Metabolites of Marine Organisms as Regulators of <Emphasis Type="Italic">O</Emphasis>-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-β-D-glucanase of Spisula sachalinensis and β-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-β-D-glucanase and β-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-β -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.__________Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 4, 2005, pp. 402–408.Original Russian Text Copyright © 2005 by Verigina, Burtseva, Ermakova, Sova, Pivkin, Zvyagintseva.     

The isolation, purification and properties of the cellobiohydrolase component of Penicillium funiculosum cellulase.

1. A cellobiohydrolase component was isolated from a Penicillium funiculosum cellulase preparation by chromatography on DEAE-Sephadex, and purified by isoelectric focusing. 2. Purified in this way, the enzyme was homogeneous as judged by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and isoelectric focusing in polyacrylamide gels. 3. Acting in isolation, the enzyme had little hydrolytic activity to highly ordered celluloses such as cotton fibre, but, when recombined in the original proportions with the other components [endo-(1 leads to 4)-beta-D-glucanase and beta-D-glucosidase] of the complex, 98% of the original activity was recovered. 4. Synergistic effects were also observed when the enzyme was acting in concert with endo-(1 leads to 4)-beta-D-glucanase from other fungal sources. 5. Less-well-ordered celluloses, such as that swollen in H3PO4, were extensively hydrolysed, the principal product being cellobiose. 6. Attack on carboxymethyl-cellulose (CM-cellulose), which is the substrate normally used to assay for endo-(1 leads to 4)-beta-D-glucanase activity, was minimal. 7. The enzyme was associated with 9% of neutral sugar, 88% of which was mannose. It was isoelectric at pH 4.36 (4 degrees C) and had a mol.wt. of 46 300 (determined by gel chromatography on a calibrated column of Ultrogel). 8. The enzyme was specific for the beta-(1 leads to 4)-linkage.     

Cloning and expression of an endo-1,6-beta-D-glucanase gene (neg1) from Neurospora crassa

A gene (neg1) encoding an endo-1,6-beta-D-glucanase from Neurospora crassa was cloned. The putative neg1 was 1443-bp long and encoded a mature endo-1,6-beta-D-glucanase protein of 463 amino acids and signal peptide of 17 amino acids. The purified recombinant protein (Neg1) obtained from Escherichia coli showed 1,6-beta-D-glucanase activity. No genes similar in sequence were found in yeasts and fungi.     

Enzymatic transformation of biologically active 1,3;1,6-β-D-glucan. Structure and activity of resulting fragments

The fragmentation of the biologically active 1,3;1,6-beta-D-glucan Antivir by endo-1,3-beta-D-glucanase LIV from crystalline styles of the marine mollusk Spisula sachalinensis was carried out. It was found that low molecular mass oligomers possessing a stabilizing effect on membranes and anti-viral activity against tobacco mosaic virus appeared in the process of enzymatic hydrolysis of Antivir. Biological activity of 1,3;1,6-beta-D-glucooligo- and polysaccharides was found to be associated with molecular mass (polymerization degree (n) not less than 14) and with presence of intralinked beta-1,6-connected monosaccharide residues. Probably, decrease in molecular mass is compensated by increase in number of intralinked beta-1,6-connected monosaccharide residues.     

Purification and partial characterization of a novel hyaluronic acid-degrading enzyme from Antarctic krill (Euphausia superba)

Summary A novel enzyme degrading hyaluronic acid has been isolated, purified and characterized from Antarctic krill (Euphausia superba). A combination of affinity chromatography (Con A-Sepharose), gel filtration (Superose 6) and fast protein liquid chromatography (Mono Q) was used for the purification. The hyaluronidase activity was determined by a radial diffusion method based on hyaluronic acid incorporated into an agarose gel. Moreover, the beta-glucuronidase and endo-(1,3)-beta-D-glucanase activities were also followed through the process using phenolphtalein mono beta-glucuronic acid and laminarin as substrates. After the final purification step on Mono Q column, the chromatogram showed three main peaks designated A, B and C. Peak C contained high hyaluronidase activity undetectable in peak A and B. The betaglucuronidase activity was associated with peak A, while the endo-(1,3)-beta-D-glucanase activity was found in peak B and slight in peak C. The hyaluronidase was purified about 85-fold. It had a pH optimum of 5.3, a temperature optimum of 37°C and a molecular weight of 80 000 Daltons. On polyacrylamide gradient gel electrophoresis the enzyme fraction showed one major band associated with hyaluronic acid decomposition, slightly contaminated with a few other components. Isoelectric focusing in combination with a hyaluronic acid zymogram demonstrated one major band at pH 6.7 with high enzyme activity. Preliminary data on enzyme specificity suggest that krill hyaluronidase is a new endo-beta-glucuronidase and support the concept of krill enzymes as a remarkable and unusually effective digestive system adapted to the Antarctic marine ecosystem.     

