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ABSTRACT: A water-soluble polysaccharide (WSP) extracted from mango (Mangifera indica L.) fruits has been suggested as a new alternative to mutan for mutanase induction in Trichoderma harzianum. Structural analyses revealed that the purified WSP was a (1→3),(1→4)-α-D-glucan with the molecular mass of ca. 760 kDa in which the (1→4)-linked and (1→3)-linked α-Glcp residues were in a ratio of 1:2.4. When the strain T harzianum CCM F-340 was grown in the presence of WSP, the maximal enzyme productivity obtained after 3 days of cultivation was 34 mU/mL. The mango WSP proved to be a very effective stimulus of mutanase expression giving a 5.1-fold higher than without WSP, transcription. It was shown that the mixture of WSP-induced mutanase and commercial dextranase had a high hydrolytic potential in the reaction with streptococcal mutan, where maximal degrees of solubilization and saccharification of this biopolymer (93.4% and 80%, respectively) were reached within 9h (solubilization) and 24h (saccharification). The mixed enzymatic preparation was also effective in degradation of streptococcal mutan and its removal from cariogenic biofilms. After 3h hydrolysis, only 18.2% of the biofilm remained adhered to the glass surface.
International journal of biological macromolecules 04/2013; · 2.37 Impact Factor
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ABSTRACT: Extracellular α-(1 → 3)-glucanase (mutanase, EC 3.2.1.84) produced by Trichoderma harzianum CCM F-340 was purified to homogeneity by ultrafiltration followed by ion exchange and hydrophobic interaction chromatography, and final chromatofocusing. The enzyme was recovered with an 18.4-fold increase in specific activity and a yield of 4.3%. Some properties of the α-(1 → 3)-glucanase were investigated. The molecular mass of the enzyme is 67 kDa, as estimated by SDS/PAGE, its isoelectric point 7.1, and the carbohydrate content 3%. The pH and temperature optima are 5.5 and 45°C, respectively. The enzyme is stable over a pH range of 4.5-6.0 and up to 45°C for 1 h. The Km and Vmax under standard assay conditions are 0.73 mg/ml and 11.39 x 10(-2) µmol/min/mg protein, respectively. The enzyme activity is stimulated by addition of Mg(2+) and Na(+), and significantly inhibited by Hg(2+). The α-(1 → 3)-glucanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-(1 → 3)-glucans. The 20-residue N-terminal sequence of the enzyme is identical with those of other α-(1 → 3)-glucanases from the genus Trichoderma, and highly similar to those from other fungi. The purified α-(1 → 3)-glucanase was effective in preventing artificial dental plaque formation. The easy purification from fermentation broth and high stability, and the effective inhibition of oral biofilm accumulation make this α-(1 → 3)-glucanase highly useful for industrial and medical application.
Acta biochimica Polonica 03/2013; · 1.49 Impact Factor
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ABSTRACT: The chemical structure of a water insoluble α-glucan isolated from the cell wall of Aspergillus wentii was described on the basis of total acid hydrolysis, methylation analysis, and 1D and 2D NMR studies (TOCSY, DQF-COSY, NOESY and HSQC) as well as other instrumental techniques. It was established that the analyzed preparation contained a linear polymer composed almost exclusively of (1→3)-linked α-d-glucose, with a molecular mass of about 850kDa. The polymer was divided into subunits separated by a short spacers of (1→4)-linked α-d-glucoses. Each subunit contained about 200 glucose residues.
Carbohydrate polymers. 01/2013; 91(2):603-8.
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ABSTRACT: Laetiporus sulphureus is an edible wood-rotting basidiomycete fungus whose fruiting bodies contain substances with verified therapeutic evidences and large amounts of α-(1 → 3)-glucan which is used as an effective inducer of microbial α-(1 → 3)-glucanases. However, production of mature fruiting bodies of this species under artificially controlled conditions has not been reported until now. Here, we provide the first report of successful initiation and development of L. sulphureus fruiting bodies in large-scale experiments. Twelve Laetiporus strains were isolated from a natural habitat. A synthetic log production system with a substrate composed of a mixture of sawdust enriched with organic and inorganic additives was developed. It was found that shocking the fungus mycelium with cold water or low temperature was the only suitable method for forced fruiting of L. sulphureus strains. Primordia of two strains were initiated already after 5-6 days from induction, and after another 2 days, they began to develop into fruiting bodies. Carpophores appeared fastest on substrates with high organic supplementation (40-45 %) and a low moisture content (40 %). The resulting mature fruiting bodies reached a weight of 200-300 g. The method of cultivation presented in this paper opens the way to commercial production of this valuable basidiomycete.
