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.

Human milk antibodies with polysaccharide kinase activity.

It was shown for the first time that a small fraction of milk secretory IgA (sIgA) is tightly bound to oligosaccharides (oligoSACs) and polysaccharides (polySACs). The ability of sIgA to phosphorylate oligo- and polysaccharides was shown to be an intrinsic property of this antibody. In contrast to known kinases, sIgAs with polysaccharide kinase activity can transfer phosphoryl group to oligo- and polysaccharides not only from [gamma-(32)P]ATP but can also use [(32)P]orthophosphate as a substrate of phosphorylation reaction.

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.

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.

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.

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.

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.

Multiple enzymic activities of human milk lactoferrin.

Lactoferrin (LF) is a Fe3+-binding glycoprotein, first recognized in milk and then in other human epithelial secretions and barrier fluids. Many different functions have been attributed to LF, including protection from iron-induced lipid peroxidation, immunomodulation and cell growth regulation, DNA binding, and transcriptional activation. Its physiological role is still unclear, but it has been suggested to be responsible for primary defense against microbial and viral infection.

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.

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.

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.

Biochemical and genetic characterization of a novel enzyme of pentitol metabolism: D-arabitol-phosphate dehydrogenase.

An enzyme with a specificity that has not been described previously, D-arabitol-phosphate dehydrogenase (APDH), has been purified from cell lysate of Enterococcus avium. SDS/PAGE indicated that the enzyme had a molecular mass of 41+/-2 kDa, whereas a molecular mass of 160+/-5 kDa was observed under non-denaturing conditions, implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have a narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into xylulose 5-phosphate and ribulose 5-phosphate, respectively, in the oxidative reaction.

Structural insights into the beta-mannosidase from T. reesei obtained by synchrotron small-angle X-ray solution scattering enhanced by X-ray crystallography.

A molecular envelope of the beta-mannosidase from Trichoderma reesei has been obtained by combined use of solution small-angle X-ray scattering (SAXS) and protein crystallography. Crystallographic data at 4 A resolution have been used to enhance informational content of the SAXS data and to obtain an independent, more detailed protein shape. The phased molecular replacement technique using a low resolution SAXS model, building, and refinement of a free atom model has been employed successfully.

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.

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.