Characterization of a new α-l-fucosidase isolated from Fusarium proliferatum LE1 that is regioselective to α-(1 → 4)-l-fucosidic linkage in the hydrolysis of α-l-fucobiosides.

Here, we report the biochemical characterization of a novel α-l-fucosidase with broad substrate specificity (FpFucA) isolated from the mycelial fungus Fusarium proliferatum LE1. Highly purified α-l-fucosidase was obtained from several chromatographic steps after growth in the presence of l-fucose. The purified α-l-fucosidase appeared to be a monomeric protein of 67 ± 1 kDa that was able to hydrolyze the synthetic substrate p-nitrophenyl α-l-fucopyranoside (pNPFuc), with Km = 1.1 ± 0.1 mM and kcat = 39.8 ± 1.8 s-1. l-fucose, 1-deoxyfuconojirimycin and tris(hydroxymethyl)aminomethane inhibited pNPFuc hydrolysis, with inhibition constants of 0.2 ± 0.05 mM, 7.1 ± 0.05 nM, and 12.2 ± 0.1 mM, respectively. We assumed that the enzyme belongs to subfamily A of the GH29 family (CAZy database) based on its ability to hydrolyze practically all fucose-containing oligosaccharides used in the study and the phylogenetic analysis. We found that this enzyme was a unique α-l-fucosidase that preferentially hydrolyzes the α-(1 â†’ 4)-L-fucosidic linkage present in α-L-fucobiosides with different types of linkages. As a retaining glycosidase, FpFucA is capable of catalyzing the transglycosylation reaction with alcohols (methanol, ethanol, and 1-propanol) and pNP-containing monosaccharides as acceptors. These features make the enzyme an important tool that can be used in the various modifications of valuable fucose-containing compounds.

The method of integrated kinetics and its applicability to the exo-glycosidase-catalyzed hydrolysis of p-nitrophenyl glycosides.

In the present work we suggest an efficient method, using the whole time course of the reaction, whereby parameters kcat, Km and product KI for the hydrolysis of a p-nitrophenyl glycoside by an exo-acting glycoside hydrolase can be estimated in a single experiment. Its applicability was demonstrated for three retaining exo-glycoside hydrolases, ß-xylosidase from Aspergillus awamori, ß-galactosidase from Penicillium sp. and α-galactosidase from Thermotoga maritima (TmGalA). During the analysis of the reaction course catalyzed by the TmGalA enzyme we had observed that a non-enzymatic process, mutarotation of the liberated α-d-galactose, affected the reaction significantly.

α-Galactobiosyl units: thermodynamics and kinetics of their formation by transglycosylations catalysed by the GH36 α-galactosidase from Thermotoga maritima.

Broad regioselectivity of α-galactosidase from Thermotoga maritima (TmGal36A) is a limiting factor for application of the enzyme in the directed synthesis of oligogalactosides. However, this property can be used as a convenient tool in studies of thermodynamics of a glycosidic bond. Here, a novel approach to energy difference estimation is suggested. Both transglycosylation and hydrolysis of three types of galactosidic linkages were investigated using total kinetics of formation and hydrolysis of pNP-galactobiosides catalysed by monomeric glycoside hydrolase family 36 α-galactosidase from T. maritima, a retaining exo-acting glycoside hydrolase. We have estimated transition state free energy differences between the 1,2- and 1,3-linkage (ΔΔG(‡)0 values were equal 5.34 ± 0.85 kJ/mol) and between 1,6-linkage and 1,3-linkage (ΔΔG(‡)0=1.46 ± 0.23 kJ/mol) in pNP-galactobiosides over the course of the reaction catalysed by TmGal36A. Using the free energy difference for formation and hydrolysis of glycosidic linkages (ΔΔG(‡)F-ΔΔG(‡)H), we found that the 1,2-linkage was 2.93 ± 0.47 kJ/mol higher in free energy than the 1,3-linkage, and the 1,6-linkage 4.44 ± 0.71 kJ/mol lower.

