Hooked on α-d-galactosidases: from biomedicine to enzymatic synthesis

<div><p></p><p>α-d-Galactosidases (EC 3.2.1.22) are enzymes employed in a number of useful bio-based applications. We have depicted a comprehensive general survey of α-d-galactosidases from different origin with special emphasis on marine example(s). The structures of natural α-galactosyl containing compounds are described. In addition to 3D structures and mechanisms of action of α-d-galactosidases, different sources, natural function and genetic regulation are also covered. Finally, hydrolytic and synthetic exploitations as free or immobilized biocatalysts are reviewed. Interest in the synthetic aspects during the next years is anticipated for access to important small molecules by green technology with an emphasis on alternative selectivity of this class of enzymes from different sources.</p></div

Polaribacter staleyi sp. nov., a polysaccharide-degrading marine bacterium isolated from the red alga Ahnfeltia tobuchiensis

A Gram-stain-negative, rod-shaped, motile by gliding and yellow-pigmented bacterium, designated strain 10Alg 139T, was isolated from the Pacific red alga Ahnfeltiato buchiensis. The phylogenetic analysis based on 16S rRNA gene sequences showed that the novel strain belonged to the genus Polaribacter , a member of the family Flavobacteriaceae , the phylum Bacteroidetes , with highest sequence similarity to Polaribacter butkevichii KMM 3938T (99.3 %) and 93.3–98.6 % to other recognized Polaribacter species. The prevalent fatty acids of strain 10Alg 139T were iso-C15 : 0 3-OH, C15 : 0 3-OH, iso-C15:0, iso-C13 : 0, C15 : 0 and C15 : 1ω6c. The polar lipid profile consisted of the major lipids phosphatidylethanolamine, two unidentified aminolipids and four unidentified lipids. The main respiratory quinone was menaquinone 6. The DNA G+C content of the type strain is 31.8 mol%. The new isolate and the type strains of recognized species of the genus Polaribacter were readily distinguished based on a number of phenotypic characteristics. A combination of the genotypic and phenotypic data showed that the isolate from alga represents a novel species of the genus Polaribacter , for which the name Polaribacter staleyi sp. nov. is proposed. The type strain is 10Alg 139T (=KCTC 52773T=KMM 6729T)

Stereochemical course of hydrolytic reaction catalyzed by alpha-galactosidase from cold adaptable marine bacterium of genus Pseudoalteromonas

The recombinant α-galactosidase of the marine bacterium (α-PsGal) was synthesized with the use of the plasmid 40Gal, consisting of plasmid pET-40b (+) (Novagen) and the gene corresponding to the open reading frame of the mature α-galactosidase of marine bacterium Pseudoalteromonas sp. KMM 701, transformed into the E. coli Rosetta(DE3) cells. In order to understand the mechanism of action, the stereochemistry of hydrolysis of 4-nitrophenyl α-D-galactopyranoside (4-NPGP) by α-PsGal was measured by 1H NMR spectroscopy. The kinetics of formation of α- and β-anomer of galactose showed that α-anomer initially formed and accumulated, and then an appreciable amount of β-anomer appeared as a result of mutarotation. The data clearly show that the enzymatic hydrolysis of 4-NPGP proceeds with the retention of anomeric configuration, probably, due to a double displacement mechanism of reaction

Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel <em>Crenomytilus grayanus</em>

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with β-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties