Characterization Of Xylanolytic Enzymes In Fecal Microbiome From Rhinopithecus Bieti

There exist in the gastrointestinal tract(GIT) of herbivorous animals rich enzymes relating to the degradation of lignocelluloses. However, different animals have different enzymes. The Rhinopithecus bieti is a first-class governmentally-protected animal in China. As a typical herbivorous primate, as a result of its different feeding types and species, the GIT of R. bieti may contain new microorganism xylanase gene resources different from other animals.This study was carried out combining traditional pure culturing technique and metagenomic technology, obtaining abundant genes of xylanolytic enzymes from R.bieti fecal microbes, which may lay a foundation for developing of the new type xylanase in the GIT and the exploration of R. bieti’s microbiological resources, and may provide a theory foundation for further studying the degradation mechanism of lignocellulose in the GIT of R. bieti.The following results were obtained:1. Using the genomic DNA extracted from the feces of wild and half-wild R.bieti as template, the xylanase gene fragments were amplified by degenerate primer of GH10 xylanase to construct library. From fecal microbial clone library of wild and half-wild R. bieti 26 and 28 GH10 xylanase gene fragments were acquired, which showed the identity of 58%-95% and 63%-81% with xylanase sequences in public databases, respectively. Contrast results showed that microbial composition of GH10 xylanase from two different environments was similar, including the Firmicutes,Bacteroidetes and uncultured bacteria. The GH10 xylanase gene of microbial origin of Bacillus, Bacteroides, Butyrivibrio were only found in wild R.bieti clone libraries,while that of GH10 xylanase gene from microbial sources of Paludibacter, Roseburia only found in half-wild R.bieti clone libraries.2. This study got rich genes of xylanolytic enzyme from R. bieti fecal microbes by the technology of pure culture and metagenomic, and found that the XynRBM26 and XynRBM encoded GH10 xylanases, and XylRBM26 encoded GH43 xylosidase.Sequence analysis showed that these genes had low identities with known genes(55%–84%), suggesting their novelty. Domain analysis indicated that XylRBM26 was a dual-domain protein, which provided valuable materials for studying the relationships between xylosidase structure and function3. The three genes, XynRBM26, XynRBM and XylRBM26 were expressed in Escherichia coli BL21, the recombinant enzymes were purified and characterized.a. Purified XynRBM26 showed optimal xylanase activity at pH 5.5 and was stable over a pH range of 5.0–7.0 and exhibited > 1 h half-life at 50 ℃. Purified XynRBM26 exhibited good salt tolerance, retaining greater than 86% xylanase activity in the presence of 5 M NaCl. Furthermore, XynRBM26 was resistant to trypsin, no xylanase activity was lost after incubating with trypsin at 37 °C for 60 min.These favorable properties made XynRBM26 a potential utility in feedstuff fields.b. Purified recombinant XynRBM was apparently optimal at 50 °C, and retained13.5% of the maximum activity at 0 °C and 46.9% of the maximum activity at 10 °C.After being processed at 50 °C for 60 min, XynRBM maintained 95.6% activity. The enzyme was also stable when trypsin was added to the reaction mixture for 1 hour,reaching the activity of 120%. Furthermore, the enzyme could effectively hydrolyze oat xylan, beechwood xylan, 4-Nitrophenyl-β-D-xylopyranoside, and4-Nitrophenyl-α-L-arabinofuranoside. These favorable properties made XynRBM a potential candidate for applications in aquaculture, and could provide good model materials for study of the relationships of xylosidase structure and substrate specificity.c. Purified recombinant XynRBM exhibited optimal activity at 50℃ and exhibited > 1 h half-life at 50 °C. The enzyme remained approximately 50% of its original activity even if the concentration of xylose was as high as 500 mM. NaCl,Pb2+ and Ca2+ had little effect on its activity. In addition, the enzyme could hydrolysed xylotetraose, xylotriose, and xylobiose to xylose. The enzyme has value for the basic research on the mechanism of tolerance to Pb2+ and Ca2+, and also has potential application prospects in the production of xylose and the processing of marine products.