木聚糖是自然界中含量丰富的异质多糖，使用双催化域双功能木聚糖酶-辅酶有助于其高效水解并降低酶的使用成本，因而应用前景广阔。然而，目前关于此类酶的研究很少，因此对新酶的鉴定和功能研究亟待加强。B. intestinalis能有效代谢木聚糖，申请人通过分析其比较转录组学研究结果，从中鉴定了两个双催化域双功能酶并进行异源表达，酶学特性初步分析表明两者分别为双功能阿拉伯呋喃糖苷酶/木聚糖酶Bi569和双功能木聚糖酶/阿魏酸酯酶Bi76，本项目拟对这两个酶及其截短突变体等的酶学性质进行研究，并分析两个双功能酶的协同水解作用，阐明全酶及其各催化域的生化功能。鉴于Bi569比商品酶活性高但耐热性差，本项目拟对其耐热性进行定向进化，在保持高酶活的同时累积有利于热稳定性的有益突变，以期获得耐热高活性双功能酶。研究结果将丰富该类双功能酶的功能数据库，并有助于揭示肠道菌对木聚糖的生化代谢机理和开发新型酶制剂。

Xylan is a kind of heterogeneous polysaccharide which represents one of the most abundant and renewable resource in the nature. The use of bifunctional endoxylanase/accessory enzyme with two catalytic domains is helpful not only to the efficient hydrolysis of xylan, but also to the reduction of the cost of enzyme preparation, which make it have great potentialities of development and extensive prospects of application. However, there are only a few studies on this kind of bifunctional enzymes, the identification and function research of novel bifunctional endoxylanase/accessory enzymes urgently await to strengthen. The gut microbe, Bacteroides intestinalis, can effectively metabolize xylan. By analyzing the results of comparative transcriptome study, we identified two bifunctional endoxylanase/accessory enzymes with two catalytic domains. Subsequently, the encoding genes for the two bifunctional enzymes were subcloned and expressed in Escherichia coli BL21(DE3). The preliminary analysis of enzyme properties revealed that the two enzymes were a bifunctional α-L-arabinofuranosidase/endoxylanase Bi569 and a bifunctional endoxylanase/feruloyl esterase Bi76, respectively. In this study, we plan to make truncated mutants for both bifunctional enzymes, and then investigate the enzyme properties of the full length enzymes and their truncated mutants. The synergistic effect of two bifunctional enzymes will be analyzed. In comparison to commercial xylanases, Bi569 exhibited higher catalytic activity towards wheat arabinoxylan, but its thermostability was not good. In order to obtain heat-resistant and highly active bifunctional α-L-arabinofuranosidase/endoxylanase, directed evolution will be applied to improve the thermostability of Bi569. On the basis of maintaining the high catalytic activity of Bi569, a series of mutations that are beneficial for improving thermostability will be accumulated. Based on these results, this study will not only help us to gain new insights into bifunctional endoxylanase/accessory enzymes, but also facilitate the exploitation of novel high efficient enzyme preparation and the revelation of the biochemical metabolism of xylan by B. intestinalis.