[Extracellular beta-D-glucosidase of the Penicillium canescens marine fungus].

Extracellular beta-D-glucosidase was isolated in a homogeneous state from the Penicillium canescens marine fungus. According to SDS-electrophoresis, the molecular weight of the enzyme was 64 kDa and the maximal activity was observed at pH 5.2 and 70 degrees C. Glucosidase catalyzed the hydrolysis of beta-glycosidic bonds both in glycosides and in glucose disaccharides and had transglycosylation activity. The enzyme can be used for the deglycosylation of natural glycosides and in enzymatic synthesis of new carbon-containing compounds.

[Distribution of O-glycosylases in marine fungi of the Sea of Japan and the Sea of Okhotsk: characterization of exocellular N-acetyl-beta-D-glucosaminidase of the marine fungus Penicillium canescens].

The capacity to produce exocellular enmzymes was studied for 92 samples of fungi from various marine habitats in the Sea of Okhotsk (78 strains) and the Sea of Japan (14 strains). Strains producing highly active glycanases and glycosidases were found. Synthesis of O-glycosylhydrolases was stimulated by addition of laminaran to the nutrient medium. Highly purified N-acetyl-beta-D-glucosaminidase was isolated from the marine fungus Penicillium canescens. The molecular weight of the enzyme determined by SDS-Na-electrophoresis was 68 kDa. The enzyme displayed maximum activity at pH 4.5 and temperature 45 degrees C. Inactivation half-time of the enzyme at 50 degrees C was 25 min. N-acetyl-beta-D-glucosaminidase hydrolyzed both beta-glucosaminide and beta-galactosaminide bonds and possessed a high transglycosylazing activity.

[Metabolites of marine organisms as regulators of O-glycosylhydrolases].

The ability of metabolites contained in culture liquid of 62 strains of marine fungi to affect the activity of two digestive enzymes of marine mollusks--endo-1,3-beta-D-glucanase of Spisula sachalinensis and beta-D-glucosidase of Littorina kurila--was studied. It was found that 66 and 71% of specimens activated, 18 and 7% inhibited, and 16 and 22% did not affect the activity of endo-1,3-beta-D-glucanase and beta-D-glucosidase, respectively. It is demonstrated that the metabolites of brown algae and marine sponges can be used for a targeted regulation of enzyme biosynthesis by marine fungi. The protein inhibitor of endo-1,3-beta-D-glucanases isolated from the brown alga Laminaria cichorioides blocked the biosynthesis of almost all O-glycosylhydrolases in five strains of marine fungi studied. The presence in culture medium of halistanol sulfate from the marine sponge of the family Halichondriidae either did not affect or activated the biosynthesis of enzymes involved in carbohydrate metabolism by marine fungi.

Filamentous marine fungi as producers of O-glycosylhydrolases: beta-1,3-glucanase from Chaetomium indicum.

Ninety fungal strains (42 species) isolated from marine habitats were studied for their ability to produce extracellular enzymes. Cultural filtrates of these strains were shown to contain a series of glycosidases (beta-glucosidases, N-acetyl-beta-glucosaminidases, beta-galactosidases alpha-mannosidases) and glucanases (1,3-beta-glucanases, amylases) which varied with habitat. The level of activity depended on the species of fungi. Several promising strains capable of producing both individual enzymes and a set of enzymes for splitting carbohydrate-containing compound have been isolated. Optimal conditions for growth of Chaetomium indicum and for biosynthesis of beta-1,3-glucanase were determined. beta-1,3-Glucanase was isolated using ion-exchange chromatography, ultrafiltration, and gel filtration. The presence of 2 enzyme forms was shown; both forms were exo-beta-1,3-glucanases.

Filamentous fungi associated with holothurians from the sea of Japan, off the primorye coast of Russia.

Holothurians (Holothurioidea, Echinodermata) are known to contain triterpene glycosides, which show antifungal activity. Nevertheless, fungi can be isolated from all organs of holothurians. During 1995-1996, mycelial fungi from several Far-Eastern holothurians--Apostichopus japonicus, Eupentacta fraudatrix, Cucumaria japonica--were collected from the Sea of Japan near the coast of Primorye (Russia) and studied. Twenty-seven species of marine fungi, mostly facultative ones belonging to the mitosporic fungi, were isolated from the holothurians and identified. Fungi isolated from the holothurian surface were more diverse and abundant than those from internal organs and coelomic fluids. Of the holothurians studied, Cucumaria japonica was poorest in abundance and diversity of fungi. The fungi Cladosporium brevicompactum and C. sphaerospermum were common in the holothurian coelom. Because of their high proteolytic activity, these fungi may be pathogenic to holothurians. The detritovorus holothurian A. japonicus was shown to modify the fungal assemblages within the marine bottom sediments.

[N-acetyl-beta-D-hexosaminidase secreted by the marine fungus Phoma glomerata]. / Sekretiruemaia N-atsetil-beta-D-geksozaminidaza morskogo griba Phoma glomerata.