Fungal pathogens secrete an inhibitor protein that distinguishes isoforms of plant pathogenesis-related endo-β-1,3-glucanases

Plant endo-β-1,3-glucanases and chitinases inhibit the growth of some fungi and generate elicitor-active oligosaccharides while depolymerizing polysaccharides of mycelial walls. Overexpression of the endo-β-1,3-glucanases and/ or chitinases in transgenic plants provides, in some cases, increased protection against fungal pathogens. However, most of the phytopathogenic fungi that have been tested in vitro are resistant to endo-β-1,3-glucanases and chitinases. Furthermore, some phytopathogenic fungi whose growth is inhibited by these enzymes are able to overcome the effect of these enzymes over a period of hours, indicating an ability of those fungi to adapt to the enzymes. Evidence is presented indicating that fungal pathogens secrete proteins that inhibit selective plant endo-β-1,3-glucanases.A glucanase inhibitor protein (GIP-1) has been purified to homogeneity from the culture fluid of the fungal pathogen of soybeans, Phytophthora sojae f. sp. glycines (Psg), and two basic pathogenesis-related endo-β-1,3-glucanases (EnGL_soy-A and EnGL_soy-B) have been purified from soybean seedlings. GIP-1 inhibits EnGL_soy-A but not EnGL_soy-B. Moreover, GIP-1 does not inhibit endo-β-1,3-glucanases secreted by Psg itself nor does GIP-1 inhibit PR-2c, a pathogenesis-related endo-β-1,3-glucanase of tobacco. Evidence is presented that Psg secretes other GIPs that inhibit other endo-β-1,3-glucanase(s) of soybean. Furthermore, GIP-1 does not exhibit proteolytic activity but does appear to physically bind to EnGL_soy-A. The results reported herein demonstrate specific interactions between gene products of the host and pathogen and establish the need to consider fungal proteins that inhibit plant endo-β-1,3-glucanases when attempting to use the genes encoding endo-β-1,3-glucanases to engineer resistance to fungi in transgenic plants.     

不同海水浓度和培养时间对海洋真菌抗菌活性的影响

用滤纸片琼脂扩散法研究不同海水浓度和培养时间对海洋真菌抗菌活性的影响,旨在为大规模培养海洋真菌和提高获得抗菌物质的几率提供理论依据。结果显示：用不同海水浓度培养基发酵海洋真菌,在供试的10株海洋真菌中,5株海洋真菌的抗菌活性和抗菌谱有明显差异;培养时间不同,6株海洋真菌的抗菌活性差别较大。实验结果表明,不同海水浓度和培养时间对海洋真菌抗菌活性有显著影响。     

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.     

Sequencing of a 1,3-1,4-beta-D-glucanase (lichenase) from the anaerobic fungus Orpinomyces strain PC-2: properties of the enzyme expressed in Escherichia coli and evidence that the gene has a bacterial origin.