MIRCEN Journal of Applied Microbiology and Biotechnology 12/2012; · 1.08 Impact Factor
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ABSTRACT: Mutanases hydrolyze d-glucosidic linkages of α-1,3-linked polysaccharides which are important components of dental plaque. Therefore, these enzymes can be useful in preventive oral hygiene. A gene encoding mutanase was cloned from soil-isolated Paenibacillus curdlanolyticus MP-1 and expressed in Escherichia coli, and the resulting recombinant enzyme was characterized. The nucleotide sequence of the mutanase gene consisted of 3786 nucleotides encoding a protein of 1261 amino acids with a theoretical molecular weight of 131.62kDa. The deduced amino acid sequence exhibited a high degree of similarity with mutanases of Paenibacillus sp. KSM-M126 and Paenibacillus humicus NA1123, with 84% and 80% identity, respectively. The recombinant enzyme was purified 17.5-fold to homogeneity with a recovery of 37%. The purified mutanase showed optimal activity at pH 6.0 and 45°C, and was completely stable at pH 4.0-9.5 and up to 45°C. The enzyme was specific for α-1,3-glucosidic linkages and effectively solubilized fungal α-1,3-glucans and streptococcal mutans, releasing nigerooligosaccharides. The mutanase did not hydrolyze a synthetic substrate readily hydrolyzed by exoglucanases and the enzyme activity was not suppressed in the presence of deoxynojirimycin, an inhibitor of exo-type enzymes. These results suggest an endohydrolytic mode of action.
Protein Expression and Purification 09/2012; 86(1):68-74. · 1.59 Impact Factor
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ABSTRACT: Water-insoluble, alkali-soluble polysaccharides (ASPs) were isolated from three fruiting bodies of the macromycete fungus Ganoderma lucidum. The structure of ASPs was determined using composition analysis, methylation analysis, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy. The analysis of the biological activity of the carboxymethylated (CM) (1→3)-α-d-glucans was based on an assessment of their cytotoxic, mitochondrial metabolism-modulating, and free radical scavenging effects against a tumor cell line (human cervical carcinoma HeLa), and two normal human cell lines (colon myofibroblasts CCD-18Co and epithelial cells CCD 841 CoTr). The chemical and spectroscopic investigations indicated that the ASPs from G. lucidum were (1→3)-α-d-glucans. After carboxymethylation (1→3)-α-d-glucans were tested in the range of 25-250μg/mL concentrations. All the tested CM-(1→3)-α-d-glucans decreased the cellular metabolism of tumor and normal cells after 24h of incubation. The CM-(1→3)-α-d-glucans had no toxic effects on cervical carcinoma cells but reduced the viability of normal cells. The cytotoxic activity of the CM-(1→3)-α-d-glucans was concentration- and cell-type-dependent with normal cells more sensitive to their action than tumor cells. Generally, the CM-(1→3)-α-d-glucans tested did not have a free radical scavenging effect. It was concluded that the carboxymethylated derivatives of (1→3)-α-d-glucans isolated from the G. lucidum fruiting bodies are biologically active and after further detailed studies may be regarded as a dietary or therapeutic supplements.
International journal of biological macromolecules 08/2012; 51(5):1014-23. · 2.37 Impact Factor
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ABSTRACT: Mutanases are enzymes that catalyze hydrolysis of α-1,3-glucosidic bonds in various α-glucans. One of such glucans, mutan, which is synthesized by cariogenic streptococci, is a major virulence factor for induction of dental caries. This means that mutan-degrading enzymes have potential in caries prophylaxis. In this study, we report the purification, characterization, and partial amino acid sequence of extracellular mutanase produced by the MP-1 strain of Paenibacillus curdlanolyticus, bacterium isolated from soil. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular mass 134 kD, while native gel filtration chromatography confirmed that the enzyme was a monomer of 142 kD. Mutanase showed a pH optimum in the range from pH 5.5 to 6.5 and a temperature optimum around 40-45°C. It was thermostable up to 45°C, and retained 50% activity after 1 hr at 50°C. The enzyme was fully stable at a pH range of 4 to 10. The enzyme activity was stimulated by the addition of Tween 20, Tween 80, and Ca²⁺, but it was significantly inhibited by Hg²⁺, Ag⁺, and Fe²⁺, and also by p-chloromercuribenzoate, iodoacetamide, and ethylenediamine tetraacetic acid (EDTA). Mutanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-1,3-glucans. It also showed binding activity to insoluble α-1,3-glucans. The N-terminal amino acid sequence was NH₂-Ala-Gly-Gly-Thr-Asn-Leu-Ala-Leu-Gly-Lys-Asn-Val-Thr-Ala-Ser-Gly-Gln. This sequence indicated an analogy of the enzyme to α-1,3-glucanases from other Paenibacillus and Bacillus species.
