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α-1,3-Glucanase from Streptomyces thermodiastaticus HF3-3 (Agl-ST) has been classified in the glycoside hydrolase (GH) family 87. Agl-ST is a multi-modular domain consisting of an N-terminal β-sandwich domain (β-SW), a catalytic domain, an uncharacterized domain (UC), and a C-terminal discoidin domain (DS). Although Agl-ST did not hydrolyze α-1,4-glycosidic bonds, its amino acid sequence is more similar to GH87 mycodextranase than to α-1,3-glucanase. It might be categorized into a new subfamily of GH87. In this study, we investigated the function of the domains. Several fusion proteins of domains with green fluorescence protein (GFP) were constructed to clarify the function of each domain. The results showed that β-SW and DS domains played a role in binding α-1,3-glucan and enhancing the hydrolysis of α-1,3-glucan. The binding domains, β-SW and DS, also showed binding activity toward xylan, although it was lower than that for α-1,3-glucan. The combination of β-SW and DS domains demonstrated high binding and hydrolysis activities of Agl-ST toward α-1,3-glucan, whereas the catalytic domain showed only a catalytic function. The binding domains also achieved effective binding and hydrolysis of α-1,3-glucan in the cell wall complex of Schizophyllum commune.

Extracellular α-1,3-glucanase HF90 (AglST2), with a sodium dodecyl sulfate (SDS)-PAGE-estimated molecular mass of approximately 91 kDa, was homogenously purified from the culture filtrate of Streptomyces thermodiastaticus HF3-3. AglST2 showed a high homology with mycodextranase in an amino acid sequence and demonstrated specificity with an α-1,3-glycosidic linkage of homo α-1,3-glucan. It has been suggested that AglST2 may be a new type of α-1,3-glucanase. The optimum pH and temperature of AglST2 were pH 5.5 and 60°C, respectively. AglST2 action was significantly stimulated in the presence of 5-20% (w/v) NaCl, and 1 mM metal ions Mn2+ and Co2+. On the other hand, it was inhibited by 1 mM of Ag+, Cu2+, Fe2+ and Ni2+. Regarding the stability properties, AglST2 retained more than 80% of its maximum activity over a pH range of 5.0-7.0 at up to 60°C and in the presence of 0-20% (w/v) NaCl. Based on these results, the properties of AglST2 were comparable with those of AglST1, which had been previously purified and characterized from S. thermodiastaticus HF3-3 previously. The N-terminal amino acid sequence of AglST2 showed a good agreement with that of AglST1, suggesting that AglST1 was generated from AglST2 by proteolysis during cultivation. MALDI-TOF mass analysis suggested that AglST1 might be generated from AglST2 by the proteolytic removal of C-terminus polypeptide (approximately 20 kDa). Our investigation thus revealed the properties of AglST2, such as tolerance against high temperature, salts, and surfactants, which have promising industrial applications.

Thermally stable α-1,3-glucanase HF65 was purified from culture filtrate of Streptomyces thermodiastaticus HF3-3. The molecular mass of this enzyme was estimated to be 65 kDa and 45.7 kDa by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography, respectively. The purified enzyme retained more than 50% of maximum activity even after incubation at 65°C more than 2 h. Moreover, α-1,3-glucanase HF65 was stable in the presence of chemicals like SDS, benzethonium chloride, and sodium fluoride at a concentration of 1%. The enzyme also exhibited salt tolerance at a concentration up to 20%. The observed stability of α-1,3-glucanase HF65 to salt and surfactants is a great advantage for its addition to commercial oral care products. Interestingly, the N-terminal amino acid sequence did not show any similarity to those of known α-1,3-glucanases, while the sequence of internal eight amino acid residues of this enzyme was homologous with those of mycodextranases. Nevertheless, the enzyme exhibited high specificity against α-1,3-glucan. According to these results, the enzyme purified from S. thermodiastaticus HF3-3 was classified as α-1,3-glucanase which was highly homologous to mycodextranase in amino acid sequence.

An enzyme hydrolyzing nigeran (alternating alpha-1,3- and alpha-1,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by percipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50 degrees C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50 degrees C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the alpha-1,4-glucosidic linkages in nigeran.

A Gram-positive bacterium, Bacillus circulans , isolated from soil was found to produce an enzyme hydrolyzing nigeran (mycodextran, alternating α-1,3- and α-1,4-linked glucan). The molecular weight of the purified enzyme was 120,000 and its isoelectric point was 8.30. The optimum pH and temperature for the enzyme activity were 6.0 and 50°C, respectively. The enzyme was stable in the pH range from 6.0 to 7.0 and up to 50°C. The K m (mg/ml) for nigeran was 1.37. The enzyme specifically hydrolyzed the nigeran into nigerose and nigeran tetrasaccharide by an endo-type action, indicating that it is a mycodextranase (EC 3.2.1.61) cleaving only the α-1,4-glucosidic linkages in nigeran. The N-terminal amino acid sequence of the purified enzyme of B. circulans (APTVYEAESAAKTGGV) was different from that of the mycodexstranase purified from Streptomyces sp. J-13-3 (XDPGDPTDPDPSGVGATLPF).

