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Two beta-glucosidases (I and II) were isolated from Schizophyllum commune, and their physical and chemical properties studied. The two enzymes have very similar sequences, as shown by HPLC analysis of tryptic digests and partial amino acid sequencing. As judged by their circular dichroism spectra, they have almost identical secondary structure. The estimates for alpha-helix, beta-sheet, and other structures were 21%, 40% and 39%, respectively, for beta-glucosidase I and 27%, 32% and 41% for beta-glucosidase II. Their near-ultraviolet spectra were identical. beta-Glucosidase I was more highly glycosylated than beta-glucosidase II, having 2 mol N-acetylglucosamine/mol enzyme 36, mol mannose/mol enzyme and 1.2 mol glucose/mol enzyme vs 1.2, 17 and 3 mol/mol, respectively, in beta-glucosidase II. The native glycosylated form of beta-glucosidase I had a molecular mass of 102 kDa, and that of beta-glucosidase II, 96 kDa. As estimated from sensitivity to N-glycanase, beta-glucosidase II sugars were mainly asparagine linked, but much of the sugar in beta-glucosidase I was not removed by this treatment and was apparently serine or threonine linked. Kinetic analysis showed that both forms had similar Km values (0.3-2.1 mM) for oligosaccharides of 2-6 residues, but the kcat values of beta-glucosidase II were lower by 30-75% than those of beta-glucosidase I. The substrate dependence of kcat/Km indicated that both enzymes had binding sites for three glucose residues. The pH optimum of beta-glucosidase I was higher than that of beta-glucosidase II (5.8 vs 5.1). Both had similar specificities for several (R)-beta-D-glucosides tested. Both enzymes were competitively inhibited by their glucose product, but beta-glucosidase II was consistently less inhibited than beta-glucosidase I. Cellobiase activity was much more markedly inhibited than the activity with higher oligosaccharides, and the result of this, plus the lower hydrolytic rate with cellobiose, resulted in an accumulation of cellobiose as higher oligosaccharides were digested. Glucono-delta-lactone inhibited both enzymes and the hydrolysis of all oligosaccharide substrates similarly (Ki = 4 microM). We conclude that the catalytic site is identical in both enzymes, but subtle structural differences are reflected in a differential activity on the higher oligosaccharides and in the differential effects of the glucose product as an inhibitor. Furthermore, ethanol had a stimulatory effect on beta-glucosidase I but inhibited beta-glucosidase II, which presumably reflects differential effects of ethanol on the conformations of the two species.

Three identical clones coding for a partial sequence of the Schizophyllum commune beta-glucosidase were isolated from a cDNA library in lambda gt11, using polyclonal antibody to the enzyme. The identity was confirmed by comparison of the amino-terminus of a peptide from a protease lys-C digest with the sequence inferred from the cDNA sequence. A comparison of the sequence with that reported for a beta-glucosidase from Candida pelliculosa revealed a region in the latter with 43% identity in amino acid sequence. There was also a similarity in the S. commune beta-glucosidase to an active site sequence proposed for a S. commune endoglucanase, suggesting the possibility of a common catalytic mechanism for these two glucolytic enzymes.

New gene selection techniques (Recombinant DNA) are currently available to exploit useful properties of various biological systems hitherto regarded as interesting but of little or no immediate commercial value. The application of genetic engineering techniques to problems in the Pulp and Paper Industry are many. As a first step these techniques are being used to provide much needed fundamental information on the cellular and molecular mechanisms involved in the expression of extra-cellular enzymes that degrade lignocellulosic pulping wastes. The information gleaned from the studies on cellulolytic fungi and bacteria can be used to genetically engineer a yeast or bacterium capable of converting pulping wastes into ethanol and other useful by-products.