Preparative Biochemistry & Biotechnology 07/2012; 42(4):335-47. · 0.47 Impact Factor
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ABSTRACT: Mutanase (α-(1→3)-glucanase) is a little-known inductive enzyme that is potentially useful in dentistry. Here, it was shown that the cell wall preparation (CWP) obtained from the fruiting body or vegetative mycelium of polypore fungus Laetiporus sulphureus is rich in α-(1→3)-glucan and can be successfully used for mutanase induction in Trichoderma harzianum. The content of this biopolymer in the CWP depended on the age of fruiting bodies and increased along with their maturation. In the case of CWP prepared from vegetative mycelia, the amount of α-(1→3)-glucan depended on the mycelium age and also on the kind of medium used for its cultivation. All CWPs prepared from the individually harvested fruiting body specimens induced high mutanase activity (0.53-0.82 U/mL) in T. harzianum after 3 days of cultivation. As for the CWPs obtained from the hyphal mycelia of L. sulpureus, the maximal enzyme productivity (0.34 U/mL after 3 days of incubation) was recorded for CWP prepared from the 3 week-old mycelium cultivated in Sabouraud medium. Statistically, a high positive correlation was found between the total percentage content of α-(1→3)-glucan in the CWP and the mutanase activity.
International Journal of Molecular Sciences 01/2012; 13(8):9584-98. · 2.60 Impact Factor
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ABSTRACT: Streptococcal mutans synthesized under different conditions by growing cultures or by their glucosyltransferases were shown to exhibit a great structural and property diversity. Culturing and environmental factors causing structural differences in mutans were specified. All of the obtained biopolymers (76 samples) were water-insoluble and most of them (72) had a structure with a predominance of α-(1→3)-linked glucose (i.e., the content of α-(1→3)-linkages in the glucan was always higher than 50%, but did not exceed 76%). An exception were four glucans containing more than 50% of α-(1→6)-sequences. In these structurally unique mutans, the ratio of α-(1→3)- to α-(1→6)-bonds ranged from 0.75 to 0.97. Aside from one polymer, all others had a heavily branched structures and differed in the number of α-(1→3), α-(1→6), and α-(1→3,6) linkages and their mutual proportion. The induction of mutanase production in shaken flask cultures of Trichoderma harzianum by the structurally diverse mutans resulted in enzyme activities ranging from 0.144 to 1.051 U/mL. No statistical correlation was found between the total percentage content of α-(1→3)-linkages in the α-glucan and mutanase activity. Thus, despite biosynthetic differences causing structural variation in the mutans, it did not matter which mutan structures were used to induce mutanase production.
Molecules 01/2012; 17(10):11800-15. · 2.39 Impact Factor
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ABSTRACT: This study, for the first time, investigated the in vitro inhibitory effects of Potentilla recta extracts and subfractions obtained with solvents of different polarity (aqueous, 50% ethanol, diethyl ether, ethyl acetate and n-butanol) against cariogenic Streptococcus spp. strains. It was found that the tested samples inhibited the growth of oral streptococci. Furthermore, all five P. recta preparations exhibited an inhibitory effect on water-insoluble α-(1→3)-,α-(1→6)-linked glucan (mutan) and artificial dental plaque formation. The ethyl acetate fraction showed the highest antibiofilm activities especially against S. sobrinus GCM 20381, with minimum mutan and biofilm inhibition concentrations of 6.25 and 25 µg/mL, respectively. The phytochemical profile of active constituents in the investigated samples was analysed. The high polyphenolics (total phenol, phenolic acids, tannins, proantocyanidins, flavonoids) content were found. The ethyl acetate fraction showed the highest concentration of total polyphenol content which may correlate with the high cariogenic activity of this subfraction. The results demonstrate that P. recta extracts and subfractions could become useful supplements for pharmaceutical products as new anticariogenic agents in a wide range of oral care products. Further studies are necessary to clarify the precise bioactive constituents of P. recta responsible for the anticariogenic properties.
