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Characterization of a Novel Xylanase Gene from Rumen Content of Hu Sheep

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Abstract

A novel xylanase gene, xyn-lxy, was cloned from a metagenomic fosmid library, which was previously constructed from the rumen contents of Hu sheep and was functionally characterized in Escherichia coli. The open reading frame was composed of 1923 bp and encoded for 640 amino acids, including a catalytic domain of glycosyl hydrolase family 10 and carbohydrate-binding module 9. The gene showed 97 % identity with uncultured bacterium Contig1552 but low similarity with xylanases from known cellulolytic-degrading microorganisms in the rumen. The recombinant XYN-LXY showed a specific activity of 664.7 U mg−1. The optimal temperature and pH of the enzyme were 50 °C and 6.0, respectively. Specifically, XYN-LXY was exclusively activated by Mn2+ among all of the cations and reducing agents tested in this study. An enzymatic hydrolysis assay revealed that XYN-LXY degraded birchwood xylan into xylooligosaccharide with a low degree of polymerization. After incubation for 4 h, the concentration of the dominant product, xylobiose, was 2.297 ± 0.175 mg ml−1 (74.07 % of total product) followed by xylose with a concentration of 0.656 ± 0.010 mg ml−1 (21.14 % of total product). The XYN-LXY exhibited deep degradation effects on the xylan substrate, which were rarely observed with endo-xylanase, making it a promising candidate for industrial application, especially in biofuel production.

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Acknowledgments

The authors acknowledge the financial support from the Innovation Team Program of Zhejiang province (2011R50025) and the Importation and Development of High-Caliber Talents Project of Beijing Municipal Institutions (CIT&CD20130324).

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The authors declare that they have no competing interests.

Authors’ Contribution

Qian Wang drafted the manuscript. Yang Luo and Bo He carried out the studies and contributed to the drafting of the manuscript. Jia-Kun Wang, Jian-Xin Liu, and Lin-Shu Jiang participated in the project design and manuscript preparation. All authors read and approved the final manuscript.

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    1. Institute of Dairy Science, College of Animal Science, Zhejiang University, Yuhangtang Road 866#, Hangzhou, Zhejiang, 310058, China

      Qian Wang, Yang Luo, Bo He, Jian-Xin Liu & Jia-Kun Wang

    2. Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore City, 117604, Singapore

      Qian Wang

    3. Beijing Key Laboratory for Dairy Cow Nutrition, Beijing University of Agriculture, Beinong Road 7, Huilongguan, Changping District, Beijing, 102206, China

      Lin-Shu Jiang

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    Supplemental Fig. S1

    Screening of positive transformants by PCR. Lane 1-4: different E. coli transformants. Plasmids isolated fromS different colonies served as templates for PCR amplification using Xyn-lxy-F and Xyn-lxy-R primers. (DOC 85 kb)

    Supplemental Fig. S2

    Lineweaver-Burk plot of XYN-LXY. 0.4 to 15 mg/ml birchwood xylan was used as the substrate for the determination of K m and V max . (DOC 26 kb)

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    Wang, Q., Luo, Y., He, B. et al. Characterization of a Novel Xylanase Gene from Rumen Content of Hu Sheep. Appl Biochem Biotechnol 177, 1424–1436 (2015). https://doi.org/10.1007/s12010-015-1823-8

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