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Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum

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Abstract

A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His6 fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49°C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58°C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg−1, respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.

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Acknowledgments

This research was conducted using funds partially provided by grant NSC 98-2313-B-002-033-MY3 from the National Science Council and grant 97AS-2.1.2-AD-U1(3) from the Council of Agriculture, Republic of China.

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Authors and Affiliations

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

    Cheng-Kang Pai & Ming-Liang Li

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

    Zong-Yuan Wu, Ming-Ju Chen, Yi-Fang Zeng, Jr-Wei Chen & Je-Ruei Liu

  3. Biotechnology Development Center, Ye Cherng Industrial Products Co., LTD, Yang Mei, Taiwan

    Chung-Hang Duan

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  1. Cheng-Kang Pai
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  2. Zong-Yuan Wu
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  5. Jr-Wei Chen
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Correspondence to Je-Ruei Liu.

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Figure S1

HPLC analysis of the amount of acetic acid released from the substrates by XynS20E. (A) Calibration curve with acetic acid standards. (B) Acetic acid standard (12 mM); (C) birchwood xylan (0.5%, w/v) in 100 mM sodium phosphate buffer (pH 8.2) incubated at 52°C for 20 min; (D) birchwood xylan (0.5%, w/v) in 100 mM sodium phosphate buffer (pH 8.2) incubated with 0.1 μg of the purified recombinant XynS20E at 52°C for 20 min (final reaction volume of 300 μl). (DOC 290 kb)

Figure S2

(A) Michaelis–Menten Plot relating the reaction rate V 0 (initial velocity) to the substrate concentration [S]. (B) Lineweaver–Burk plot, plotting the inverse of substrate concentration against the inverse of the initial velocity. (DOC 81 kb)

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Pai, CK., Wu, ZY., Chen, MJ. et al. Molecular cloning and characterization of a bifunctional xylanolytic enzyme from Neocallimastix patriciarum . Appl Microbiol Biotechnol 85, 1451–1462 (2010). https://doi.org/10.1007/s00253-009-2175-5

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  • DOI: https://doi.org/10.1007/s00253-009-2175-5

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