腾冲热泉嗜热细菌新资源的挖掘及木聚糖酶活性菌株筛选与酶学特性研究

Hot spring is a stable and close microbial ecosystem with high temperature. The high temperature of hot spring leads the microbes to adapt a unique physiological mechanism and create the specific genes, which help them to survive the high temperature and also make them as a potential source of many unique enzymes..Xylans are almost as ubiquitous as cellulose, forms a major component of plant cell walls and hard woods. Endo-β-1,4 xylanases (EC 3.2.1.8, 1,4-β-D-xylan sylanohydrolase) is the major enzyme which degrade xylan into D-xylose and xylooligosacchariedes. In recent years, xylanases have attracted much attention and extensively employed in food, animal feed, textile, waste treatment, and biofuel industries. Howerver, many commercially available xylanases in the market lack thermostability and can’t meet industrial demands. Therefore, there are many demands for xylanases with thermostability. Understanding the present situation in the present work an attempt will be made to isolate novel microbes from Tengchong hot springs as well as screen the isolates with thermostable activity of xylanases. .In the present study, two rational and holistic approaches applied to isolate bacteria community and screen the isolates with xylanase activity effectively from hot spring environment will be established in order to improve the success rate and obtaining large numbers of pure-culture bacteria as well as those of demonstrating xylanase activity which are hardly or uncultured before from Tengchong hot springs. The diversity of bacteria community will be analyzed based on 16S rRNA. Thermophile bacteria which produced thermostable xylanase was isolated, screened and identified. The novel isolates will be classified and identified by using polyphasic taxonomy approachs..The genome of novel strain with xylanase activity was sequenced, assembled and then the genes of xylanase were annotated. Base on the annotated gene sequence, the primer was designed and the whole gene of xylanase was cloned and expressed. .Among isolated microbes with xylanase activity, the strains with high thermostable xylanase activity will be selected for fermentation to produce the xylanase and the xylanase was purified. The xylanase proteins produced from xylanase gene isolates were investigated according to their charateristics. Compared with the characteristics and structure of xylanase as well as the genome information of its host, the xylanases and their host strains were evaluated for industrial application. This study will help to understand the distribution and improve the isolation rate of prokaryotic microbes with culture-dependent from Tengchong hot springs habitats. This work will be served as groundwork to exploit the novel products including thermostable enzymes and other bioactive compounds with special characteristics produced by thermophiles, as well as to develop the potential biotechnological applications in various industrial fields.

热泉生境具有高温特点，蕴含丰富的嗜热微生物。这些微生物具有特殊的生理机制和独特基因，是发现未知或具有特殊功能酶类的理想对象。木聚糖酶是重要的工业酶制剂，其应用广泛。高温热泉生境原核微生物资源的研究，为耐高温酶的筛选和开发提供了资源。本项目以腾冲热泉群为研究对象，系统地开展热泉生境嗜热细菌新资源的挖掘、耐高温木聚糖酶活性菌株筛选与酶学性质研究。拟结合前期对腾冲热泉微生物的免培养研究结果，探索适合腾冲热泉生境嗜热细菌的分离技术和产木聚糖酶活性菌株筛选技术，获得更多难培养或未培养微生物和产酶菌株，拓展微生物与木聚糖酶资源；采用基因组研究方法，从产酶新物种中克隆表达木聚糖酶基因，同时选取高酶活性菌株，发酵优化产酶，对两种木聚糖酶源进行酶学特性研究与结构分析，评价酶与菌株的应用潜力，为耐高温木聚糖酶开发与应用提供资源与保障。本项目的实施可加强对我国高温热泉生境微生物与酶资源的挖掘、开发及利用。

高温热泉生境蕴含有丰富的嗜热微生物，具有特殊的生理机制和独特的基因，是发现未知或具有特殊功能酶类的理想地。木聚糖酶是重要的工业酶制剂，但当前存在着热稳定性差的局限性。本研究以腾冲热泉群为研究对象，通过纯培养分离、基因组学和酶学性质等方法系统地开展热泉生境嗜热原核微生物资源的分离、耐高温木聚糖酶筛选与酶学性质研究。本研究取得的主要成果：（1）研究热泉环境微生物群落的底物代谢差异、功能多样性指数、主成分分析和聚类分析，了解不同热泉环境微生物群落多样性与代谢特征。结果表明8个热泉环境微生物群落分为三种代谢类型。（2）根据微生物群落代谢特征，设计出四类13种分离培养基分离腾冲热泉中嗜热原核微生物资源。共分离出634株嗜热原核微生物，属于Firmicutes、Proteobacteria、Deinococcus-Thermus、Actinobacteria和Chloroflex的7个纲，11个目，19个科，32个属以及19个潜在的新分类单元。（3）筛选木聚糖酶活性菌株，直接筛选、玉米芯、麸皮和桦木屑为底物的富集处理1和富集处理2获得木聚糖酶活性菌株阳性率分别为9.1%、11.4%和45.7%。Geobacillus sp. YIM 71344发酵所产木聚糖酶的酶学性质，表明该酶最适反应温度和pH为80℃、pH6.0和pH9.0；可在70℃和pH6.0-12.0耐受下保持较高酶活力，可特异性降解不同来源的木聚糖底物。（4）Meiothermus sp. YIM 71147和Geobacillus sp. YIM 71344进行基因组测序，发现这两株菌中有相当数量的基因与木聚糖的降解有关。从中预测到3个木聚糖酶基因：xynGL001395、xynGL002415和xynGL002429，这3个木聚糖酶基因均属于GH10家族，具有典型的(α/β)8结构。（5）克隆xynGL002415、xynGL002429基因，构建工程菌BL21(DE3)XynGL002415和BL21(DE3)XynGL002429。对基因工程菌BL21(DE3)XynGL002415编码的木聚糖酶XynGL002415进行了纯化与酶学性质研究，该酶最适反应温度为70℃，最适pH9.0。具有广谱pH耐受性。可特异性降解不同来源的木聚糖底物。本研究的实施可加强对我国高温热泉生境微生物与酶资源的挖掘、开发及利用。