A 971-bp cDNA, designated licA, was obtained from a library of Orpinomyces sp. strain PC-2 constructed in Escherichia coli. It had an open reading frame of 738 nucleotides encoding LicA (1,3-1,4-beta-D-glucanase; lichenase) (EC 3.2.1.73) of 245 amino acids with a calculated molecular mass of 27,929 Da. The deduced amino acid sequence had high homology with bacterial beta-glucanases, particularly in the central regions and toward the C-terminal halves of bacterial enzymes. LicA had no homology with plant beta-glucanases. The genomic DNA region coding for LicA was devoid of introns. More than 95% of the recombinant beta-glucanase produced in E. coli cells was found in the culture medium and periplasmic space. A N-terminal signal peptide of 29 amino residues was cleaved from the enzyme secreted from Orpinomyces, whereas 21 amino acid residues of the signal peptide were removed when the enzyme was produced by E. coli. The beta-glucanase produced by E. coli was purified from the culture medium. It had a molecular mass of 27 kDa on sodium dodecyl sulfate-polyacrylamide gels. The Km and Vmax values with lichenin as the substrate at pH 6.0 and 40 degrees C were 0.75 mg/ml and 3,790 micromol/min/mg, respectively. With barley beta-glucan as the substrate, the corresponding values were 0.91 mg/ml and 5,320 micromol/min/mg. This enzyme did not hydrolyze laminarin, carboxymethylcellulose, pustulan, or xylan. The main products of lichenin and barley beta-glucan hydrolysis were triose and tetraose. LicA represented the first 1,3-1,4-beta-D-glucanase reported from fungi. The results presented suggest that licA of Orpinomyces had a bacterial origin.     

O-glycosylhydrolases of marine filamentous fungi. beta-1,3-Glucanases of 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.     

Distribution of intracellular fucoidan hydrolases among marine bacteria of the family Flavobacteriaceae

A search for fucoidan-degrading enzymes and other O-glycosylhydrolases has been performed among 51 strains of marine bacteria of the family Flavobacteriaceae isolated from red, green, and brown algae, as well as from the sea urchin Strongylocentrotus intermedius and the holothurian Apostichopus japonicus. Over 40% of the studied strains synthesized fucoidanases. The marine bacteria Mesonia algae KMM 3909(T) (an isolate from green alga Acrosiphonia sonderi), as well as Maribacter sp. KMM 6211 and Gramella sp. KMM 6054 (associants of the sea urchin S. intermedius), were the best producers of fucoidanases. Xylose effectively induced the biosynthesis of fucoidanases in these strains. None of the 15 strains of marine bacteria belonging to the genus Arenibacter produced polysaccharide hydrolases.     

O-glycosylhydrolases of embryos of the sea urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> and effect of some natural substances on their biosynthesis

Embryos of the sea urchin Strongylocentrotus intermedius have been found to contain o-glycosyl hydrolases: highly active 1,3-β-D-glucanase and β-D-mannosidase as well as a lower activity of β-D-glucosidase and β-D-galactosidase. Dynamics of changes of the enzyme activities has been studied at various stages of the sea urchin development. There has also been studied effects of some substances (natural fucoidans, β-1.3; 1.6-glucans formed by enzymatic synthesis as well as protein inhibitor of marine mollusc endo-1,3-β-D-glucanases) on development of the embryos and biosynthesis of 1,3-β-D-glucanase and α-D-mannosidase.     

The action of Laminaria japonica extracts on 1,3-beta-D-glucanase, a digestive enzyme of the Strongylocentrotus intermedius sea urchin

Nutritional attractiveness of the brown alga Laminaria japonica for the sea urchin Strongylocentrotus intermedius was studied. The composition of L. japonica was analyzed after one and two years of its life under natural conditions, in its seedlings, and in the alga partially degraded by natural factors. Substances extracted with various solvents were tested for the presence of inhibitors and activators of 1,3-beta-D-glucanase, a digestive enzyme of the sea urchin. Ethanolic extract of freshly harvested L. japonica was found to suppress the enzyme activity. Substances present in ethanolic extracts of the alga after one or two years of its life cycle and in the alga, partly degraded by natural factors, activated the sea urchin enzyme. This fact is in agreement with earlier natural observations concerning the nutritional attractiveness of such L. japonica samples for Strongylocentrotus intermedius.     

