[1,3-beta-Glucanases of actinomycetes]. / 1,3-beta-Gliukanazy aktinomitsetov.

Different actinomyces species (25 strains) were studied and Actinomyces cellulosae 41 was found to be the most active one in its capacity to cause lysis of Saccharomyces cerevisiae intact cells as well as in the production of 1,3-beta-glucanase. The enzyme preparation containing 1,3-beta-glucanase can be obtained from the filtrate of the actinomycete cultural broth after 60 h of growth by precipitation with four volumes of ethanol. The optimal pH for the action of 1,3-beta-glucanase from A. cellulosae is 5.5. Hydrolysis of laminarin (5 mg of the substrate) yields 2.4 mg of reducing sugars or 13.3 microM (recalculated per glucose). There is a direct correlation between the amount of produced reducing sugars and the enzyme concentration up to 10 microM/ml (recalculated per glucose). The enzyme hydrolysate contains glucose and oligosaccharides with a different degree of polymerization. Therefore, the enzyme is endo-1,3-beta-glucanase. It produces less quantities of the disaccharide than those of glucose and trisaccharide. The enzyme yields only traces of glucose upon prolonged hydrolysis of p-nitrophenyl-beta-D-glucoside (PNPG) and shows a weak capacity for transglycosylation when laminarin is used as a donor and PNPG as an acceptor.

[beta-Glucanases of Geotrichum candidum]. / beta-Gliukanazy Geotrichum candidum.

The formation of (1-4)-, (1-3)- and (1-6)-beta-glucanases and beta-glucosidases was studied during the growth of the fungus Geotrichum candidum under the conditions of submerged cultivation in a medium optimal for the production of cellulolytic enzymes. Endo-(1-4)-beta-glucanases and C1 enzyme, as well as (1-3)- and (1-6)-beta-glucanases appeared in the medium as soon as by the 45th hour of growth. However, the maximal concentration of the enzymes in the medium was observed at different periods of the fermentation: between 75th and 105th, 70th and 95th, 55th and 100th, 80th and 105th hours, respectively. The content of the enzymes abruptly decreased by the 160th hour of the growth. The activity of beta-glucosidases, which was low at the beginning of the growth, sharply increased by the 70th hour and remained at the same level by the 160th hour of the growth. The accumulation of beta-glucanases was an uneven process, consistent with irregular changes in the content of DNA and protein in the biomass. The isoelectric points of beta-glucanases and beta-glucosidases were studied in the filtrate of the cultural broth after 96 h of the cultivation. The high activity of endo-(1-4)-beta-glucanase was found at the pH 4.6, 4.1 and 3.8; its low activity was detected at the pH 6.4, 3.2, 1.6 and 1.3. Other glucanases behaved also as acid proteins. During isoelectric focusing, (1-3)-beta-glucanase showed the peaks of activity at the pH 4.4, 4.0, 3.8 and 2.9; (1-6)-beta-glucanase, at the pH 5.0, 3.7, 3.5, 3.1 and 2.0; beta-glucosidases were distributed over a broad pH range from 6.7 to 2.0, with the maximal activity at the pH 6.2, 4.8 and 3.7.

[Endo-1,3-beta-glucanase from Actinomyces cellulosae]. / Endo-1,3-beta-gliukanaza Actinomyces cellulosae.

Endo-1.3-beta-glucanase (EC 3.2.1.39) from Actinomyces cellulosae was purified 960-fold by ion-exchange chromatography on DEAE-cellulose and DEAE-Sephadex A-50 and by gel-filtration through Acrylex P-60. The enzyme was electrophoretically homogeneous and had molecular weight of about 30 000 as determined by polyacrylamide gel disc-electrophoresis in the presence of SDS and gel-filtration through Sephadex G-200. The isoelectric point lies at 4.0; the pH optimum for the non-purified enzyme preparation is 5.5, that for the purified one is 6.0. The enzyme is stable within the pH range of 7.0-8.0 and is rapidly inactivated in acid media. The enzyme does not hydrolyze laminaribiose, lichenane, barley glucan, pustulane, KM-cellulose, but splits laminarane to form high molecular weight oligosaccharides, a small number of low molecular weight oligosaccharides and a negligible amount of glucose. Studies with glucose oxidase showed that splitting of 1.3-beta-glucan results in a beta-form of glucose.

[Preparation of protoplasts and localization of ribonuclease, beta-1,3-glucanase and glucosoisomerase in the fungal mycelium of Penicillium brevi-compactum]. / Poluchenie protoplastov i lokalizatsiia ribonukleazy, beta-1,3-gliukanazy i gliukozoizomerazy v mitselii griba Penicillium brevi-compactum.

Optimal conditions for the formation and isolation of protoplasts from the fungus Penicillium brevi-compactum were investigated. Localization of ribonuclease, glucosoisomerase and beta-1,3-glucanase in the mycelium was examined. To produce protoplasts, the mycelium was treated for 3-4 hours at 40 degrees C with the incubation mixture, containing chitinase from Actinomyces kurssanovii, lytic enzymes from Act. cellulose, lysozyme and 0.8 M mannitol as an osmotic stabilizer. The levels of activities of RNase, beta-1,3-glucanase, and glucosoisomerase were measured in the fungal mycelium before preparation of protoplasts, in the incubation mixture after their preparation, and in the protoplast lysate. The protoplast formation facilitated the release of RNase, beta-1,3-glucanase and glucosoisomerase from the fungal mycelium into the incubation mixture.

[Enzymatic degradation of cell walls of yeast cells at different growth phases]. / Fermentativnoe rasshcheplenie kletochnykh stenok drozhzhei, nakhodiashchikhsia v razlichnykh fazakh rosta

The enzymes of Actinomyces cellulosae were fractionated by precipitation with ammonium sulphate and gel filtration on Sephadex G-75. Fractionation yielded protease, beta-1,3-glucanase and an enzyme of a low molecular weight and unknown nature involved in lysis by 70% of yeast cells at early exponential growth phase without the participation of the other two enzymes. The protease did not accomplish lysis of the cells; beta-1,3-glucanase was involved in degradation of the cell walls of old yeast cells.