Transglycosylating and hydrolytic activities of the beta-mannosidase from Trichoderma reesei.

A purified beta-mannosidase (EC 3.2.1.25) from the fungus Trichoderma reesei has been identified as a member of glycoside hydrolase family 2 through mass spectrometry analysis of tryptic peptides. In addition to hydrolysis, the enzyme catalyzes substrate transglycosylation with p-nitrophenyl beta-mannopyranoside. Structures of the major and minor products of this reaction were identified by NMR analysis as p-nitrophenyl mannobiosides and p-nitrophenyl mannotriosides containing beta-(1-->4) and beta-(1-->3) linkages. The rate of donor substrate hydrolysis increased in presence of acetonitrile and dimethylformamide, while transglycosylation was weakly suppressed by these organic solvents. Differential ultraviolet spectra of the protein indicate that a rearrangement of the hydrophobic environment of the active site following the addition of the organic solvents may be responsible for this hydrolytic activation.

Novel precipitated fluorescent substrates for the screening of cellulolytic microorganisms.

New substrates, 2-(2'-benzothiazolyl)-phenyl (BTP) cellooligosaccharides with degree of polymerization (d.p.) 2-4 (BTPG(2-4)) were synthesized for the screening of microbial cellulolytic activity in plate assays. The substrates were very efficient that was shown for several cellulolytic bacteria, including yeast-like isolates from Kamchatka hot springs. Three tested bacterial strains and eighteen of 30 of the yeast isolates showed ability to degrade cellulose with cellobiohydrolase, beta-glucosidase and endo-cellulase activities measured with standard substrates. The structures of 2-(2'-benzothiazolyl)-phenyl oligosaccharides were solved by NMR- and mass-spectrometry. The usefulness of the 2-(2'-benzothiazolyl)-phenyl substrates were also shown during purification of the B. polymyxa cellulolytic complex, which consists of at least three types of the enzymes: cellobiohydrolase, endo-beta-d-glucanase and beta-glucosidase.

Biochemical analysis of Thermotoga maritima GH36 alpha-galactosidase (TmGalA) confirms the mechanistic commonality of clan GH-D glycoside hydrolases.

Organization of glycoside hydrolase (GH) families into clans expands the utility of information on catalytic mechanisms of member enzymes. This issue was examined for GH27 and GH36 through biochemical analysis of GH36 alpha-galactosidase from Thermotoga maritima (TmGalA). Catalytic residues in TmGalA were inferred through structural homology with GH27 members to facilitate design of site-directed mutants. Product analysis confirmed that the wild type (WT) acted with retention of anomeric stereochemistry, analogous to GH27 enzymes. Conserved acidic residues were confirmed through kinetic analysis of D327G and D387G mutant enzymes, azide rescue, and determination of azide rescue products. Mutation of Asp327 to Gly resulted in a mutant that had a 200-800-fold lower catalytic rate on aryl galactosides relative to the WT enzyme. Azide rescue experiments using the D327G enzyme showed a 30-fold higher catalytic rate compared to without azide. Addition of azide to the reaction resulted in formation of azide beta-d-galactopyranoside, confirming Asp327 as the nucleophilic residue. The Asp387Gly mutation was 1500-fold catalytically slower than the WT enzyme on p-nitrophenyl alpha-d-galactopyranoside. Analysis at different pH values produced a bell-shaped curve of the WT enzyme, but D387G exhibited higher activity with increasing pH. Catalyzed reactions with the D387G mutant in the presence of azide resulted in formation of azide alpha-d-galactopryanoside as the product of a retaining mechanism. These results confirm that Asp387 is the acid/base residue of TmGalA. Furthermore, they show that the biochemical characteristics of GH36 TmGalA are closely related to GH27 enzymes, confirming the mechanistic commonality of clan GH-D members.

