Biochemical Characterization of a Novel Endo-1,3-ß-Glucanase from the Scallop Chlamys farreri.

Endo-1,3-ß-glucanases derived from marine mollusks have attracted much attention in recent years because of their unique transglycosylation activity. In this study, a novel endo-1,3-ß-glucanase from the scallop Chlamys farreri, named Lcf, was biochemically characterized. Unlike in earlier studies on marine mollusk endo-1,3-ß-glucanases, Lcf was expressed in vitro first. Enzymatic analysis demonstrated that Lcf preferred to hydrolyze laminarihexaose than to hydrolyze laminarin. Furthermore, Lcf was capable of catalyzing transglycosylation reactions with different kinds of glycosyl acceptors. More interestingly, the transglycosylation specificity of Lcf was different from that of other marine mollusk endo-1,3-ß-glucanases, although they share a high sequence identity. This study enhanced our understanding of the diverse enzymatic specificities of marine mollusk endo-1,3-ß-glucanases, which facilitated development of a unique endo-1,3-ß-glucanase tool in the synthesis of novel glycosides.

Molecular characterization of endo-1,3-ß-glucanase from Cellulosimicrobium cellulans: effects of carbohydrate-binding module on enzymatic function and stability.

An endo-1,3-ß-glucanase was purified from Tunicase®, a crude enzyme preparation from Cellulosimicrobium cellulans DK-1, and determined to be a 383-residue protein (Ala1-Leu383), comprising a catalytic domain of the glycoside hydrolase family 16 and a C-terminal carbohydrate-binding module family 13. The Escherichia coli expression system of the catalytic domain (Ala1-Thr256) was constructed, and the protein with N-terminal polyhistidine tag was purified using a Ni-nitrilotriacetic acid column. We analyzed enzymatic properties of the recombinant catalytic domain, its variants, and the Tunicase®-derived full-length endo-1,3-ß-glucanase. Substitution of Glu119 with Ala and deletion of Met123, both of the residues are located in the catalytic motif, resulted in the loss of hydrolytic activity. In comparison between the full-length enzyme and isolated catalytic domain, their hydrolytic activities for soluble substrates such as laminarin and laminarioligosaccharides were similar. In contrast, the hydrolytic activity of the full-length enzyme for insoluble substrates such as curdlan and yeast-glucan was significantly higher than that of the catalytic domain. It should be noted that the acid stabilities for the hydrolysis of laminarin were clearly different. Secondary structure analysis using circular dichroism showed that the full-length enzyme was more acid stable than was the catalytic domain, possibly because of domain interactions between the catalytic domain and the carbohydrate-binding module.

Catalytic properties and amino acid sequence of endo-1→3-ß-D-glucanase from the marine mollusk Tapes literata.

A specific 1→3-ß-D-glucanase with molecular mass 37 kDa was isolated in homogeneous state from crystalline style of the commercial marine mollusk Tapes literata. It exhibits maximal activity within the pH range from 4.5 to 7.5 at 45°C. The 1→3-ß-D-glucanase catalyzes hydrolysis of ß-1→3 bonds in glucans as an endoenzyme with retention of bond configuration, and it has transglycosylating activity. The K(m) for hydrolysis of laminaran is 0.25 mg/ml. The enzyme is classified as a glucan endo-(1→3)-ß-D-glucosidase (EC 3.2.1.39). The cDNA encoding this 1→3-ß-D-glucanase from T. literata was sequenced, and the amino acid sequence of the enzyme was determined. The endo-1→3-ß-D-glucanase from T. literata was assigned to the 16th structural family (GHF 16) of O-glycoside hydrolases.

Characterization of a new beta(1-3)-glucan branching activity of Aspergillus fumigatus.

