Kirill N. Neustroev

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 IgM and IgG antibodies from patients with multiple sclerosis

IgG and IgM antibodies from the sera of patients with multiple sclerosis (MS) were found to possess amylolytic activity hydrolyzing alpha-(1-->4)-glucosyl linkages of maltooligosaccharides, glycogen, and several artificial substrates. Individual IgM fractions isolated from 54 analyzed patients with the clinically definite diagnoses of MS had approximately three orders of magnitude higher specific amylolytic activity than that for healthy donors, whereas IgG from only a few patients had high amylolytic activity. Strict criteria were used to prove that the amylolytic activity of IgMs and IgGs is their intrinsic property and is not due to any enzyme contamination. Fab fragments produced from IgM and IgG fractions of the MS patients displayed the same amylolytic activity. IgMs from various patients demonstrated different modes of action in hydrolyzing maltooligosaccharides.

Enzymatic properties of α-galactosidase from Trichoderma reesei in the hydrolysis of galactooligosaccharides

Enzymatic properties of the α-galactosidase (α-galactoside galactohydrolase, EC 3.2.1.22) from Trichoderma reesei in the hydrolysis of natural galactooligosaccharides and α-O-methyl D-galactopyranoside have been investigated in a wide range of substrate concentrations. The hydrolyses of α-O-methyl D-galactopyranoside and melibiose were inhibited by substrate at concentrations higher than 100 mM while in the hydrolysis of raffinose and stachyose such an effect was not observed. It was shown by 1H and 13C NMR spectroscopy and HPLC techniques that inhibition by the excess of α-O-methyl D-galactopyranoside or melibiose strongly correlated with formation of transglycosylation products. The product of autocondensation reaction with α-O-methyl D-galactopyranoside as substrate was found to be α-O-methyl galactopyranosyl-1,6-D-galactopyranoside. The stereochemical course of stachyose hydrolysis has been determined. The enzyme catalyses the hydrolysis with retention of anomeric configuration and is assumed to operate via a double displacement mechanism. Simultaneous hydrolysis of stachyose and raffinose effected by the α-D-galactosidase was studied by direct 1H NMR measurements. Cleavage of the terminal galactose residue of stachyose was found to be the rate-limiting step. Formation constants of enzyme-substrate complex for stachyose and raffinose were calculated. The suggested model can be used for simulating the two-substrate system and predicting the extent of stachyose hydrolysis.

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.

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.

Enzymatic activity and β-galactomannan binding property of β-mannosidase from Trichoderm reesei

An extracellular 105-kDa β-mannosidase (β-d-mannoside-mannohydrolase, E.C. 3.2.1.25) was purified to homogeneity from culture filtrate of Trichoderma reesei. Specific activity of the β-mannosidase toward p-nitrophenyl-β-d-mannopyranoside was 3.2 U/mg at the optimal pH 3.5 (Km = 0.12 mM, kcat = 2.95 × 10−3 μmol min/μg. An additional β-galactomannan (GM) binding site of the enzyme was found on the basis of kinetic studies. The enzyme GM dissociation constant (KD) was 1.21 mg/ml. β-1,4-mannooligosaccharides inhibited the binding of the enzyme to galactomannan. The inhibition constant of the sorption decreased with increasing of the β-1,4-mannooligosaccharide length. Mannose, the competitive inhibitor of the β-mannosidase in hydrolysis of p-nitrophenyl-β-d-mannopyranoside, did not inhibit sorption of the enzyme on β-GM. Chitin, xylan, raw starch, and microcrystalline cellulose had no affinity to the β-mannosidase. The enzyme hydrolyzed β-1,4-mannooligosaccharides with the rate depending on the chain length and liberated mannose from soluble and insoluble fractions of β-GM from locust beans with initial rates of 0.3 and 0.05 μmol min/ml U, respectively.

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.

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

Abstract 1- O -Acetyl-β- d -galactopyranose (AcGal), a new substrate for β-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-β- d -galactopyranose catalyzed by the β- 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 β-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the β- d -galactosidase in the reaction of AcGal and methyl β- d -galactopyranoside ( 1 ) as substrates was investigated by 1 H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was β- d -galactopyranosyl-(1→6)-1- O -acetyl-β- d -galactopyranose (with a yield of ∼70%). In the case of 1 as a substrate, the main transglycosylation product was methyl β- d -galactopyranosyl-(1→6)-β- d -galactopyranoside. Methyl β- d -galactopyranosyl-(1→3)-β- d -galactopyranoside was found to be minor product in the latter reaction.

A model for cleavage ofO-glycosidic bonds in glycoproteins

The present work investigated the possibility of cleavage of α-linkages between mannose or galactose and serine/threonine residues by α-mannosidase and α-galactosidase. The study was carried out initially with model synthetic compounds imitating theO-glycosidic bond in glycoproteins, and further with glucoamylase. It was shown that α-mannosidase and α-galactosidase can hydrolyse these linkages after proteolytic digestion of glucosamylase.