[Beta-glucanase and chitinase biosynthesis in a culture of a mycophilic strain of Trichoderma viride]. / Biosintez beta-gliukanaz i khitinazy v kul'ture mikofil'nogo shtamma Trichoderma viride.

The production of extracellular 1,3-, 1,6-beta-glucanases and chitinase was studied during submerged cultivation of a Trichoderma viride strain 3/78 on various carbon sources: glycerol, glucose, lactose, sucrose, laminaran, starch, pustulan, chitin, and Agaricus bisporus fruit bodies. The synthesis of these enzymes and cellulase was studied also under the conditions of depression at low concentrations (10(-2) and 10(-3)M) of the first five aforementioned carbon sources as well as cellobiose, gentiobiose, N-acetyl-beta-D-glucosamine and 0.1% chitooligosaccharides and A. bisporus cell walls. The experiments were conducted with the washed mycelium of this strain grown for 2 days in a medium with glycerol as a carbon source. The results indicated that 1,3- and 1,6-beta-glucanases of the strain were of the constitutive nature and were repressed by such carbon sources as glycerol and glucose. Chitinase and cellulase were shown to be inducible enzymes. Chitinase was induced by N-acetyl-beta-D-glucosamine, chitooligosaccharides and A. bisporus cell walls as well as by lactose when the fungus was grown on this carbon source. Cellulase biosynthesis was induced by lactose, cellobiose and gentiobiose.

[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.

[Purification and some properties of 1,3-1,4-beta-glucanase from Bacillus subtilis]. / Ochistka i nekotorye svoistva 1,3-1,4-beta-gliukanazy iz Bacillus subtilis.

Using chromatography on cellulose, SE-Sephadex G-50 and gel filtration on acrylex P-60, 1.3 -- 1.4-beta-glucanase from Bac. subtilis, strain 103 was obtained and purified 142-fold. The specific activity of the purified enzyme was 18.5 units per mg of protein. The homogeneity of 1.3 -- 1.4-beta-glucanase was determined by gel filtration on acrylex P-60, polyacrylamide gel electrophoresis, isoelectrofocusing and ultracentrifugation. Using electrophoresis in Na-SDS and gel filtration on acrylex P-60, the molecular weight of the enzyme was found to be equal to 30 000 and 33 000, respectively. The isoelectric point for the enzyme lies at pH 5.4. The enzyme does not contain tryptophane, free SH-groups or carbohydrates.

[Preparation of cellulolytic enzymes from Geotrichum candidum]. / Poluchenie tselliuloliticheskikh fermentov iz Geotrichum candidum.

The ability of different fungi cultivated on cellulose to synthesize extracellular cellulase was examined. The fungus Geotrichum candidum str. 3c was the most active among the fungi tested, including Trichoderma fungi. The formation of cellulolytic enzymes in the culture filtrate of G. candidum occurred, when the entire glucose and the major portion of reducing substances were utilized. The formation of cellulases depended on the content of ammonium ions. The enzyme accumulation reached the highest level at the stationary phase of the culture growth and depended on the inoculum amount. When the inoculum was used as a submerged culture at a concentration of 3%, accumulation of C1-enzyme, endo-(1 leads to 4)beta-glucanases, and beta-glucosidases reached maximum by 44, 52 and 56 hours of cultivation. The fats that are frequently used as a foam catcher inhibited the cellulase formation by the abouve strain. From the filtrate cellulolytic enzymes were isolated via spray drying and precipitation with 3 ethanol volumes to yield 6.5 and 5.0 g/l, respectively. The cellulases when stored at 5 degrees C did not lose their activity for a year and became inactivated by 40-50% in 5 years.

[Effect of fungal cellulases on enzymic activity of malt]. / Vliianie preparatov gribnykh tselliulaz ns fermentativnuiu atkivnost' soloda.

In order to increase malt enzymic activity, cellulases from the fungus Geotrichum candidum were used. Malt milk was processed for 1--3 hr at 40 degrees C and pH 6.0--6.5 by the fungal preparation or culture fluid filtrate used in such quantities in which there were 0.5 to 9.0 units of cellulase activity per g malt. During the process enzymic activities of malt increased significantly: dextrinating activity--by 73%, amylolytic activity--by 60--100%, saccharifying activity--by 3.4 times, and proteolytic activity--by 2 times.

[Purification of beta-glucanase on cellulose]. / Ochistka beta-gliukanazy na tselliuloze.

The paper describes a method for purifying beta-glucanse (lychenase) which includes enzyme absorption on the microcrystalline cellulose column or ground filter paper at pH 5.5, and elution with phosphate buffer pH 7.6. Specific activity of beta-glucanase eluted from the column may be increased by 8--23 times, depending on the purification conditions.