A new HPLC method was developed to separate linear from beta(1-6)-branched beta(1-3)-glucooligosaccharides. This methodology has permitted the isolation of the first fungal beta(1-6)/beta(1-3)-glucan branching transglycosidase using a cell wall autolysate of Aspergillus fumigatus (Af). The encoding gene, AfBGT2 is an ortholog of AfBGT1, another transglycosidase of A. fumigatus previously analyzed (Mouyna, I., Hartland, R. P., Fontaine, T., Diaquin, M., Simenel, C., Delepierre, M., Henrissat, B., and Latgé, J. P. (1998) Microbiology 144, 3171-3180). Both enzymes release laminaribiose from the reducing end of a beta(1-3)-linked oligosaccharide and transfer the remaining chain to another molecule of the original substrate. The AfBgt1p transfer occurs at C-6 of the non-reducing end group of the acceptor, creating a kinked beta(1-3;1-6) linear molecule. The AfBgt2p transfer takes place at the C-6 of an internal group of the acceptor, resulting in a beta(1-3)-linked product with a beta(1-6)-linked side branch. The single Afbgt2 mutant and the double Afbgt1/Afbgt2 mutant in A. fumigatus did not display any cell wall phenotype showing that these activities were not responsible for the construction of the branched beta(1-3)-glucans of the cell wall.

Production, purification and characterization of a thermostable ß-1,3-glucanase produced by Moniliophthora perniciosa.

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L(-1) and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

Functional characterisation and product specificity of Endo-ß-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1.

Endo-ß-1,3-glucanase from alkalophilic bacterium, Bacillus lehensis G1 (Blg32) composed of 284 amino acids with a predicted molecular mass of 31.6 kDa is expressed in Escherichia coli and purified to homogeneity. Herein, Blg32 characteristics, substrates and product specificity as well as structural traits that might be involved in the production of sugar molecules are analysed. This enzyme functions optimally at the temperature of 70 °C, pH value of 8.0 with its catalytic activity strongly enhanced by Mn2+. Remarkably, the purified enzyme is highly stable in high temperature and alkaline conditions. It exhibits the highest activity on laminarin (376.73 U/mg) followed by curdlan and yeast ß-glucan. Blg32 activity increased by 62% towards soluble substrate (laminarin) compared to insoluble substrate (curdlan). Hydrolytic products of laminarin were oligosaccharides with degree of polymerisation (DP) of 1 to 5 with the main product being laminaritriose (DP3). This suggests that the active site of Blg32 could recognise up to five glucose units. High concentration of Blg32 mainly produces glucose whilst low concentration of Blg32 yields oligosaccharides with different DP (predominantly DP3). A theoretical structural model of Blg32 was constructed and structural analysis revealed that Trp156 is involved in multiple hydrophobic stacking interactions. The amino acid was predicted to participate in substrate recognition and binding. It was also exhibited that catalytic groove of Blg32 has a narrow angle, thus limiting the substrate binding reaction. All these properties and knowledge of the subsites are suggested to be related to the possible mode of action of how Blg32 produces glucooligosaccharides.

Crystallization and preliminary crystallographic analysis of endo-1,3-beta-glucanase from alkaliphilic Nocardiopsis sp. strain F96.

Endo-1,3-beta-glucanase, an enzyme that hydrolyzes the 1,3-beta-glycosyl linkages of beta-glucan, belongs to the family 16 glycosyl hydrolases, which are widely distributed among bacteria, fungi and higher plants. Crystals of a family 16 endo-1,3-beta-glucanase from the alkaliphilic Nocardiopsis sp. strain F96 were obtained by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1), with unit-cell parameters a = 34.59, b = 71.84, c = 39.67 A, beta = 90.21 degrees, and contained one molecule per asymmetric unit. The Matthews coefficient (VM) and solvent content were 1.8 A3 Da(-1) and 31.8%, respectively. Diffraction data were collected to a resolution of 1.3 A and gave a data set with an overall Rmerge of 6.4% and a completeness of 99.3%.

Formation, separation and characterization of three beta-1,3-glucanases from Sclerotium glucanicum.