Phytotherapy Research 03/2011; 25(3):343-50. · 2.09 Impact Factor
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ABSTRACT: alpha-1,3-Glucanases (mutanases) are currently of great interest due to their potential use in the field of dental care. These enzymes have been reported in several bacteria, yeasts and fungi, but up to now, characterization of this family of proteins has been relatively poor. In this study, we identify and characterize a mutanase gene from Trichoderma harzianum CCM F-340. Sequence analysis, on the nucleotide and amino acid levels reveals that this alpha-1,3-glucanase is highly homologous to alpha-1,3-glucanases from T harzianum isolate CBS 243.71 and T asperellum CECT 20539. T. harzianum CCM F-340 mutanase is a 634-aa residue protein with a calculated molecular mass of 67.65 kDa, composed of two distinct, highly conserved domains (a long N-terminal catalytic domain and a short C-terminal polysaccharide-binding domain) separated by a less conserved Pro-Ser-Thr-rich linker region. The mutanase gene was expressed in an E. coli BL21 (DE3) host, under the transcriptional control of T7 promoter. The purified enzyme migrated as a band of about 68 kDa after SDS-polyacrylamide gel electrophoresis, which coincided with the predicted size based on the amino acid sequence. Our data indicate that this enzyme is highly conserved in Trichoderma and can be produced in active form in such heterologous expression system.
Polish journal of microbiology / Polskie Towarzystwo Mikrobiologów = The Polish Society of Microbiologists 01/2011; 60(4):293-301. · 0.76 Impact Factor
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ABSTRACT: The aerial parts of selected Potentilla species (P. anserina, P. argentea, P. erecta, P. fruticosa, P. grandiflora, P. nepalensis, P. norvegica, P. pensylvanica, P. crantzii and P. thuringiaca) were investigated in order to determine their contents of polyphenolic compounds. The results showed that P. fruticosa has relatively high concentrations of tannins (167.3 +/- 2.0 mg/g dw), proanthocyanidins (4.6 +/- 0.2 mg/g dw) and phenolic acids (16.4 +/- 0.8 mg/g dw), as well as flavonoids (7.0 +/- 1.1 mg/g dw), calculated as quercetin. Furthermore, we investigated the in vitro inhibitory effects of aqueous extracts from these species against cariogenic Streptococcus spp. strains. It was found that the tested samples moderately inhibit the growth of oral streptococci. However, all the preparations exhibited inhibitory effects on water-insoluble alpha-(1-->3)-, alpha-(1-->6)-linked glucan (mutan) and artificial dental plaque formation. The extract from P. fruticosa showed the highest anti-biofilm activities, with minimum mutan and biofilm inhibition concentrations of 6.25-25 and 50-100 microg/mL, respectively. The results indicate that the studied Potentilla species could be a potential plant material for extracting biologically active compounds, and could become a useful supplement for pharmaceutical products as a new anticariogenic agent in a wide range of oral care products.
Molecules 07/2010; 15(7):4639-51. · 2.39 Impact Factor
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ABSTRACT: The cell wall material from fruiting bodies of Laetiporus sulphureus has been suggested as a new alternative to mutan for the mutanase induction in Trichoderma harzianum. Structural analyses revealed that the alkali-soluble wall fraction from this polypore fungus contained 56.3% of (1-->3)-linked alpha-glucans. When the strain T. harzianum F-340 was grown on a cell wall preparation from L. sulphureus, the maximal enzyme productivity obtained after 3 days of cultivation was 0.71 U/ml. This yield was about 1.8-fold higher than that achieved on mutan, known so far as the best, but expensive and inaccessible, inducer of mutanase production. Cell-wall-induced mutanase showed a high hydrolytic potential in reaction with a dextranase-pretreated mutan, where maximal degrees of saccharification and solubilization of this biopolymer (80% and 100%, respectively) were reached in 3 h at 45 degrees C. The mutanase preparation was also effective in degradation of streptococcal mutan and its removal from oral biofilms, especially in a mixture with dextranase.