[Physiological and biochemical properties of Actinomyces kurssanovii, active producter of chitinase]. / Fiziologo-biokhimicheskie svoistva Actinomyces kurssanovii aktivnogo produtsenta khitinazy.

Chitinase biosynthesis by Actinomyces kurssonovii 75 was studied under conditions of periodic cultivation in a laboratory fermenter. The activity of components of the chitinolytic complex correlated with the growth phases of the culture. The activity of chitobiase (beta-N-acetylglucoseaminidase) predominated in the cultural broth in the exponential growth phase of the culture; it decreased later by 40-50 per cent, while the activity of chitinase became maximum. The biosynthesis of chitinolytic enzymes by the growing actinomycete was accompanied with a rapid hydrolysis of demineralized crab shells, and a gradual increase in the pH of the medium to 9.0. A chitinolytic preparation obtained from the supernatant of the cultural broth of Act kurssanovii hydrolysed ground chitin by 70-80 per cent during 5 days. Fe2+ and Ca2+ ions increased the activity of the preparation by 25 and 30 per cent respectively; Mn2+ ions decreased the activity by 40 per cent.

[beta-Glucosidases from fungus Geotrichum candidum]. / beta-Gliukozidazy iz griba Geotrichum candidum

beta-Glucosidases from Geotrichum candidum 3C cellulase preparation were separated from C1 enzymes and beta-1,4-glucanases by means of DEAE-Sephadex A-50 chromatography, gel filtration through P-150 Biogel and chromatography on CM-cellulose, and then were fractionated by isoelectric focusing using carrier ampholites with pH ranges 3-6 and 4-6. beta-Glucosidases with pI 3.8, 4.2, 4.6, 5.1, 5.6 and 6.2 were found in cellulase preparation from G. candidum 3C. Molecular weight of beta-glucosidases with pI 3.8, 4.2, 4.6 and 6.2, isolated under isoelectric focusing, were estimated by means of gel filtration through Sephadex G-200 to be 35000, 123000, 188000 and 223000 respectively. beta-Glucosidases with pI 3.8, 4.6, 5.6 and 6.2 hydrolyzed cellobiose and did not attack p-nitrophenyl-beta-D-glucopyranoside; those with pI 4.2 and 5.6 hydrolyzed p-nitrophenyl-beta-D-glucopyranoside and plant glucoside, protodioscin, and did not split cellobiose. All the beta-glucosidases studied did not hydrolyze laminaribose, beta-D-methylsylopyranoside, alder O-methylglucuronoxylane, o-nitrophenyl-beta-D-galactopyranoside and p-nitrophenyl-alpha-D-glucopyranoside. beta-Cellobiase with pI 6.2 hydrolzed lactoses, cellobioses with pI 3.8 and pI 5.6 splited gentiobiose. beta-Glucosidase with pI 4.6 did not attack any substrate studied, except cellobiose.

[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.

[Production of chitinase by Actinomyces kurssanovii and its properties]. / Obrazovanie i svoistva khitinazy Actinomyces kurssanovii

Actinomyces kurssanovii, a culture producing large amounts of chitinase and chitobiase, was cultivated on a medium of the following composition (%): demineralized crab shells, 3.0; K2HPO4, 0.5; peptone, 0.2; yeast extract, 0.1; MgSO4-H2O, 0.09. The maximum amount of the enzymes was synthesized after growth in a fermenter of the actinomycete during 48 hours. The highest activity of chitinase is manifested at pH 7.0 and depends on ionic composition of the buffer, being higher in veronal buffer than in phosphate or tris//HC1 buffers. The chitinase and chitobiase of the strain decompose completely colloid chitin and chitin in demineralized crab shells with the formation of N-acetyl-D-glucosamine.

[Biosynthesis of chitinase by Achromobacter liquefaciens]. / Biosintez khitinazy kul'turoi Achromobacter liquefaciens

Bacterial cultures under study synthesize exocellular chitinase on a medium containing chitin or demineralized crab shells as a source of carbon and nitrogen. Conditions for biosynthesis of chitinase by the cells of Achromobacter liquefaciens 301a were investigated under periodic and continuous conditions of cultivation. The preparation of chitinase isolated from the cultural broth of A. liquefaciens 301a hydrolysed colloid and native chitin at the optimum pH 6.5 and temperature 40degreesC. The terminal products of the reaction are chitobiose and N-acetylglucosamine.

[Chitinase formation by actinomycetes in deep cultures]. / Obrazovanie khitinazy aktinomitsetami pri glubinom kul'tivirovanii

Actinomycetes were cultivated in the medium containing demineralized crab shells as a source of carbon. Chitinase was found in filtrates of the cultural broth of all 44 studied strains. The activity of the extracellular chitinase was determined both by decomposition of ground chitin (CHI-activity) and colloid chitin (CHI-activity). The ratio between CHI and CHX in filtrates of the cultural broth of the most active nine strains varied from one to two.