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Characterization of an α-l-rhamnosidase from Aspergillus kawachii and its gene

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Abstract

An α-l-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on l-rhamnose as the sole carbon source. The α-l-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 °C. Further, it was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60 °C for 1 h. Its T 50 value was determined to be 72 °C. The enzyme was able to hydrolyze α-1,2- and α-1,6-glycosidic bonds. The specific activity of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii α-l-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the α-l-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins.

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

    Yuichiro Mese, Nahoko Nishibori, Kazuo Masaki, Tsutomu Fujii & Haruyuki Iefuji

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

    Yuichiro Mese & Haruyuki Iefuji

  4. Suishin-Yamane Honten Co.,Ltd., 1-5-58 Higashi-cho, Mihara, Hiroshima, 723-0011, Japan

    Takashi Handa & Yuichi Yamane

Authors
  1. Takuya Koseki
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  2. Yuichiro Mese
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  3. Nahoko Nishibori
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  4. Kazuo Masaki
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  5. Tsutomu Fujii
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  6. Takashi Handa
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  7. Yuichi Yamane
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  8. Yoshihito Shiono
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  9. Tetsuya Murayama
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  10. Haruyuki Iefuji
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Correspondence to Takuya Koseki.

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ESM Fig. 1

Alignment of the amino acid sequences of A. kawachii Ak-RhaA (accession no. AB374267), A. aculeatus RhaA (accession no. AF284761), A. aculeatus RhaB (accession no. AF284762), Bacillus sp. GL1 RhaB, and hypothetical proteins from A. oryzae (accession no. XP_001820356) and A. fumigatus (XP_749916). Identical residues among the six enzymes have been indicated by asterisks. Residues with conserved substitutions and semi-conserved substitutions have been indicated by colons and dots, respectively. The N-terminal amino acid sequence determined by using a protein sequencer has been underlined. Key residues involved in the enzyme catalysis and/or substrate recognition of Bacillus sp. GL1 RhaB have been indicated in boxes. (PDF 64.9 KB)

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Koseki, T., Mese, Y., Nishibori, N. et al. Characterization of an α-l-rhamnosidase from Aspergillus kawachii and its gene. Appl Microbiol Biotechnol 80, 1007–1013 (2008). https://doi.org/10.1007/s00253-008-1599-7

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  • DOI: https://doi.org/10.1007/s00253-008-1599-7

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