The appearance of beta-1,3-glucanases in supernatants of Sclerotium glucanicum cultures was followed by SDS-PAGE and shown to be dependent on cultivation time. Three beta-1,3-glucanases were isolated and purified. Glucanase I and III appeared homogeneous on SDS-PAGE with molecular masses of 85 and 33.5 kDa, respectively. Enzyme I was an endo-splitting beta-1,3-glucanase. In hydrolyzing laminarin it released glucose, laminaritriose and laminaribiose as major endproducts and smaller amounts of higher oligosaccharides. Enzyme III was an exo-beta-1,3-glucanase removing glucose from laminarin and gentiobiose and glucose from scleroglucan. For laminarin as substrate the Km of enzyme I and III was 2.5 and 3.33 mg/ml, respectively. Enzyme II was only partially purified and found to be also an exo-beta-1,3-glucanase, releasing glucose as the only hydrolysis product from laminarin. It did not attack scleroglucan. Its molecular weight was determined to be 78 kDa. Optimum pH and temperature of the three enzymes were determined. The three activities were significantly inhibited by 1 mM Hg2+.

1,3-beta-D-glucanases from Pisum sativum seedlings. I. Isolation and purification.

Two buffer-soluble endo-1,3-beta-D-glucanases (EC 3.2.1.6) have been purified to within 1% of electrophoretic homogeneity from etiolated Pisum sativum stem tissues. Purified glucanase I and II differ in physical properties, such as electrophoretic mobility in sodium dodecyl sulfate polyacrylamide gels (Mr values were 22 000 and 37 000, respectively) and isoelectric focusing, (pI values were 5.4 and 6.8, respectively). Although the enzymes have similar pH optima (5.5--6.0), Km values for various substrates (0.6--7.4 mg/ml) and thermal inactivation profiles, they are localized in different tissues and they differ markedly in the rates with which they attack the internal linkages of long- vs. short-chain substrates. Glucanase I is concentrated in apical regions of the stem and is most effectively assayed reductometrically (as laminarinase), while glucanase II is localized in mature regions and is relatively more active in viscometric assays (as carboxymethyl-pachymanase).

Identification and enzymatic characterization of an endo-1,3-ß-glucanase from Euglena gracilis.

Euglena produces paramylon as a storage polysaccharide, and is thought to require ß-1,3-glucan degrading enzymes to release and utilize the accumulated carbohydrate. To investigate ß-1,3-glucan degradation in Euglena, endo-1,3-ß-glucanases were partially purified from Euglena gracilis by hydrophobic, gel filtration and anion-exchange chromatography. Tryptic digests and mass-spectrometric analysis identified three proteins in the purified fraction as a member of glycoside hydrolase family (GH) 17 and two members of GH81. These genes were cloned from an Euglena cDNA pool by PCR. EgCel17A fused with a histidine-tag at the carboxy terminus was heterologously produced by Aspergillus oryzae and purified by immobilized metal affinity chromatography. Purified EgCel17A had a molecular weight of about 40kDa by SDS-PAGE, which was identical to that deduced from its amino acid sequence. The enzyme showed hydrolytic activity towards ß-1,3-glucans such as laminarin and paramylon. Maximum activity of laminarin degradation by EgCel17A was attained at pH 4.0-5.5 and 60°C after 1h incubation or 50°C after 20h incubation. The enzyme had a Km of 0.21mg/ml and a Vmax of 40.5units/mg protein for laminarin degradation at pH 5.0 and 50°C. Furthermore, EgCel17A catalyzed a transglycosylation reaction by which reaction products with a higher molecular weight than the supplied substrates were initially generated; however, ultimately the substrates were degraded into glucose, laminaribiose and laminaritriose. EgCel17A effectively produced soluble ß-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-ß-glucanase to be identified from E. gracilis.

Purification and properties of a 1,3-beta-glucanase from Penicillium oxalicum autolysates.

High 1,3-beta-glucanase activity was detected during autolysis in a culture medium containing Penicillium oxalicum. It was due to the combined action of four enzymes. The purification process for the major enzyme produced a homogeneous band in the SDS polyacrylamide gel that corresponded to a molecular weight of 79,400 daltons. The enzyme pI was 6.3 and it was only active against 1,3-beta-glucans, with a S0.5 of 0.23 mg ml-1 against laminarin. The enzymatic optima were found at pH 4 and 55 degrees C, and instability was evident when pH and temperature were altered. The enzyme was not active against oxidated laminarin and was barely inhibited by glucono-D-lactone. Hg2+, Ag+ and Fe2+ were effective inhibitors. The enzyme was adsorbed by concanavalin-A-sepharose.

Pilot scale production of a Trichoderma reesei endo-beta-glucanase by brewer's yeast.

