Novel carbohydrase "complex" from solid-state cultures of the aerobic fungus Penicillium capsulatum

A multiactivity preparation was isolated from solid-state cultures of Penicillium capsulatum. It migrated as a single band (pI 4.02) on isoelectric focusing, on nondenaturing polyacrylamide gradient gel electrophoresis (M_r 242,500), and on SDS-polyacrylamide gradient gel electrophoresis (M_r 135,000). It also migrated as a single band when pretreated with 6 M urea (or 6 M guanidine-HCl) plus octyl glucoside and electrophoresed in the presence of urea. Thus, the purified entity is a dimer of similar, if not identical, subunits. Transmission electron microscopy of negatively stained preparations revealed bipartite projections with length to width ratios of 2:1 and obvious division into two main masses. Each of the latter exhibited three smaller submasses. The calculated molecular weights of the native particle and subunits were consistent with the values obtained by electrophoresis. The purified preparation catalysed the hydrolysis of p-nitrophenyl-β-D-glucoside, p-nitrophenyl-β-D-xyloside, cellobiose, laminarin, β-glucan, lichenan, and carboxyl reduced pneumococcal type III polysaccharide. We conclude that each subunit is composed of three enzymes: β-glucosidase, laminarinase, and lichenase. However, the possibility that each subunit is a single protein with three domains, each displaying one of the above activities, is not precluded. Glucose accumulated during the hydrolysis of β-glucan (or laminarin), but oligomeric intermediates did not. This would imply exoaction. By contrast, the rate of reduction in viscosity of a solution of the β-glucan greatly exceeded the rate of release of reducing sugars therefrom. This is typical of endoaction. We reconcile this apparent contradiction by proposing that the products of the endoacting β-glucanase and laminarinase components are immediately acted upon by the exoacting β-glucosidase component to yield glucose, with obvious advantages to the cell. Complexes with the ability to effect complete conversion of polymers to monomers should also have considerable commercial potential.