Journal of Microbiology and Biotechnology 08/2008; 18(7):1335-41. · 1.38 Impact Factor
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ABSTRACT: The strain Paenibacillus curdlanolyticus MP-1 was used to obtain mutan-hydrolyzing enzymes. Different methods of precipitation and concentration of the post culture liquid were tested. All these methods produced satisfactory results in regard to the overall activity of mutanase and yielded active preparations of the enzyme. The best precipitation was obtained with propanol -98% of the initial enzyme activity was preserved with a purification of 2-fold. Salting out with ammonium sulfate at 50% saturation gave mutanase recovery of 77% and a purification of around 2-fold. Ultrafiltration yielded an about 10-fold concentrated preparation of the enzyme with a yield of 98%. Lyophilization and concentration of the culture broth (in the range from 5 to 20 times) in a vacuum evaporator yielded active crude preparations with mutanase recovery of 97%.
Preparative Biochemistry & Biotechnology 02/2008; 38(4):389-96. · 0.47 Impact Factor
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Brazilian Journal of Microbiology - BRAZ J MICROBIOL. 01/2005; 36(2).
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ABSTRACT: Crude mutanase preparations of Trichoderma harzianum were obtained from the culture supernatant by means of ammonium sulfate salting out, ultrafiltration, freeze-drying, concentration under reduced pressure, and fractional precipitation with organic solvents (methanol, ethanol, propanol, isopropanol, acetone). Ammonium sulfate was the worst precipitant, causing a fall in total mutanase activity by 47%. Other methods of enzyme recovery from the post-culture fluid yielded in most cases very good results in regard to specific and overall activities of the enzymatic preparations.
Biotechnology Letters 02/2001; 23(6):427-431. · 1.68 Impact Factor
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ABSTRACT: Potentilla L. (Rosaceae) species have been used in traditional medicine in Asia, Europe and Northern America. This study analyzed the biological activity of aqueous extracts of Potentilla species (Rosaceae): Dasiphora fruticosa (syn. P. fruticosa), P. norvegica, P. pensylvanica, P. thuringiaca, P. crantzii and P. nepalensis. The activities were tested using MTT, NR and DPPH assays on normal human colon epithelium (CCD 841 CoTr) and colon myofibroblast (CCD-18Co) cells. Moreover, cell morphology using the May-Grünwald-Giemsa method, IL-6 by ELISA, and nitric oxide (NO) analysis with the Griess method in culture supernatants were performed after 24 h. Extracts were tested at dose levels between 25 and 250 microg/mL. For ELISA, 15 microg/mL was chosen. All extracts suppressed the metabolism of myofibroblasts, while epithelial cells' mitochondrial dehydrogenase activity decreased after incubation with extracts. All extracts showed a free radical scavenging (DPPH) effect in a concentration-dependent manner. The most potent was the extract from D. fruticosa, while the least action was observed for P. thuringiaca. Potentilla extracts stimulated, IL-6 production in tested cells but the level of the cytokine was found to decrease in epithelial cells. Pre-incubation of cells with LPS resulted in increased IL-6 secretion. Modulation of NO production after extract addition and cell pre-incubation with LPS was also observed. Potentilla extracts may be interesting natural factors modulating the main features of cells forming the colon wall, and thus may be potentially useful in the prophylaxis or healing of colon disorders.
Acta poloniae pharmaceutica 70(3):523-31. · 0.66 Impact Factor
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ABSTRACT: Trichoderma harzianum (CCM F-470) was found to produce large amounts of extracellular mutanase (0.33 U ml−1, 1.85 U mg protein−1) when grown aerobically on the optimized mutan medium in fermenter culture with an automatic pH control. The mutanase from this source was purified to homogeneity by a rapid procedure, using ion-exchange chromatography, hydrophobic interaction chromatography and chromatofocusing. The enzyme was recovered with a 94-fold increase in specific activity and a yield of 73%. The molecular weight of the purified enzyme proved to be 67 kDa, as estimated by SDS-PAGE, and to be 274 kDa, as determined by size-exclusion HPLC. These results indicate that the native mutanase is probably a tetramer protein. The isoelectric point was at 7.11, and the carbohydrate content in the purified enzyme was 4.42%. The pH and temperature optima were 5.5 and 40°C, respectively. The enzyme remained stable over a pH range of 4.5-6.0 and up to 35° for 1 h. The values of Km and Vmax under standard assay conditions were 1.2 × 10−3 g ml−1 and 8.48 × 10−2 U mg−1, respectively.
Mycological Research.