Cloning and expression of a Bacillus subtilis Endo-1,3-1,4-beta-D-glucanase gene in Escherichia coli K12

EcoRI fragments of DNA from Bacillus subtilis NCIB 8565, a high producer of an endo-1,3-1,4-beta-D-glucanase, were 'shot-gun' cloned in the plasmid vector pBR325. A 3.5 kb insert, carrying single restriction sites for AvaI, BglII, ClaI, PvuI and PvuII, was shown to direct the synthesis of beta-glucanase in Escherichia coli K12. Enzyme activity was demonstrated in extracellular fractions of E. coli harbouring the beta-glucanase gene; however, the largest proportion (greater than 50%) of total enzyme activity was periplasmic in location. beta-Glucanase activity and cellular location were independent of the orientation of the 3.5 kb fragment in pBR325.     

Brown seaweed protein as an inhibitor of marine mollusk endo-(1-->3)-beta-D-glucanases

Aqueous ethanol extracts from brown seaweed were found to contain substances inhibiting endo-(1-->3)-beta-D-glucanases, the digestive enzymes of marine mollusks. The inhibitors were detected in 70% of the brown seaweeds investigated. An irreversible protein inhibitor with high specificity for endo-(1-->3)-beta-D-glucanases of marine mollusks was isolated from the brown seaweed, Laminaria cichorioides. As determined by gel filtration, the molecular weight of the inhibitor was 46 kDa. The value of [I]50 (10(-8) M) for the inhibitor was comparable with the corresponding value for natural alpha-amylase inhibitors from terrestrial plants. Chemical modification results indicated that tryptophan, dicarboxylic acid, histidine and probably tyrosine residues of inhibitor molecule are important for interaction of the inhibitor with the enzyme.     

Purification of the cellulase complex produced byPenicillium camemberti and its partial characterization

Three cellulase components (FP-ase, CMC-ase and cellobiase) were purified by affinity binding on Avicel followed by Sephadex G-25, DEAE-Sepharose, DEAE-cellulose and Sephadex G-100 chromatography from the culture filtrate of the newly isolated strain Penicillium camemberti. The isolated enzymes had the properties of cellobiohydrolase, endo-1,4-beta-D-glucanase and cellobiase and their respective molar masses were 99, 87 and 61 kDa as determined by molecular sieve chromatography on Sephadex G-100. The amino acid composition of each fraction was also determined.     

Endo-beta-1,3-galactanase from winter mushroom Flammulina velutipes

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.     

Induction of endo-1,4-beta-xylanase and beta-galactosidase in the original and recombinant strains of the fungus Penicillium canescens

The induction of the synthesis of secreted enzymes endo-1,4-beta-xylanase (EC 3.2.1.8) and beta-galactosidase (EC 3.2.1.23) in the original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for the induction: the synthesis of beta-galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4-beta-xylanase was observed at 5 to 10 mM. An increase in the number of endo-1,4-beta-xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of beta-galactosidase; the synthesis of endo-1,4-beta-xylanase in the high-copy-number recombinant producing beta-galactosidase was affected to a lesser extent. The amount of the enzymes synthesized did not depend on the saccharide used as a sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Multiple forms of endo-1,4-beta-D-glucanase in the extracellular cellulase of Penicillium pinophilum

The influence of the composition of the growth medium on the production of endo-1,4-beta-D-glucanase (CM-cellulase) activity by P. pinophilum was studied in shake flask cultures using Avicel PH101 as the carbon source. It was observed that the culture conditions had a profound effect on the level of endoglucanase (CM-cellulase) produced by P. pinophilum. However, isoelectric focusing of the endoglucanase activity obtained from shake flask and fermenter cultures using the same growth medium revealed that the enzyme system found in both cultures was identical qualitatively, and contained seven or eight different endoglucanase components. All the endoglucanase components appeared simultaneously in the early stages of culture and prolonged incubation resulted only in an increase in the concentration of these enzymes. Protease levels were found to be low in both types of culture but were particularly so in the growth medium which contained corn steep liquor. The proteases were unable to release low molecular weight peptides when P. pinophilum cellulase protein was used as a substrate. The results were interpreted to indicate that the multiplicity of endoglucanase components found in cultures of P. pinophilum is most likely the result of expression of a number of specific genes rather than by post-secretional modification of one or more endoglucanase(s) synthesized by the fungus.