Endo-beta-glucanase I (EGI) of Trichoderma reesei was produced in laboratory and pilot scale using recombinant strains of "bottom-fermenting" Saccharomyces cerevisiae. The gene eg/1 was integrated in the chromosome or an expression cassette was inserted on a multicopy plasmid. Expression levels were compared in a laboratory scale bioreactor. The best EGI-producing strain was cultivated in pilot scale. Adsorbent treatment was used to remove endogenous yeast proteins and other impurities from the culture filtrate during concentration. Effective pilot scale one-step purification of the EGI protein was obtained using DEAE-Sepharose, on which EGI was weakly bound. The purified enzyme reacted with antibodies prepared against T. reesei EGI and catalyzed the hydrolysis of both insoluble and soluble substrates.

Purification and determination of the action pattern of Haliotis tuberculata laminarinase.

The major laminarinase activity (EC 3.2.1.39) from the gastropodean marine mollusc Haliotis tuberculata was purified to homogeneity by cation exchange chromatography and its action pattern was investigated by HPAEC-PAD analysis of the degradation of various laminarin samples. It consists of a 60 kDa protein capable of depolymerizing the unbranched portions of the beta-(1-->3), beta-(1-->6)-glucan, down to laminaritriose. The enzyme operates via a molecular mechanism retaining the anomeric configuration. As the purified protein does not cleave the beta-(1-->6) linkages, it can be used for the structural analysis of laminarins.

Purification and characterisation of two endo-(1 linked to 3)-beta-D-glucanases from Telescopium telescopium.

The crystalline style of the gastropod Telescopium telescopium contains two (1 linked to 3)-beta-D-glucanases and a beta-D-glucosidase. The two glucanases (I and II) have been purified and shown to be endo-enzymes. Both enzymes attack laminarin, carboxymethylpachyman, and lichenin, but have no action towards carboxymethyl-cellulose. The main products of hydrolysis of laminarin are D-glucose and beta-(1 linked to 3)-linked oligosaccharides of d.p. 2, 3, and 4. Glucanases I and II are similar to each other, although they differ in molecular weight and kinetic properties.

Purification, characterization, and action-pattern studies on the endo-(1 linked to 3)-beta-D-glucanase from Rhizopus arrhizus QM 1032.

The extracellular (1 linked to 3)-beta-D-glucanase [(1 linked to 3)-beta-D-glucan glucanohydrolase, EC 3.2.1.6] produced by Rhizopus arrhizus QU 1032 was purified 305-fold in 70% overall yield. This preparation was found to be homogeneous by ultracentrifugation (sedimentation velocity and equilibrium studies), electrophoresis on acrylamide gel with normal, sodium dodecyl sulfate, and urea-acetic acid gels, and upon isoelectric focusing. The amino acid composition of the enzyme has been determined and it possesses a carbohydrate moiety compose of mannose and galactose (in the ratio approximately 5:1) that is linked to the protein through a 2-acetamido-2-deoxyglucose residue. The molecular weight, as determined by equilibrium sedimentation, is 28,800 and this number was confirmed by electrophoresis on gels of sodium dodecyl sulfate. The enzyme does not possess subunit structure. It hydrolyzes its substrates with retention of configuration and possesses transglycosylating ability. The rates of hydrolysis of a wide variety of substrates were determined, and its action pattern on a series of oligosaccharides containing mixed (1 linked to 3)-, (1 linked to 4)-, and (1 linked to 6)-beta-D-glucopyranosyl residues was investigated. The enzyme favors stretches of beta-D-(1 linked to 3) linkages, but it can hydrolyze beta-D-(1 linked to 4) linkages that are flanked on the non-reducing side with stretches of beta-D-(1 linked to 3) links. The enzyme will not act on (1 linked to 6)-beta-D-glucosyl linkages located in stretches of beta-D-(1 linked to 3) and will not act on (1 linked to 3) beta-D-glycosidic linkages involving sugars other than D-glucose.

An endo-(1 leads to 3)-beta-D-glucanase from Mucor hiemalis.

