Applied Microbiology and Biotechnology

, Volume 89, Issue 6, pp 1761–1771 | Cite as

Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae

  • Cristiane Akemi Uchima
  • Gaku Tokuda
  • Hirofumi Watanabe
  • Katsuhiko Kitamoto
  • Manabu Arioka
Biotechnologically Relevant Enzymes and Proteins

Abstract

Neotermes koshunensis is a lower termite that secretes endogenous β-glucosidase in the salivary glands. This β-glucosidase (G1NkBG) was successfully expressed in Aspergillus oryzae. G1NkBG was purified to homogeneity from the culture supernatant through ammonium sulfate precipitation and anion exchange, hydrophobic, and gel filtration chromatographies with a 48-fold increase in purity. The molecular mass of the native enzyme appeared as a single band at 60 kDa after gel filtration analysis, indicating that G1NkBG is a monomeric protein. Maximum activity was observed at 50 °C with an optimum pH at 5.0. G1NkBG retained 80% of its maximum activity at temperatures up to 45 °C and lost its activity at temperatures above 55 °C. The enzyme was stable from pH 5.0 to 9.0. G1NkBG was most active towards laminaribiose and p-nitrophenyl-β-d-fucopyranoside. Cellobiose, as well as cello-oligosaccharides, was also well hydrolyzed. The enzyme activity was slightly stimulated by Mn2+ and glycerol. The Km and Vmax values were 0.77 mM and 16 U/mg, respectively, against p-nitrophenyl-β-d-glucopyranoside. An unusual finding was that G1NkBG was stimulated by 1.3-fold when glucose was present in the reaction mixture at a concentration of 200 mM. These characteristics, particularly the stimulation of enzyme activity by glucose, make G1NkBG of great interest for biotechnological applications, especially for bioethanol production.

Keywords

Glucose-stimulated β-glucosidase Neotermes koshunensis Heterologous expression Aspergillus oryzae 

Supplementary material

253_2010_2963_MOESM1_ESM.doc (666 kb)
Online Resource 1Heterologous protein production in a time course analyses (DOC 666 kb)
253_2010_2963_MOESM2_ESM.doc (256 kb)
Online Resource 2TLC analysis from cello-oligosaccharides hydrolyzed by the purified G1NkBG (DOC 256 kb)
253_2010_2963_MOESM3_ESM.doc (46 kb)
Online Resource 3Comparative table between the properties of β-glucosidases from different microorganisms (DOC 45 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Cristiane Akemi Uchima
    • 1
  • Gaku Tokuda
    • 2
  • Hirofumi Watanabe
    • 3
  • Katsuhiko Kitamoto
    • 1
  • Manabu Arioka
    • 1
  1. 1.Department of BiotechnologyUniversity of TokyoTokyoJapan
  2. 2.Center of Molecular BiosciencesUniversity of the RyukyusOkinawaJapan
  3. 3.National Institute of Agrobiological ScienceTsukubaJapan

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