ABSTRACT
The review covers recent literature on the ocean as both a source of biotechnological tools and as a source of bio-inspired materials. The emphasis is on marine biomacromolecules namely hyaluronic acid, chitin and chitosan, peptides, collagen, enzymes, polysaccharides from algae, and secondary metabolites like mycosporines. Their specific biological, physicochemical and structural properties together with relevant applications in biocomposite materials have been included. Additionally, it refers to the marine organisms as source of inspiration for the design and development of sustainable and functional (bio)materials. Marine biological functions that mimic reef fish mucus, marine adhesives and structural colouration are explained.
ABSTRACT
The dextransucrase DSR-OK from the Gram-positive bacterium Oenococcus kitaharae DSM17330 produces a dextran of the highest molar mass reported to date (â¼109 g/mol). In this study, we selected a recombinant form, DSR-OKΔ1, to identify molecular determinants involved in the sugar polymerization mechanism and that confer its ability to produce a very-high-molar-mass polymer. In domain V of DSR-OK, we identified seven putative sugar-binding pockets characteristic of glycoside hydrolase 70 (GH70) glucansucrases that are known to be involved in glucan binding. We investigated their role in polymer synthesis through several approaches, including monitoring of dextran synthesis, affinity assays, sugar binding pocket deletions, site-directed mutagenesis, and construction of chimeric enzymes. Substitution of only two stacking aromatic residues in two consecutive sugar-binding pockets (variant DSR-OKΔ1-Y1162A-F1228A) induced quasi-complete loss of very-high-molar-mass dextran synthesis, resulting in production of only 10-13 kg/mol polymers. Moreover, the double mutation completely switched the semiprocessive mode of DSR-OKΔ1 toward a distributive one, highlighting the strong influence of these pockets on enzyme processivity. Finally, the position of each pocket relative to the active site also appeared to be important for polymer elongation. We propose that sugar-binding pockets spatially closer to the catalytic domain play a major role in the control of processivity. A deep structural characterization, if possible with large-molar-mass sugar ligands, would allow confirming this hypothesis.
Subject(s)
Bacterial Proteins/metabolism , Dextrans/metabolism , Glucosyltransferases/metabolism , Oenococcus/metabolism , Bacterial Proteins/chemistry , Binding Sites , Biosynthetic Pathways , Glucosyltransferases/chemistry , Models, Molecular , Oenococcus/chemistry , Protein DomainsABSTRACT
The factors that define the resulting polymer length of distributive polymerases are poorly understood. Here, starting from the crystal structure of the dextransucrase DSR-M in complex with an isomaltotetraose, we define different anchoring points for the incoming acceptor. Mutation of one of these, Trp624, decreases the catalytic rate of the enzyme but equally skews the size distribution of the resulting dextran chains toward shorter chains. Nuclear magnetic resonance analysis shows that this mutation influences both the dynamics of the active site and the water accessibility. Monte Carlo simulation of the elongation process allows interpretation of these results in terms of enhanced futile encounters, whereby the less effective binding increases the pool of effective seeds for the dextran chains and thereby directly determines the length distribution of the final polymers.
Subject(s)
Dextrans/chemistry , Glucosyltransferases/chemistry , Glucosyltransferases/metabolism , Biocatalysis , Catalytic Domain , Escherichia coli/genetics , Glucosyltransferases/genetics , Leuconostoc/enzymology , Models, Chemical , Molecular Structure , Monte Carlo Method , Mutation , Oligosaccharides/chemistry , Protein EngineeringABSTRACT
A gene encoding a novel dextransucrase was identified in the genome of Oenococcus kitaharae DSM17330 and cloned into E. coli. With a kcat of 691s-1 and a half-life time of 111h at 30°C, the resulting recombinant enzyme -named DSR-OK- stands as one of the most efficient and stable dextransucrase characterized to date. From sucrose, this enzyme catalyzes the synthesis of a quasi linear dextran with a molar mass higher than 1×109g·mol-1 that presents uncommon rheological properties such as a higher viscosity than that of the most industrially used dextran from L. mesenteroides NRRL-B-512F, a yield stress that was never described before for any type of dextran, as well as a gel-like structure. All these properties open the way to a vast array of new applications in health, food/feed, bulk or fine chemicals fields.
Subject(s)
Dextrans/biosynthesis , Glucosyltransferases/metabolism , Oenococcus/enzymology , Recombinant Proteins/metabolism , Amino Acid Sequence , Biocatalysis , Carbohydrate Conformation , Databases, Protein , Glucosyltransferases/genetics , Oenococcus/genetics , Recombinant Proteins/genetics , Sucrose/chemistry , Sucrose/metabolism , ViscosityABSTRACT
Leuconostoc citreumNRRL B-742 has been known for years to produce a highly α-(1â3)-branched dextran for which the synthesis had never been elucidated. In this work a gene coding for a putative α-transglucosylase of the GH70 family was identified in the reported genome of this bacteria and functionally characterized. From sucrose alone, the corresponding recombinant protein, named BRS-B, mainly catalyzed sucrose hydrolysis and leucrose synthesis. However, in the presence of sucrose and a dextran acceptor, the enzyme efficiently transferred the glucosyl residue from sucrose to linear α-(1â6) dextrans through the specific formation of α-(1â3) linkages. To date, BRS-B is the first reported α-(1â3) branching sucrase. Using a suitable sucrose/dextran ratio, a comb-like dextran with 50% of α-(1â3) branching was synthesized, suggesting that BRS-B is likely involved in the comb-like dextran produced byL. citreumNRRL B-742. In addition, data mining based on the search for specific sequence motifs allowed the identification of two genes putatively coding for branching sucrases in the genome ofLeuconostoc fallaxKCTC3537 andLactobacillus kunkeeiEFB6. Biochemical characterization of the corresponding recombinant enzymes confirmed their branching specificity, revealing that branching sucrases are not only found inL. citreumspecies. According to phylogenetic analyses, these enzymes are proposed to constitute a new subgroup of the GH70 family.