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Applied Microbiology and Biotechnology

, Volume 93, Issue 5, pp 1989–1998 | Cite as

Identification, cloning, and characterization of β-glucosidase from Ustilago esculenta

  • Masahiro Nakajima
  • Tetsuro Yamashita
  • Machiko Takahashi
  • Yuki Nakano
  • Takumi TakedaEmail author
Biotechnologically relevant enzymes and proteins

Abstract

Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.

Keywords

Ustilago esculenta GH 3 β-glucosidase Laminarioligosaccharides Saccharification Plant cell wall 

Notes

Acknowledgments

This work was supported in part by the Japanese Society for the Promotion of Science (21612009).

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Masahiro Nakajima
    • 1
  • Tetsuro Yamashita
    • 2
  • Machiko Takahashi
    • 1
  • Yuki Nakano
    • 1
  • Takumi Takeda
    • 1
    • 3
    Email author
  1. 1.Iwate Biotechnology Research CenterKitakamiJapan
  2. 2.Iwate UniversityMoriokaJapan
  3. 3.KitakamiJapan

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