Biochemical and kinetic analysis of the GH3 family beta-xylosidase from Aspergillus awamori X-100.

The beta-xylosidase from Aspergillus awamori X-100 belonging to the family 3 glycoside hydrolase revealed a distinctive transglycosylating ability to produce xylooligosaccharides with degree of polymerization more than 7. In order to explain this fact, the enzyme has been subjected to the detailed biochemical study. The enzymatic hydrolysis of p-nitrophenyl beta-D-xylopyranoside was found to occur with overall retention of substrate anomeric configuration suggesting cleavage of xylosidic bonds through a double-displacement mechanism. Kinetic study with aryl beta-xylopyranosides substrates, in which leaving group pK(a)s were in the range of 3.96-10.32, revealed monotonic function of log(k(cat)) and no correlation of log(k(cat)/Km) versus pKa values indicating deglycosylation as a rate-limiting step for the enzymatic hydrolysis. The classical bell-shaped pH dependence of k(cat)/Km indicated two ionizable groups in the beta-xylosidase active site with apparent pKa values of 2.2 and 6.4. The kinetic parameters of hydrolysis, Km and k(cat), of p-nitrophenyl beta-D-1,4-xylooligosaccharides were very close to those for hydrolysis of p-nitrophenyl-beta-D-xylopyranoside. Increase of p-nitrophenyl-beta-D-xylopyranoside concentration up to 80 mM led to increasing of the reaction velocity resulting in k(cat)(app)=81 s(-1). Addition of alpha-methyl D-xylopyranoside to the reaction mixture at high concentration of p-nitrophenyl-beta-D-xylopyranoside (50 mM) caused an acceleration of the beta-xylosidase-catalyzed reactions and appearance of a new transglycosylation product, alpha-methyl D-xylopyranosyl-1,4-beta-D-xylopyranoside, that was identified by 1H NMR spectroscopy. The kinetic model suggested for the enzymatic reaction was consistent with the results obtained.

Transferase and hydrolytic activities of the laminarinase from Rhodothermus marinus and its M133A, M133C, and M133W mutants.

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus beta-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent beta(1-4) and beta(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl beta-laminarin oligosaccharides of degree of polymerisation 2-8 were synthesized enzymatically. Catalytic efficiency values, k (cat)/K (m), of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-beta-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were beta-glucopyranosyl-beta-1,3-D-glucopy- ranosyl-beta-1,3-D-glucopyranose and beta-glucopyranosyl-beta-1, 3-D-glucopyranosyl-beta-1,3-D-glucpyranosyl-beta-1,3-D- glucopyranoxside.

Structural insights into the beta-xylosidase from Trichoderma reesei obtained by synchrotron small-angle X-ray scattering and circular dichroism spectroscopy.

The enzyme beta-xylosidase from Trichoderma reesei, a member of glycosil hydrolase family 3 (GH3), is a glycoside hydrolase which acts at the glycosidic linkages of 1,4-beta-xylooligosaccharides and that also exhibits alpha-l-arabinofuranosidase activity on 4-nitrophenyl alpha-l-arabinofuranoside. In this work, we show that the enzyme forms monomers in solution and derive the low-resolution molecular envelope of the beta-xylosidase from small-angle X-ray scattering (SAXS) data using the ab initio simulated annealing algorithm. The radius of gyration and the maximum dimension of the beta-xylosidase are 30.3 +/- 0.2 and 90 +/- 5 A, respectively. In contrast to the fold of the only two structurally characterized members of GH3, the barley beta-d-glucan exohydrolase and beta-hexosaminidase from Vibrio cholerae, which have respectively two or one distinct domains, the shape of the beta-xylosidase indicates the presence of three distinct structural modules. Domain recognition algorithms were used to show that the C-terminal part of the amino acid sequence of the protein forms the third domain. Circular dichroism spectroscopy and secondary structure prediction programs demonstrate that this additional domain adopts a predominantly beta conformation.

