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Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies

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Abstract

A gene encoding a xylanase, named xynS20, was cloned from the ruminal fungus Neocallimastix patriciarum. The DNA sequence of xynS20 revealed that the gene was 1,008 bp in size and encoded amino acid sequences with a predicted molecular weight of 36 kDa. The amino acid sequence alignment showed that the highest sequence identity (28.4%) is with insect gut xylanase XYL6805. According to the sequence-based classification, a putative conserved domain of glycosyl hydrolase family 11 was detected at the N-terminus of XynS20 and a putative conserved domain of family 1 carbohydrate-binding module (CBM) was observed at the C-terminus of XynS20. An Asn-rich linker sequence was found between the N-terminal catalytic domain and the C-terminal CBM of XynS20. To examine the activity of the gene product, xynS20 gene was cloned as an oleosin-fused protein, expressed in Escherichia coli, affinity-purified by formation of artificial oil bodies, released from oleosin by intein-mediated peptide cleavage, and finally harvested by concentration of the supernatant. The specific activity of purified XynS20 toward oat spelt xylan was 1,982.8 U mg−1. The recombinant XynS20 was stable in the mild acid pH range from 5.0 to 6.0, and the optimum pH was 6.0. The optimal reaction temperature of XynS20 was 45°C; at temperatures below 30 and above 55°C, enzyme activity was less than 50% of that at the optimal temperature.

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Acknowledgements

This research was conducted using funds partially provided by grants NSC 95-2313-B-002-118-MY3 and NSC 97-ET-7-002-006-ET from the National Science Council and grant 95AS-5.1.3-AD-U1(3) from the Council of Agriculture, Republic of China.

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Authors and Affiliations

  1. Department of Animal Science and Technology, Institute of Biotechnology, National Taiwan University, 4F, No. 81 Chang-Xing Street, Taipei, Taiwan

    Je-Ruei Liu

  2. Institute of BioAgricultural Sciences, Academia Sinica, 128 Academia Road, Taipei, Taiwan

    Chung-Hang Duan & Kuo-Joan Cheng

  3. Department of Animal Science, McGill University, Montreal, Canada

    Xin Zhao

  4. Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung, Taiwan

    Jason T. C. Tzen

  5. Department of Life Science, National Taiwan Normal University, Taipei, Taiwan

    Cheng-Kang Pai

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  1. Je-Ruei Liu
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  2. Chung-Hang Duan
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  3. Xin Zhao
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  4. Jason T. C. Tzen
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  5. Kuo-Joan Cheng
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  6. Cheng-Kang Pai
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Correspondence to Je-Ruei Liu or Chung-Hang Duan.

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Liu, JR., Duan, CH., Zhao, X. et al. Cloning of a rumen fungal xylanase gene and purification of the recombinant enzyme via artificial oil bodies. Appl Microbiol Biotechnol 79, 225–233 (2008). https://doi.org/10.1007/s00253-008-1418-1

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  • DOI: https://doi.org/10.1007/s00253-008-1418-1

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