Among the ten strains of marine fungi studied, the mycelial fungus Phoma glomerata showed maximum potency in producing N-acetyl-beta-D-glucosaminidase. The conditions for fungal growth and enzyme biosynthesis were evaluated. N-acetyl-beta-D-hexosaminidase was isolated from the culture liquid of Phoma glomerata by ion-exchange chromatography (on DEAE-cellulose and DEAE-Sephacell) and gel filtration (on Toyopearl HW-55) with a yield of 35%; the enzyme, purified 36.4-fold, had a molecular weight of 20 kDa. The homogeneity of the enzyme was confirmed by gel filtration and SDS-PAGE. Transglycosylation reactions catalyzed by the enzyme produced N-acetyl-D-glucosamine and N-acetyl-D-galactosamine with respective yields of 38 and 46%.

[O-glycosylhydrolases of marine filamentous fungi. beta-1,3-Glucanases of Trichoderma aureviride]. / O-glikozilgidrolazy morskikh mitselial'nykh gribov. beta-1,3-Glukanazy Trichoderma aureviride.

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl-D-glucosaminidase, D-glucosidase, D-galactosidase, beta-1,3-glucanase, amylase, and pustulanase. beta-1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion-exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that beta-1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.

[Amylases of the fungus Aspergillus flavipes associated with Fucus evanescens]. / Amilazy griba Aspergillus flavipes, assotsiirovannogo s Fucus evanescens.

A promising producer of extracellular amylases, Aspergillus flavipes, was selected from 245 strains of marine fungi. Depending on the conditions of growth, this strain produced diverse amylolytic complexes. When grown on medium containing peptone and yeast extract (pH 7.0), A. flavipes synthesized three forms of amylase, differing in pH optimum (5.5, 6.0, and 7.5). A single form of the enzyme was synthesized either in the absence of peptone from the medium or at the initial pH value of the medium, equal to 8.6. The activity of the isolated amylase forms decreased in the presence of proteolytic enzymes. New, highly stable forms of amylase (with pH optima of 5.5 and 7.5 and maximum activity at 60-80 degrees C) were synthesized in the presence of diisopropyl fluorophosphate, an inhibitor of proteases.

[Marine fungi in sediments of the Sea of Japan (Russian territory) and their biologically active metabolites]. / Griby gruntov Iaponskogo moria (Rossiiskoe poberezh'e) i ikh biologicheskie aktivnye metabolity.

The most abundant marine fungi encountered in various regions of the Sea of Japan belong to the genera Penicillium, Aspergillus, Wardomyces, Trichoderma, Chrysosporium, and Chaetomium. Facultative marine fungi of the genera Scytalidium, Verticillium, and Oidiodendron and obligate marine fungi of the genus Dendryphiella are much less abundant. The composition of marine sediments and the anthropogenic load on them were found to influence the abundance and species diversity of fungi, as well as the occurrence of fungal strains producing hemolytically active substances. The biodiversity of mycobiota and the abundance of hemotoxin-producing fungi in marine sediments may be used to evaluate the anthropogenic load on marine biocenoses. Hemolytic compounds were produced by 57% of the fungi isolated from marine sediments. The hemolytic activity of Chaetomium spiculipilium was revealed in the fraction of the culture liquid containing extracellular fatty acids and pigments. The fatty acid composition of this marine fungus was determined.

Enzymes of carbohydrate metabolism of mycelial fungi from marine environments. beta-1,3-glucanase of the marine fungus Chaetomium indicum.

Thirty samples of fungi belonging to 17 species living in marine environments were studied for their ability to produce extracellular enzymes. In the culture fluids, a variety of glycosidases (beta-glucosidases, N-acetyl-beta-glucosaminidase, beta-galactosidases, and alpha-mannosidases) and glucanases (amylases and beta-1,3-glucanases) were found. Several cultures were found that could be used as efficient producers of either individual enzymes or a whole complement of enzymes degrading carbohydrate-containing compounds. Optimal growth conditions for the fungus Chaetomium indicum and beta-1,3-glucanase biosynthesis were developed. beta-1,3-Glucanase was isolated by a combination of ion-exchange chromatography, ultrafiltration, and gel chromatography. The molecular mass of the enzyme determined by gel-filtration was 54 kD. The enzyme was stable at temperatures below 50 degrees C, had a temperature optimum for activity at 60 degrees C, and retained activity between pH 4.5 and 7.5. The pH dependence of the beta-1, 3-glucanase activity showed two maxima, at pH 4.4 and 5.6; this suggested the existence of two forms of the enzyme. Analysis of the products of enzymatic hydrolysis of laminaran, transglycosylating ability, and the effect of a specific natural inhibitor indicates that both forms are exo-beta-1,3-glucanases.

New diterpenic altrosides of the fungus Acremonium striatisporum isolated from a sea cucumber.

Two new diterpenic glycosides, virescenosides M (1) and N (2), have been isolated from a marine strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Their structures were determined on the basis of MS and NMR data as beta-D-altropyranosido-19-7-oxo-isopimara-8,15-diene-2alp ha, 3beta-diol (1) and beta-D-altropyranosido-19-isopimara-7, 15-diene-2alpha,3beta,6beta-triol (2). Three other altrosides (3-5), identified as virescenosides A, B, and C from the terrestrial strain Acremonium luzulae, were also isolated. The cytotoxic activity of the virescenosides was examined.