Activation of dextransucrase from Leuconostoc mesenteroides by the substrate, dextran.

Kobayashi, Mikihiko; Yokoyama, Ikuko; Matsuda, Kazuo

doi:10.1271/bbb1961.48.221

Cited by 6 publications

(6 citation statements)

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“…It has been proposed that glucan is bound to the enzyme through non‐covalent links [38]. More recently, results coming from inhibition kinetic studies using various agents suggested that the glucan binding site was separated from the sucrose binding site [47–51]. This hypothesis has also been confirmed by the identification of glucansucrase sequences and by structure–function relationship studies (see ).…”

Section: Mechanism Of Glucansucrase Actionmentioning

confidence: 89%

Glucansucrases: mechanism of action and structure–function relationships

1999

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Glucansucrases are produced principally by Leuconostoc mesenteroides and oral Streptococcus species, but also by the lactic acid bacteria (Lactococci, Lactobacilli). They catalyse the synthesis of high molecular weight D-glucose polymers, named glucans, from sucrose. In the presence of efficient acceptors, they catalyse the synthesis of low molecular weight oligosaccharides. Glucosidic bond synthesis occurs without the mediation of nucleotide activated sugars and cofactors are not necessary. Glucansucrases have an industrial value because of the production of dextrans and oligosaccharides and a biological importance by their key role in the cariogenic process. They were identified more than 50 years ago. The first glucansucrase encoding gene was cloned more than 10 years ago. But the mechanism of their action remains incompletely understood. However, in order to synthesise oligosaccharides of biological interest or to develop vaccines against dental caries, elucidation of the factors determining the regiospecificity and the regioselectivity of glucansucrases is necessary. The cloning of glucansucrase encoding genes in addition to structure-function relationship studies have allowed the identification of important amino acid residues and have shown that glucansucrases are composed of two functional domains: a core region (ca. 1000 amino acids) involved in sucrose binding and splitting and a C-terminal domain (ca. 500 amino acids) composed of a series of tandem repeats involved in glucan binding. Enzymology studies have enabled different models for their action mechanism to be proposed. The use of secondary structure prediction has led to a clearer knowledge of structure-function relationships of glucansucrases. However, mainly due to the large size of these enzymes, data on the three-dimensional structure of glucansucrases (given by crystallography and modelling) remain necessary to clearly identify those features which determine function.

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Section: Mechanism Of Glucansucrase Actionmentioning

confidence: 89%

Glucansucrases: mechanism of action and structure–function relationships

1999

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“…The values determined based on glucan produced by DSRS‐His 6 , T350K, S455K, and T350K/S455K were 40.0, 29.0, 36.4, and 16.9 mM, respectively. When a Leuconostoc dextransucrase was incubated with various concentrations of dextrans at a fixed sucrose concentration, a downward biphasic plot was obtained, giving two K m values from the two limbs of curves from Lineweaver–Burk plots [25]. With 200 mM sucrose, the K m values for the clinical dextran of DSRS‐His 6 , T350K, S455K, and T350K/S455K were 3.5 and 36.4, 2.5 and 33.3, 5.7 and 28.6, and 2.3 and 40.0 μM, respectively, for sucrose cleaving activity.…”

Section: Resultsmentioning

confidence: 99%

Changes in linkage pattern of glucan products induced by substitution of Lys residues in the dextransucrase

et al. 2005

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Dextransucrase S (DSRS) is the only active glucansucrase that has been found in Leuconostoc mesenteroides NRRL B-512F strain. Native DSRS produces mainly 6-linked glucopyranosyl residue (Glcp), while Escherichia coli recombinant DSRS was observed to produce a glucan consisting of 70% 6-linked Glcp and 15% 3,6-Glcp. Lys residues were introduced at the N-terminal end of the core domain by site-directed mutagenesis. In glucans produced by the one-point mutants T350K and S455K, the amount of 6-linked Glcp was increased to about 85% of the total glucan produced, more similar in structure to native B-512F dextran. The double mutant T350K/S455K produced adhesive, water-insoluble glucan with 77% 6-linked Glcp, 8% 3,6-linked Glcp and 4% 2,6-linked Glcp. The T350K/S455K mutant exhibited a 10-fold increase in glucosyltransferase activity over those of the parental DSRS-His 6 and its T350K and S455K mutants. This is the first report demonstrating a change in the properties of a dextransucrase or a related glucosyltransferase through simple site-directed mutagenesis to create 2,6-linked Glcp.

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“…The only difference from the batch reactions (Goulas et al, 2004) is that at steady state, the sugars produced will have molecular sizes determined from the substrate input and the residence time in the reactor, instead of that being determined only from the reaction time in the limited substrate batch conditions. An important aspect of the combined reaction scheme is that dextran degradation catalyzed by dextranase will act to inhibit dextransucrase activity by breaking down the stable aggregates of the dextransucrase subunits and dextran resulting in inactivation (Robyt, 1995;Kobayashi et al, 1984Kobayashi et al, , 1986. This phenomenon, its effect on the proposed synthesis, and means of reducing it are discussed.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

Goulas

Cooper²,

Grandison

et al. 2004

Biotech & Bioengineering

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A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity.

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Activation of dextransucrase from Leuconostoc mesenteroides by the substrate, dextran.

Cited by 6 publications

References 0 publications

Glucansucrases: mechanism of action and structure–function relationships

Glucansucrases: mechanism of action and structure–function relationships

Changes in linkage pattern of glucan products induced by substitution of Lys residues in the dextransucrase

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

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