzymes.org.There is still a lack of low-temperature xylosidase, which has been reported to have been studied in gene sequence and enzymology. It is urgent to explore the chilled xylanase and xylanase gene resources, and to study the mechanism of psychrophilic xylanase. In this study, a novel chilled xylanase Xyn27 was obtained, and the mechanism of the chilled xylanase AX543 was preliminarily analyzed and mutated. The results are as follows: a novel xylanase Xyn27 containing a signal peptide (20 residues, encoding 346 amino acids) was obtained from the genomic DNA of the genus Cladosporium by Touch-down PCR and thermal asymmetric interlacing (TAILL) PCR, and a novel xylanase, Xyn27, was obtained from the genomic DNA of the genus Cladosporium. The highest consistency with the reported GH10 xylanase X _ M _ S _ 003662144) was 83. The recombinant Xyn27 was successfully expressed in Pichia pastoris GS115. The optimal inducing condition was that Xyn27 was a chilled xylanase at 35 鈩，

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