Purification and properties of two β‐glucosidases isolated from Aspergillus niger

The cellulase complex of the fungus Aspergillus niger (strain CBS 554.65 = ATCC 16 888) was fractionated by gel filtration yielding six pronounced peaks. Only proteins from the fraction corresponding to the first peak (96 kDa) showed β‐glucosidase activity vs. the substrate 4‐nitrophenyl‐β‐D‐glucopyranoside ( p NPG). These proteins have been fractionated by chromatofocusing, yielding two β‐glucosidases (I and II) which are shown to be homogeneous in isoelectric focusing experiments (pI = 4.6 and 3.8, respectively). Kinetic experiments with p NPG, MU‐glucopyranoside and cellobiose revealed that both types of β‐glucosidases behave like aryl‐β‐glucosidases. β‐Glucosidase‐I acting on p NPG exhibits a split kinetics characterized by high and low substrateconcentration kinetics which are differentiated by different values of V and of K m . In addition, β‐glucosidase‐II is shown to be an exo‐glucohydrolase as deduced from experiments with MU‐cellobiopyranoside. Experimental features should be emphasized; usual soft‐gel ion‐exchange materials did not work in the chromatofocusing separation of the two β‐glucosidases, in contrast to the 10μ‐Si 500 = DEAE exchange material (Serva) typically used in HPLC‐experiments. Furthermore, protein content determinations based on different procedures yielded widely differing values.