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Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

The glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii (AkCel61) is a modular enzyme that consists of a catalytic domain and a carbohydrate-binding module belonging to family 1 (CBM1) that are connected by a Ser-Thr linker region longer than 100 amino acids. We expressed the recombinant AkCel61, wild-type enzyme (rAkCel61), and a truncated enzyme consisting of the catalytic domain (rAkCel61ΔCBM) in Pichia pastoris and analyzed their biochemical properties. Purified rAkCel61 and rAkCel61ΔCBM migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were demonstrated to have apparent molecular masses of 81,000 and 34,000 Da, respectively. After treatment with endoglycosidase H, both proteins showed an increase in mobility, thus, demonstrating estimated molecular masses of 78,000 and 28,000 Da, respectively. Mass spectrometry analysis revealed that rAkCel61 and rAkCel61ΔCBM expressed in P. pastoris are heterogeneous due to protein glycosylation. The rAkCel61 protein bound to crystalline cellulose but not to arabinoxylan. The rAkCel61 and rAkCel61ΔCBM proteins produced small amounts of oligosaccharides from soluble carboxymethylcellulose. They also exhibited a slight hydrolytic activity toward laminarin. However, they showed no detectable activity toward microcrystalline cellulose, arabinoxylan, and pectin. Both recombinant enzymes also showed no detectable activity toward p-nitrophenyl β-d-glucoside, p-nitrophenyl β-d-cellobioside, and p-nitrophenyl β-d-cellotrioside.

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Author notes

  1. Kiyoshi Ito

    Present address: Graduate School of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima, 890-0065, Japan

Authors and Affiliations

  1. Department of Bioresource Engineering, Faculty of Agriculture, Yamagata University, 1-23 Wakaba-machi, Tsuruoka, 997-8555, Japan

    Takuya Koseki, Yoshihito Shiono & Tetsuya Murayama

  2. Graduate School of Biosphere Science, Hiroshima University, 4-4-1 Kagamiyama, Higashi-Hiroshima, 739-8528, Japan

    Yuichiro Mese & Haruyuki Iefuji

  3. Department of Biotechnology, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Shinya Fushinobu

  4. National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashi-Hiroshima, 739-0046, Japan

    Kazuo Masaki, Tsutomu Fujii, Kiyoshi Ito & Haruyuki Iefuji

Authors
  1. Takuya Koseki
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  2. Yuichiro Mese
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  4. Kazuo Masaki
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  9. Haruyuki Iefuji
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Corresponding author

Correspondence to Takuya Koseki.

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Supplemental figure

Multiple alignment of amino acid sequences of AkCel61 and other family 61 proteins. AkCel61, A. kawachii Cel61; ABF50850, A. nidulans AN1602.2; VvEgII, V. volvacea EgII; CAD21296, N. crassa putative endoglucanase. Identical amino acids at conserved positions are boxed in black, and the N-glycosylation recognition sites are boxed in gray (PDF 39 kb).

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Koseki, T., Mese, Y., Fushinobu, S. et al. Biochemical characterization of a glycoside hydrolase family 61 endoglucanase from Aspergillus kawachii . Appl Microbiol Biotechnol 77, 1279–1285 (2008). https://doi.org/10.1007/s00253-007-1274-4

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  • DOI: https://doi.org/10.1007/s00253-007-1274-4

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