Mycodextranase (EC 3.2.1.61) is an alpha-glucanase that cleaves alpha-1,4-bonds of alternating alpha-1,3- and alpha-1,4-linked D-glucan (nigeran). The gene encoding mycodextranase from Streptomyces sp. J-13-3 was cloned by hybridization with a degenerate oligonucleotide probe from the amino-terminal amino acid sequence of the enzyme and its nucleotide structure was analyzed. The open reading frame consisted of 1,803 base pairs encoding a signal peptide of 60 amino acids and a mature protein of 540 amino acids with a calculated molecular weight of 56,078. The deduced amino acid sequence showed weak similality to a chitinase homolog from Streptomyces lividans and a chitinase from Xanthomonas sp.

The chemical constitution of the carbohydrate portion of mycodextranase, an exocellular endo-alpha(1 yields 4) D-glucanase of Penicillium melinii, has been investigated. At least 80% of the carbohydrate, consisting exclusively of mannose and glucose, is released from protein by treatment of the enzyme with 0.05 M potassium hydroxide plus 1 M sodium borohydride or 0.5 M sodium hydroxide at 50 degrees. There is concomitant destruction of 60% of the threonine and 15% of the serine of the treated enzyme and an increase in absorption, at 241 nm, of the treated protein's spectrum, indicative of an O-glycosidic beta-hydroxyamino acyl linkage between untreated protein and its associated carbohydrate. Mannose is the monosaccharide involved in this linkage. Smith degradation, methylation, and glycosidase digestions of the carbohydrate indicate that it is present in mycodextranase as side chains of mannose, glucosyl alpha(1 yields 2)-mannose, and mannosyl alpha(1 yields 2)-glucosyl alpha(1 yields 2)-mannose units with each enzyme molecule bearing a calculated average of 25 side chains. Separation of pronase glycopeptides by gel filtration on Sephadex G-25 revealed that 96% of the carbohydrate is present in the highest molecular weight fraction which contains 60% of the threonine of mycodextranase but only 3.5% of the aromatic acids judged by its absorbance at 280 nm. Further fractionation of this glycopeptide component on Sephadex G-75 indicates carbohydrate is restricted to two fractions, one containing 71% by weight of the threonine and serine of mycodextranase and 56% of its carbohydrate. These results suggest carbohydrate chains of mycodextranase are clustered in a few threonin-rich regions along the polypeptide chain rather than being separated from each other by nonglycosylated areas.

Mycodextranase is an α-glucanase which splits only the α-1,4 linkages in a substrate having alternating α-1,3 and α-1,4 links. It ïs a fungal enzyme of rather infrequent occurrence, adaptive in nature, and in its properties strongly resembling other fungal glucanases. The products of its action on mycodextran are a tetramer and nigerose. The hypothesis is advanced that this glucanase, and others, contain a site which binds a dimeric portion of the polymer; in mycodextranase, the bound dimer is nigerose, the α-1,3-disaccharide.

A specific fungal α-(1 → 4)-glucanase, mycodextranase, useful for the large scale production of nigerose, has been purified to homogeneity in high yield from the culture filtrate of Penicillium melinii QM 1931. It has been resolved into fast and slow forms; designations based on their differing electrophoretic mobilities at pH 4.5. The fast form constitutes about 85% of the protein and enzymatic activity. Both forms hydrolyze the glucan nigeran (mycodextran) and certain oligosaccharides of nigeran yielding nigerose and the tetrasaccharide, O-α-d-glucopyranosyl-(1 → 3)-O-α-d-glucopyranosyl-(1 → 4)-O-α-d-glucopyranosyl-(1 → 3)-d-glucose, as sole end products. Glucose is not produced. Sedimentation equilibrium studies indicate that fast and slow enzymes both have molecular weights of 40,000 ± 2,000 daltons. They have similar but not identical amino acid compositions both being rich in serine, threonine, glutamic and aspartic acids, but have a low content of cysteine and tryptophan. Both mycodextranases contain associated carbohydrate. The ultraviolet absorption spectra, urea sensitivities, and pH activity profiles for both forms are practically identical. In addition both forms display maximum catalytic activities at 70° and are fully active in 40% dimethyl sulfoxide. Of a number of general enzyme inhibitors tested, only Ag+, Hg+, N-chlorosuccinimide, and N-bromosuccinimide inactivated the fast enzyme. Some aspects of the action pattern and mechanism of the fast form have been examined in detail. It operates via an endo-multichain mechanism; both end products are formed at a constant rate during hydrolysis but nigerose does not arise from tetrasaccharide. Competitive inhibition studies show that the fast enzyme preferentially binds oligosaccharides with alternating α-(1 → 3)- and α-(1 → 4)-glucosidic linkages, and experiments with certain oligosaccharides of nigeran give results consistent with a model in which the substrate-binding site holds 8 glucopyranose units with a catalytic site located midway in this site. The enzyme has been used to study the fine structure of nigeran and the general structure of isolichenin. In the former case this has been done by identifying and quantitating the linkages present at its reducing and nonreducing termini. The data obtained are used to postulate a general mechanism for the biosynthesis of nigeran.

MYCODEXTRAN (nigeran) is an unbranched polysaccharide composed of a regular alternating sequence of α–(1rarr;3) and α–(1→4) linked glucopyranosyl units. It is produced in the mycelia of various species of moulds1. Reese and Mandels have recently reported2 the isolation and partial purification of an inducible glucanase (mycodextranase) produced in the culture filtrates of selected species of fungi grown on a medium containing mycodextran as a carbon source. The enzyme is an endoglucanase that hydrolyses nigeran to produce hexameric and tetrameric units which are subsequently hydrolysed to produce the disaccharide nigerose (O–α–D–glucopyranosyl–(1→3)–D–glucose) as the final product2.