Synthesis of arabinitol 1-phosphate and its use for characterization of arabinitol-phosphate dehydrogenase.

D-arabinitol 1-phosphate (Ara-ol1-P), a substrate for D-arabinitol-phosphate dehydrogenase (APDH), was chemically synthesized from D-arabinonic acid in five steps (O-acetylation, chlorination, reduction, phosphorylation, and de-O-acetylation). Ara-ol1-P was used as a substrate for the characterization of APDH from Bacillus halodurans. APDH converts Ara-ol1-P to xylulose 5-phosphate in the oxidative reaction; both NAD(+) and NADP(+) were accepted as co-factors. Kinetic parameters for the oxidative and reductive reactions are consistent with a ternary complex mechanism.

Chemo-enzymatic synthesis of 4-methylumbelliferyl beta-(1-->4)-D-xylooligosides: new substrates for beta-D-xylanase assays.

Transglycosylation catalyzed by a beta-D-xylosidase from Aspergillus sp. was used to synthesize a set of 4-methylumbelliferyl (MU) beta-1-->4-D-xylooligosides having the common structure [beta-D-Xyl-(1-->4)]2-5-beta-D-Xyl-MU. MU xylobioside synthesized chemically by the condensation of protected MU beta-D-xylopyranoside with ethyl 2,3,4-tri-O-acetyl-1-thio-beta-D-xylopyranoside was used as a substrate for transglycosylation with the beta-D-xylosidase from Aspergillus sp. to produce higher MU xylooligosides. The structures of oligosaccharides obtained were established by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. MU beta-D-xylooligosides synthesized were tested as fluorogenic substrates for the GH-10 family beta-D-xylanase from Aspergillus orizae and the GH-11 family beta-D-xylanase I from Trichoderma reesei. Both xylanases released the aglycone from MU xylobioside and the corresponding trioside. With substrates having d.p. 4 and 5, the enzymes manifested endolytic activities, splitting off MU, MUX, and MUX2 primarily.

Catalytic properties of IgMs with amylolytic activity isolated from patients with multiple sclerosis.

BACKGROUND: Recently, amylolytic activity was detected in IgMs isolated from the sera of the patients with multiple sclerosis. MATERIAL/METHODS: All purified samples of IgM were electrophoretically homogenous and did not contain any co-purified a-amylase and a-glucosidase activities, in accordance with a set of criteria developed for abzymes. The amylolytic activity of abzymes was studied in the hydrolysis of p-nitrophenyl a-D-maltooligosaccharides with different degrees of polymerization from 1 to 8 by TLC and reverse-phase HPLC techniques. RESULTS: All IgM samples isolated from 54 patients with clinically definite multiple sclerosis demonstrated hydrolytic activity towards the above artificial substrates. The Michaelis constant values (Km) in the hydrolysis of p-nitrophenyl a-D-maltoheptaoside were in the range of 10 p-nitrophenyl or p-nitrophenyl a-D-glucosides, thus indicating the presence of an a-D-glucosidase activity. For a number of the investigated samples, specific amylolytic activity increased depending on the length of substrates (from p-nitrophenyl maltopentaoside to p-nitrophenyl maltohexaoside); for other IgMs, the opposite dependence was observed. All IgMs studied did not exhibit any other glycoside hydrolase activities toward p-nitrophenyl glycoside substrates. CONCLUSIONS: Abzyme fractions from different donors demonstrated catalytic heterogeneity in Michaelis-Menten parameters and different modes of action in the hydrolysis of p-nitrophenyl maltooligosaccharides. Enzymatic properties of the IgMs tested varied from human a-amylases. All investigated abzyme samples did not show transglycosylating ability.

Biochemical characterization of Aspergillus awamori exoinulinase: substrate binding characteristics and regioselectivity of hydrolysis.

