Physical and kinetic properties of the family 3 β-glucosidase from Aspergillus niger which is important for cellulose breakdown

Heather F. Seidle, Ira Marten, Oded Shoseyov, Reuben E. Huber*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

A β-glucosidase (BGS) purified from Aspergillus niger cellulase powder (obtained from Sigma, St. Louis, MO, USA) was characterized. Electrophoresis, size exclusion chromatography, and dynamic light scattering indicated that the enzyme is a dimer of approximately 200 kDa. Five of the seven N-glycosylated oligosaccharides attached to BGS were composed of D-mannoses attached to a β(1-4)-N-acetyl-glucosamine-β-(1-4)-fucose- α-(1-6)-N-acetylglucosamine core. The other two were similar, but the cores of these did not have the D-fucose. The enzyme is a retaining glycosidase, and it also has a distinct preference for the β-configuration at the reducing end of cellobiose. BGS is thermostable up to 65°C but is sensitive to freezing and thawing. The extinction coefficient of BGS was found to be 1.8 cm-1mg-1. All substrates assayed resulted in Eadie-Hofstee plots that were curved at high substrate concentrations. TLC of the reaction products showed that the substrates themselves act as acceptors when present at high concentrations. The transglucosidic activity rate is different from the hydrolytic activity rate and this causes the curvature at high substrate concentrations. The enzyme produces gentiobiose when D-glucose is the acceptor. pH optima of the Vmax{h} with pNPGlc, oNPGlc, and cellobiose were between pH 4 and 4.5, and the K m values decreased at pH values between 3 and 5. Inhibition experiments indicated that the enzyme is specific for glucosyl substrates and suggested that D-gluconolactone is a transition state analog. Studies with cello-oligosaccharides and 3,4-dinitrophenyl-ceHobiose showed that BG S is an exo-hydrolase having at least five glucose subsites and that it cleaves from the nonreducing end. The properties of a family 3 β-glucosidase (BG3) sequenced by Dan et al. [Dan, S., Marton, I., Dekel, M., Bravdo, B-A., He, S., Withers, S. G., and Shoseyov, O. (2000) J. Biol. Chem. 275: 4973-4980] was also studied and was shown to have very similar properties to those of BGS. Sequence analysis of a portion of BG S verified that these are the same enzymes.

Original languageAmerican English
Pages (from-to)11-23
Number of pages13
JournalProtein Journal
Volume23
Issue number1
DOIs
StatePublished - 2004

Bibliographical note

Funding Information:
This work was supported by funds from the Natural Science and Engineering Research Council (NSERC) of Canada. The authors would like to thank Dr. Deane McIntyre of the University of Calgary for his expertise and advice concerning the identification of the unknown sugars with NMR. Special thanks to Helga Lay for synthesizing the 3,4-dinitrophenyl cellobioside.

Keywords

  • Cellulase
  • Hydrolysis
  • Kinetics
  • Transglucosidic reactions
  • β-Glucosidase

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