Substrate Binding Sites of<i>Leuconostoc</i>Dextransucrase Evaluated by Inhibition Kinetics

Kobayashi, Mikihiko; Yokoyama, Ikuko; Matsuda, Kazuo

doi:10.1080/00021369.1986.10867780

Cited by 5 publications

(6 citation statements)

References 7 publications

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“…7) Compared with these known inhibitors, the K j of CI-7, 0.25 mM, was satisfactory and this inhibition was ascribed not to any particular functional groups of the analog structure, but to the cyclic tx-I,6-linked structure. Because cyclodextran had no free hydroxyl group at the C-6 position, dextran sucrase could not transfer the glucosyl group to form new tx-I,6-linkages.…”

Section: Discussionmentioning

confidence: 93%

“…7 ,13) Inhibition kinetic analysis with a series of substrate analogs was useful to prove the different location of donor and acceptor sites of dextransucrase. 7 ) Among the inhibitors tested in the above two studies, EDTA was a potent inhibitors, i.e., noncompetitive inhibition with K/s of 1.1 mM for higher and 0.2mM for lower sucrose concentrations. 7, 13) This may be related to the activation of transferase by Ca 2 +.…”

Section: Discussionmentioning

confidence: 99%

“…7) Since cyclodextrans are composed of (X-I ,6-linked glucosyl residues, these cyclic sugars had a function as dextran analogs. Therefore, it was interesting to evaluate whether these cyclodextrans inhibited the sucrase activity or transferase activity.…”

Section: Inhibition Of Dextran Synthesismentioning

confidence: 99%

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Inhibition of Dextran and Mutan Synthesis by Cycloisomaltooligosaccharides

Kobayashi

Funane

Oguma

1995

Bioscience, Biotechnology, and Biochemistry

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Section: Discussionmentioning

confidence: 93%

Section: Discussionmentioning

confidence: 99%

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Inhibition of Dextran and Mutan Synthesis by Cycloisomaltooligosaccharides

Kobayashi

Funane

Oguma

1995

Bioscience, Biotechnology, and Biochemistry

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“…A single chain, primer-dependent mechanism of elongation was proposed for dextran elongation, as it was generally accepted for polysaccharide biosynthesis at this period (2,16,17). Working with the L. mesenteroides NRRL B-512F dextransucrase (DSR-S), Tsuchiya et al (16) proposed thus that in absence of exogenous co-substrate, impurities in the crude dextransucrase preparation or sucrose itself could act in the role of primer.…”

mentioning

confidence: 99%

Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases

Moulis¹,

Joucla²,

Harrison³

et al. 2006

J. Biol. Chem.

121

128

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Glucan formation catalyzed by two GH-family 70 enzymes, Leuconostoc mesenteroides NRRL B-512F dextransucrase and L. mesenteroides NRRL B-1355 alternansucrase, was investigated by combining biochemical and kinetic characterization of the recombinant enzymes and their respective products. Using HPAEC analysis, we showed that two molecules act as initiator of polymerization: sucrose itself and glucose produced by hydrolysis, the latter being preferred when produced in sufficient amounts. Then, elongation occurs by transfer of the glucosyl residue coming from sucrose to the non-reducing end of initially formed products. Dextransucrase preferentially produces an isomaltooligosaccharide series, whose concentration is always low because of the high ability of these products to be elongated and form high molecular weight dextran. Compared with dextransucrase, alternansucrase has a broader specificity. It produces a myriad of oligosaccharides with various ␣-1,3 and/or ␣-1,6 links in early reaction stages. Only some of them are further elongated. Overall alternan polymer is smaller in size than dextran. In dextransucrase, the A repeats often found in C-terminal domain of GH family 70 were found to play a major role in efficient dextran elongation. Their truncation result in an enzyme much less efficient to catalyze high molecular weight polymer formation. It is thus proposed that, in dextransucrase, the A repeats define anchoring zones for the growing chains, favoring their elongation. Based on these results, a semi-processive mechanism involving only one active site and an elongation by the non-reducing end is proposed for the GH-family 70 glucansucrases. Glucansucrases from Glycoside-Hydrolase (GH)2 -family 70 (EC. 2.4.1.5) are extracellular enzymes produced by lactic acid bacteria of the genus Leuconostoc, Streptococcus, or Lactobacillus (1). From sucrose, they catalyze the synthesis of high molecular weight glucans. They can also produce oligosaccharides or glucoconjugates by a transglucosylation reaction from the sucrose donor to an exogenous acceptor, and this so called "acceptor reaction" occurs at the cost of polymer synthesis (2, 3). An interesting diversity exists in the GH-family 70, where there are enzymes able to synthesize all the types of glucosidic linkages, namely ␣-1,2; ␣-1,3; ␣-1,4; or ␣-1,6 glucosidic bonds. So, depending on the enzyme specificity, a wide range of glucans can be produced, varying in terms of size, structure, degree of branches and spatial arrangements.Primary structures of at least 44 different glucansucrases are now available in GH-family 70.3 With an average predicted molecular mass of more than 160,000 Da, they all show the same organization consisting of a variable region at the N terminus, a conserved catalytic domain, and a C-terminal domain typically containing a series of homologous repeating units. In a number of streptococcal glucansucrases, as well as for the L. mesenteroides NRRL B-512F dextransucrase, the repeats have been demonstrated to play a role in enzyme glucan bindi...

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“…In the 40's, the synthesis of dextran from cell-free dextransucrase was carried out for the first time (Hehre,1946). Subsequently, different medium compositions and fermentations conditions have been used (Kobayashi, 1986;Lawford, 1979;López, 1980). Several fermentation methods: batch, fedbatch and continuous culture, as well as the different criteria of aeration have been employed.…”

mentioning

confidence: 99%

Scale-up of dextransucrase production by Leuconostoc mesenteroides in fed batch fermentation

Michelena

Martínez

Bell

et al. 2003

Braz. arch. biol. technol.

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Fed batch fermentation was carried out for the dextransucrase enzyme production from Leuconostoc mesenteroides and the production was scale-up using oxygen transfer criteriuom. It was found that in 5 L vessel fermentation capacity, the best agitation speed was 225 min-1 and aeration rate was 0.15 vvm, obtaining dextransucrase activity of 127 DSU/mL.. The maximum enzyme production velocity coincide with the maximum growth velocity between 6 and 7 h of fermentation, which confirmed that dextransucrase production was associated with microbial growth. High enzyme yields were achieved during scale up based on oxygen transfer rate.

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Substrate Binding Sites ofLeuconostocDextransucrase Evaluated by Inhibition Kinetics

Cited by 5 publications

References 7 publications

Inhibition of Dextran and Mutan Synthesis by Cycloisomaltooligosaccharides

Inhibition of Dextran and Mutan Synthesis by Cycloisomaltooligosaccharides

Understanding the Polymerization Mechanism of Glycoside-Hydrolase Family 70 Glucansucrases

Scale-up of dextransucrase production by Leuconostoc mesenteroides in fed batch fermentation

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