Enzymes That Hydrolyze Fungal Cell Wall Polysaccharides

Abstract

Abstract A specific fungal α-(1 → 4)-glucanase, mycodextranase, useful for the large scale production of nigerose, has been purified to homogeneity in high yield from the culture filtrate of Penicillium melinii QM 1931. It has been resolved into fast and slow forms; designations based on their differing electrophoretic mobilities at pH 4.5. The fast form constitutes about 85% of the protein and enzymatic activity. Both forms hydrolyze the glucan nigeran (mycodextran) and certain oligosaccharides of nigeran yielding nigerose and the tetrasaccharide, O-α-d-glucopyranosyl-(1 → 3)-O-α-d-glucopyranosyl-(1 → 4)-O-α-d-glucopyranosyl-(1 → 3)-d-glucose, as sole end products. Glucose is not produced. Sedimentation equilibrium studies indicate that fast and slow enzymes both have molecular weights of 40,000 ± 2,000 daltons. They have similar but not identical amino acid compositions both being rich in serine, threonine, glutamic and aspartic acids, but have a low content of cysteine and tryptophan. Both mycodextranases contain associated carbohydrate. The ultraviolet absorption spectra, urea sensitivities, and pH activity profiles for both forms are practically identical. In addition both forms display maximum catalytic activities at 70° and are fully active in 40% dimethyl sulfoxide. Of a number of general enzyme inhibitors tested, only Ag+, Hg+, N-chlorosuccinimide, and N-bromosuccinimide inactivated the fast enzyme. Some aspects of the action pattern and mechanism of the fast form have been examined in detail. It operates via an endo-multichain mechanism; both end products are formed at a constant rate during hydrolysis but nigerose does not arise from tetrasaccharide. Competitive inhibition studies show that the fast enzyme preferentially binds oligosaccharides with alternating α-(1 → 3)- and α-(1 → 4)-glucosidic linkages, and experiments with certain oligosaccharides of nigeran give results consistent with a model in which the substrate-binding site holds 8 glucopyranose units with a catalytic site located midway in this site. The enzyme has been used to study the fine structure of nigeran and the general structure of isolichenin. In the former case this has been done by identifying and quantitating the linkages present at its reducing and nonreducing termini. The data obtained are used to postulate a general mechanism for the biosynthesis of nigeran.