Heterologous expression and characterization of a glucose-stimulated β-glucosidase from the termite Neotermes koshunensis in Aspergillus oryzae.
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 Mn(2+) and glycerol. The K(m) and V(max) 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.
Department of Biotechnology, University of Tokyo, Tokyo, Japan.
Cristiane Akemi
CA
Uchima
Gaku
G
Tokuda
Hirofumi
H
Watanabe
Katsuhiko
K
Kitamoto
Manabu
M
Arioka
D000818:Animals; D001236:Aspergillus oryzae / Q000235:genetics* / Q000378:metabolism; D005591:Chemical Fractionation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D020049:Isoptera / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D020536:Enzyme Activators; D011994:Recombinant Proteins; D001617:beta-Glucosidase