Elena V. Eneyskaya

Enzymatic synthesis of β-xylanase substrates: transglycosylation reactions of the β-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.

Amylolytic activity of IgG and sIgA immunoglobulins from human milk.

BACKGROUND New natural amylolytic abzymes (Abs) for catalytically active antibodies from human milk have been identified and investigated. METHODS The amylolytic activity of human milk autoantibodies was studied by TLC and HPLC techniques analyzing the hydrolysis of maltooligosaccharides with different degrees of polymerization and of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside (EPS). IgG and sIgA fractions were isolated from human milk by affinity chromatography. After SDS-PAGE preparation of native IgG and sIgA and their renaturation, the amylolytic activity was in-gel assayed. RESULTS All electrophoretically homogeneous preparations of IgG and its Fab fragments as well as sIgA antibodies possessed alpha-amylolytic activity. The specific activities of these catalytic antibodies varied in the range from 1.83 up to 3.33 kat/kg, which is about one order of magnitude higher than that for IgGs from the sera of cancer patients. IgG and sIgA fractions showed Michaelis constants for hydrolysis of 4-nitrophenyl 4,6-O-ethylidene-alpha-D-maltoheptaoside in the range of 10(-4) M/l. Fractions of autoantibodies from different donors exhibited different modes of action in hydrolysis of maltooligosaccharides, maltose and p-nitrophenyl-alpha-D-glucopyranose. CONCLUSIONS IgG antibodies, their Fab fragments, and sIgA fractions isolated from human milk of healthy women possessed amylolytic activity in the hydrolysis of maltooligosaccharides and several artificial substrates.

Transglycosylation activity of α-d-galactosidase from Trichoderma reesei An investigation of the active site

The transglycosylation reaction catalyzed by alpha-D-galactosidase from the mycelial fungus Trichoderma reesei was studied using p-nitrophenyl alpha-D-galactopyranoside (PNPG). An aliphatic alcohol or the substrate itself can be an acceptor of the galactose residue in this reaction. The transglycosylation products were identified as alkyl galactosides in the case of alcohols or as galactobioside and galactotrioside in the case of PNPG. The transglycosylation rates follow a first-order equation with respect to the alcohol concentrations except for methanol. Affinities of some substrates were estimated from their Ki values in the reaction of the enzyme with PNPG. Transglycosylation of the substrate suggests a model for the enzyme active center. It is proposed that the active center includes two galactose-binding sites and a hydrophobic site.

Enzymatic properties of α-d-galactosidase from Trichoderma reesei☆

Abstract The kinetics of hydrolysis of a number of natural and synthetic substrates [melibiose, raffinose, stachyose, methyl α- d -galactopyranoside, and p-nitrophenyl α- d -galactopyranoside (PNPG)], catalyzed by α- d -galactosidase from the fungus Trichoderma reesei, has been studied. A number of N-acyl-α-d-galactopyranosylamines, which are competitive inhibitors of α- d -galactosidase, have been synthesized, and the KI values for these compounds have been obtained. The inhibiting properties of the competitive inhibitors of d -galactose, 1,5-anhydro- d -galactitol, and 2-deoxygalactose have been compared, and reasons for differences in KI values between these compounds have been discussed. It has been shown that α- d -galactosidase exhibits transglycosylating activity; the main product of transglycosylation in the reaction with PNPG is p-nitrophenyl 6-O-α-d-galactopyranosyl-α-d-galactopyranoside. The hydrolysis inhibition in the presence of a substrate has been shown to correlate with the substrate transglycosylation. Data of steady-state kinetics together with data of presteady-state kinetics obtained by the stop-flow method suggest that an intermediate galactosyl-enzyme complex is formed in the reaction and is of particular importance in the processes under study. A minimal kinetic scheme describing the experimental data obtained is proposed.

Mutagenesis and subsite mapping underpin the importance for substrate specificity of the aglycon subsites of glycoside hydrolase family 11 xylanases.

Glycoside hydrolase family (GH) 11 xylanase A from Bacillus subtilis (BsXynA) was subjected to site-directed mutagenesis to probe the role of aglycon active site residues with regard to activity, binding of decorated substrates and hydrolysis product profile. Targets were those amino acids identified to be important by 3D structure analysis of BsXynA in complex with substrate bound in the glycon subsites and the +1 aglycon subsite. Several aromatic residues in the aglycon subsites that make strong substrate-protein interactions and that are indispensable for enzyme activity, were also important for the specificity of the xylanase. In the +2 subsite of BsXynA, Tyr65 and Trp129 were identified as residues that are involved in the binding of decorated substrates. Most interestingly, replacement of Tyr88 by Ala in the +3 subsite created an enzyme able to produce a wider variety of hydrolysis products than wild type BsXynA. The contribution of the +3 subsite to the substrate specificity of BsXynA was established more in detail by mapping the enzyme binding site of the wild type xylanase and mutant Y88A with labelled xylo-oligosaccharides. Also, the length of the cord - a long loop flanking the aglycon subsites of GH11 xylanases - proved to impact the hydrolytic action of BsXynA. The aglycon side of the active site cleft of BsXynA, therefore, offers great potential for engineering and design of xylanases with a desired specificity.

An α-L-fucosidase from Thermus sp. with unusually broad specificity

An α-L-fucosidase (E.C. 3.2.1.51) exhibiting a wide aglycon specificity expressed in ability of cleaving α1 → 6-, α1 →3-, α1 → 4-, and α1 → 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The α-L-fucosidase hydrolyzes p-nitrophenyl α-L-fucopyranoside with Vmax of 12.0 ± 0.1 μM/min/mg and Km = 0.20 ± 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 ± 10 kDa consisting of four identical subunits with a molecular mass of 61.0 ± 0.5 kDa. The N-terminal sequence showed homology to some α-L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl α-L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the α-L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl α-L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.

Transglycosylating and hydrolytic activities of the β-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.

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.

The carbohydrate moiety of α-galactosidase from Trichoderma reesei

Abstracta-Galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by b-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: a1-6 D-mannopyranosyl- a-D-glycopyranoside and a1-6 D-glucopyranosyl- a-D-galactopyranoside and one trimer: a-D-glucopyranosyl- a1-2 D-mannopyranosyl- a1-6 D-galac-topyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo- b-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo- b- N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of a-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of a-glactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described.

Enzymatic synthesis of 4-methylumbelliferyl (1→3)-β-d-glucooligosaccharides—new substrates for β-1,3-1,4-d-glucanase

Abstract The transglycosylation reactions catalyzed by β-1,3- d -glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1→3)-β- d -gluco-oligosaccharides having the common structure [β- d -Glcp-(1→3)]n-β- d -Glcp-MeUmb, where n=1–5. The β-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)-β- d -glucan donor substrates, while MeUmb-β- d -glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [β- d -Glcp-(1→3)]2-β- d -Glcp-MeUmb (MeUmbG3) 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 β-1,3-1,4- d -glucanase from Rhodothermus marinus, which releases MeUmb from β-di- and β-triglucosides and from acetal-protected β-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG2.