牦牛瘤胃元基因组文库中木聚糖酶基因的分析

Analysis of xylanases derived from the metagenomic BAC clone library of yak rumen

摘要:

摘要:【目的】了解牦牛瘤胃微生物木聚糖酶多样性及其降解特征，为木聚糖降解提供新的基因资源。【方法】根据对已构建的瘤胃微生物元基因组细菌人工染色体(BAC) 克隆文库高通量测序结果的注释，筛选其中编码木聚糖酶的基因并进行多样性分析;对其中一个木聚糖酶基因及其连锁的木糖苷酶基因进行克隆表达和酶学性质表征，分析其协同作用。【结果】共筛选到14 个木聚糖酶基因，均编码GH10 家族木聚糖酶，其氨基酸序列之间的相似性为20.5%－91.3%;其中7 个木聚糖酶基因所在的不同的DNA 片段(contig) 上存在木糖苷酶基因，编码的木糖苷酶属于GH43 或GH3 糖苷水解酶家族。将其中一对连锁的木聚糖酶(Xyn32) 和木糖苷酶基因(Xyl33) 分别克隆、表达和纯化。纯化后的木聚糖酶比活为1.98 IU /mg，但不具有阿魏酸酯酶活性;木糖苷酶比活为0. 07 U /mg，且具有α-阿拉伯呋喃糖苷酶活性。体外实验证明，木糖苷酶Xyl33 对与之连锁的木聚糖酶Xyn32 的木聚糖降解具有协同作用。

Abstract:

Abstract:［Objective］ The diversity of rumen microbial xylanases and their degradation characteristics were studied and the resources of new xylanases genes and enzymes were provided．［Methods］According to the result of the highthroughput sequencing of the rumen microbial metagenome bacterial artificial chromosome (BAC) clones library，the diversity of xylanase genes was analyzed and screened． Then one xylanase and its downstream xylosidase gene screened was cloned and expressed in Escherichia coli． The enzyme characterization of the recombinant xylanase and xylosidase and their synergistic effect were studied．［Results］All 14 xylanases screened from the library belong to GH10 family proteins．These xylanases shared the amino acid sequence similarity between 20. 5% and 91.3%．Intriguingly，7 xylanase genes in different contigs were found to be followed by xylosidase gene． The specific enzyme activity of the xylanase (Xyn32) was 1.98 U /mg and no ferulic acid esterase activity was detected．The specific enzyme activity of the coupled xylosidase (Xyl33) was 0.07 IU /mg，and xylosidase (Xyl33) also displayed the activity of arabinofuranosidase．In addition，the in vitro experiment confirmed the synergistic effect between the coupled xylanase and xylosidase．