Studies on Cellulases of a Phytopathogenic Fungus, Pyricularia oryzae Cavara

III. Multiplicity of β-Glucosidase, and Purification and Properties of a Second Component

Abstract

To determine the relationship between the induction patterns of three components of β-glucosidase of Pyricularia oryzae and carbon sources in the growth medium, various culture conditions were examined. Avicel, hydroxyethylcellulose and methyl-β-D-glucoside as the carbon source induced both β-glucosidase components, GB-1 and GB-2, whereas cellobiose and gentiobiose induced only one component, GB-1. Thus, these two components were induced independently and hence thought to be isozymes.
The GB-2 was purified to homogeneity by ion exchange and gel filtration chromatog-raphies from two different cultures on methyl-β-D-glucoside and Avicel. The specific activity of GB-2 when salicin was used as substrate was approximately 5.9mg glucose/min/mg protein. GB-2 was found to be an oligomeric glycoprotein, which consisted of two subunits with molecular weight of approximately 120, 000, comprising a relatively large number of acidic amino acids and mannose, as is the case with GB-1.
These two isozymes were clearly different in thermostability, GB-2 being more thermolabile than GB-1. However, the same carboxyl group (pKa 4.2-4.8) was found to be strongly implicated in the formation and dissociation of the enzyme-substrate complex for both of the enzymes, from the analysis of kinetic parameters as a function of pH.