Physical and Kinetic Properties of the Family 3 β-Glucosidase from Aspergillus niger Which Is Important for Cellulose Breakdown

Abstract

A beta-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 beta(1-4)-N-acetyl-glucosamine-beta-(1-4)-fucose-alpha-(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 beta-configuration at the reducing end of cellobiose. BGS is thermostable up to 65 degrees C but is sensitive to freezing and thawing. The extinction coefficient of BGS was found to be 1.8 cm(-1) mg(-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 Km 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-cellobiose showed that BGS is an exo-hydrolase having at least five glucose subsites and that it cleaves from the nonreducing end. The properties of a family 3 beta-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 BGS verified that these are the same enzymes.

Purification and characterization of three β-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28

“…35) While normal hydrolysis takes place at low substrate concentrations, the same enzyme was reported to carry out a transglucosidic reaction at high substrate concentrations, where the second substrate molecule (such as pNP-Glc) or the product (glucose) competes with water for the glycosyl moiety covalently bound to the enzyme. 36) Thus, the apparent HGT-BG activities of our P1.2 in the presence of 20% glucose could also include transglucosidic reaction between the high concentration of glucose and the covalently bound glucose that accelerated the breakdown of pNP-Glc.…”

“…Nonetheless, the kinetic parameters for hydrolytic reaction of our P1.2 (derived from reactions at low substrate concentrations) are in the same range as the corresponding parameters reported for the A. niger β-glucosidase with transglucosidic activity. 35,37) Although there have been many reports on β-glucosidases and some HGT-BGs from Aspergillus spp. and other fungi, only a small number of them were fully characterized in terms of kinetic studies (Supplemental Table 1).…”

Metabolic engineering and thermodynamic characterization of an extracellular -glucosidase produced by Aspergillus niger

“…K m is a measure of affinity of an enzyme for a substrate and low values of K m indicate high affinity of the enzyme for the substrate (Hamilton et al, 1998). Seidle et al (2004) calculated K m and V max of 1 mM and 40 U/mg respectively for pNPG hydrolysis with a β-glucosidase from A. niger. K m and V max values of 3.3 mM and 43.68 µmol/min.…”

Immobilization of β-glucosidase on Eupergit C for Lignocellulose Hydrolysis

“…enzyme molecules immobilized by single point attachment: a previous report on penicillin G acylase showed that the half life of the multipoint immobilized enzyme was 100-fold higher than that of single-point immobilized enzyme (Mateo et al 2002). It is further noted that A. niger b-glucosidase is a dimeric enzyme composed of two identical $100 kDa subunits (Seidle et al 2004): presumably, thermal stabilization requires attachment of both monomers. A shift in pH optima following immobilization has been reported for several enzymes and is generally related to the ionization of groups on the carrier (Bissett & Sternberg 1978).…”