[Analysis of xylanases derived from the metagenomic BAC clone library of yak rumen]
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High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits
Published on: September 20, 2016
Min Wang1, Furong Chen, Shan Zhang
1State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. minwang2009@126.com
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 high-throughput 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 (Xy133) was 0.07 IU/mg, and xylosidase (Xy133) also displayed the activity of arabinofuranosidase. In addition, the in vitro experiment confirmed the synergistic effect between the coupled xylanase and xylosidase.