An endo-(1 leads to 3)-beta-D-glucanase (EC 3.2.1.6), isolated from the culture filtrate of the fungus Mucor hiemalis, was purified by ammonium sulfate fractionation, gel filtration, and column chromatography on O-(carboxymethyl)cellulose. The optimum pH, optimum temperature, and Km value of the enzyme were pH 5.0, 50 degrees, and 0.048%, respectively. The enzyme was strongly inactivated by Pb2+, Cu2+, and Hg2+ ions and also inhibited by Zn2+ and Fe3+ ions. The enzyme was specific for laminaran and the action pattern of the enzyme was of the endo-type. The molecular weight of the enzyme, as determined by gel filtration, was 30,000.

Purification of some glycoside hydrolases by affinity chromatography.

Two glycoproteins have been isolated from the cell walls of baker's yeast. One is a glucan-protein complex which has been partially characterised as having a branched carbohydrate structure composed of chains of (1 leads to 3)-linked beta-D-glucosyl residues, some of which are attached by (1 leads to 6)-linkages to the main chain. Immobilization of this glycoprotein was achieved by covalent attachment to Sepharose, and the product was used to isolate a number of (1 leads to 3)-beta-D-glucan hydrolases from Helix pomatia, malted barley, and Basidiomycete QM806. The second glycoprotein, a mannan-protein complex, after immobilization, has been used in the purification of an alpha-D-mannosidase from jack-bean meal.

Purification and characterization of a (1-->3)-beta-D-glucan endohydrolase from rice (Oryza sativa) bran.

A (1-->3)-beta-glucanase with an apparent M(r) of 29,000 and an isoelectric point of 4.0 has been purified 2000-fold from extracts of rice bran, using fractional precipitation with ammonium sulfate, anion exchange chromatography, size-exclusion chromatography, chromatofocussing, and hydrophobic interaction chromatography. The enzyme can be classified with the EC 3.2.1.39 group, because it releases laminarabiose and higher laminara-oligosaccharides from linear (1-->3)-beta-D-glucans with an action pattern that is typical of (1-->3)-beta-D-glucan endohydrolases. However, the introduction of substituents or branching in the (1-->3)-beta-D-glucan substrates causes a marked decrease in the rate of hydrolysis. Thus, substituted or branched (1-->3)-beta-D-glucans of the kind commonly found in fungal cell walls are less susceptible to hydrolysis than essentially linear (1-->3)-beta-D-glucans. Kinetic analyses indicate an apparent Km of 42 microM, a kcat constant of 67 s-1, and a pH optimum of 5.0 during hydrolysis of the (1-->3)-beta-D-glucan, laminaran, from Laminaria digitata. The first 60 NH2-terminal amino acid residues of the purified rice (1-->3)-beta-glucanase contain blocks of amino acids that are conserved in other cereal (1-->3)-beta-glucanases. Although the precise tissue location and function of the enzyme in rice bran are not known, it is likely that it is concentrated in the aleurone layer and that it plays a preemptive role in the protection of ungerminated grain against pathogen attack.

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase.

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).

Polysaccharide hydrolases from leaves of Boscia senegalensis: properties of endo-(1-->3)-beta-D-glucanase.

The leaves of Boscia senegalensis are traditionally used in West Africa in cereal protection against pathogens, pharmacologic applications, and food processing. Activities of alpha-amylase, beta-amylase, exo-(1-->3, 1-->4)-beta-D-glucanase, and endo-(1-->3)-beta-D-glucanase were detected in these leaves. The endo-(1-->3)-beta-D-glucanase (EC 3.2.1.39) was purified 203-fold with 57% yield. The purified enzyme is a nonglycosylated monomeric protein with a molecular mass of 36 kDa and pI > or = 10.3. Its optimal activity occurred at pH 4.5 and 50 degrees C. Kinetic analysis gave Vmax, kcat, and Km values of 659 U/mg, 395 s(-1), and 0.42 mg/mL, respectively, for laminarin as substrate. The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance liquid chromatography revealed that the enzyme hydrolyzes not only soluble but also insoluble (1-->3)-beta-glucan chains in an endo fashion. This property is unusual for endo-acting (1-->3)-beta-D-glucanase from plants. The involvement of the enzyme in plant defense against pathogenic microorganisms such as fungi is discussed.