1H-NMR analysis was applied to investigate the hydrolytic activity of Aspergillus awamori inulinase. The obtained NMR signals and deduced metabolite pattern revealed that the enzyme cleaves off only fructose from inulin and does not possess transglycosylating activity. Kinetics for the enzyme hydrolysis of inulooligosaccharides with different degree of polymerization (d.p.) were recorded. The enzyme hydrolyzed both beta2,1- as well as beta2,6-fructosyl linkages in fructooligosaccharides. From the k(cat)/K(m) ratios obtained with inulooligosaccharides with d.p. from 2 to 7, we deduce that the catalytic site of the inulinase contains at least five fructosyl-binding sites and can be classified as exo-acting enzyme. Product analysis of inulopentaose and inulohexaose hydrolysis by the Aspergillus inulinase provided no evidence for a possible multiple-attack mode of action, suggesting that the enzyme acts exclusively as an exoinulinase.

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).

Enzymatic synthesis of beta-xylanase substrates: transglycosylation reactions of the beta-xylosidase from Aspergillus sp.

A beta-D-xylosidase with molecular mass of 250+/-5 kDa consisting of two identical subunits was purified to homogeneity from a cultural filtrate of Aspergillus sp. The enzyme manifested high transglycosylation activity in transxylosylation with p-nitrophenyl beta-D-xylopyranoside (PNP-X) as substrate, resulting in regio- and stereoselective synthesis of p-nitrophenyl (PNP) beta-(1-->4)-D-xylooligosaccharides with dp 2-7. All transfer products were isolated from the reaction mixtures by HPLC and their structures established by electrospray mass spectrometry and 1H and 13C NMR spectroscopy. The glycosides synthesised, beta-Xyl-1-->(4-beta-Xyl-1-->)(n)4-beta-Xyl-OC6H4NO2-p (n=1-5), were tested as chromogenic substrates for family 10 beta-xylanase from Aspergillus orizae (XynA) and family 11 beta-xylanase I from Trichoderma reesei (XynT) by reversed-phase HPLC and UV-spectroscopy techniques. The action pattern of XynA against the foregoing PNP beta-(1-->4)-D-xylooligosaccharides differed from that of XynT in that the latter released PNP mainly from short PNP xylosides (dp 2-3) while the former liberated PNP from the entire set of substrates synthesised.

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp.

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.

Purification, characterization, gene cloning and preliminary X-ray data of the exo-inulinase from Aspergillus awamori.

Extracellular exo-inulinase has been isolated from a solid-phase culture of the filamentous fungus Aspergillus awamori var. 2250. The apparent molecular mass of the monomer enzyme was 69 +/- kDa, with a pI of 4.4 and a pH optimum of 4.5. The enzyme hydrolysed the beta-(2-->1)-fructan (inulin) and beta-(2-->6)-fructan (levan) via exo-cleavage, releasing fructose. The values for the Michaelis constants K(m) and V(max) in the hydrolysis of inulin were 0.003 +/- 0.0001 mM and 175 +/- 5 micromol.min(-1).mg(-1). The same parameters in the hydrolysis of levan were 2.08 +/- 0.04 mg/ml and 1.2 +/- 0.02 micromol/min per mg, respectively. The gene and cDNA encoding the A. awamori exo-inulinase were cloned and sequenced. The amino acid sequence indicated that the protein belongs to glycoside hydrolase family 32. A surprisingly high similarity was found to fructosyltransferase from Aspergillus foetidus (90.7% on the level of the amino acid sequence), despite the fact that the latter enzyme is unable to hydrolyse inulin and levan. Crystals of the native exo-inulinase were obtained and found to belong to the orthorhombic space group P2(1)2(1)2(1) with cell parameters a=64.726 A (1A=0.1 nm), b=82.041 A and c=136.075 A. Crystals diffracted beyond 1.54 A, and useful X-ray data were collected to a resolution of 1.73 A.