Purification, surfactant stabilization, molecular weight studies, and divalent metal ion kinetics of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

dc.contributor.author Miller, Arthur
dc.contributor.department Biochemistry, Biophysics and Molecular Biology
dc.date 2018-08-15T06:06:23.000
dc.date.accessioned 2020-07-02T06:08:49Z
dc.date.available 2020-07-02T06:08:49Z
dc.date.copyright Tue Jan 01 00:00:00 UTC 1985
dc.date.issued 1985
dc.description.abstract <p>Dextransucrase from Leuconostoc mesenteroides NRRL B-512F, an enzyme that synthesizes an (alpha)-(1(--->)6)-linked glucan with (alpha)-(1(--->)3)-linked branches from the glucosyl moiety of sucrose, was stabilized against thermal inactivation and against inactivation caused by dilution and manipulation by the addition of nonionic detergents, mild ionic detergents, poly(ethylene glycol), or high-molecular-weight dextran;Dextransucrase was purified by a sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and DEAE-Trisacryl M chromatography. The purified enzyme had a specific activity of 170 U/mg protein and no detectable carbohydrate;The enzyme consisted of two forms of differing molecular weight. A 177-kDa form was biosynthesized initially and converted during aging to a 158-kDa form by an unknown but probably proteolytic mechanism. By using radiation inactivation and SDS polyacrylamide gel electrophoresis, it was determined that dextran is not synthesized by the cooperative action of two or more peptides (subunits), but by single dextransucrase peptides of 177 and 158 kDa;The sucrose binding site of B-512F dextransucrase was an inhibitory divalent metal ion site also, with a higher affinity for Zn('2+), Ni('2+), and Co('2+) than for Ca('2+). A second metal ion site with a high affinity for Ca('2+) was found. Ca('2+) was an activator at this site, whereas Zn('2+), Ni('2+), and Co('2+) were inhibitors. Ca('2+) was a nonessential activator; i.e., it was not required for dextran synthesis;The periodic acid-Schiff stain, when used to detect glycansucrase activity on polyacrylamide gels, stained many proteins that were neither glycoproteins nor active glycansucrases. This artifactual staining, which increased during the storage of stained gels, could be prevented by proteolytically removing all protein from the gels before staining, or considerably slowed by the action of crosslinking agents on the stained gels.</p>
dc.format.mimetype application/pdf
dc.identifier archive/lib.dr.iastate.edu/rtd/8729/
dc.identifier.articleid 9728
dc.identifier.contextkey 6343487
dc.identifier.doi https://doi.org/10.31274/rtd-180813-8707
dc.identifier.s3bucket isulib-bepress-aws-west
dc.identifier.submissionpath rtd/8729
dc.identifier.uri https://dr.lib.iastate.edu/handle/20.500.12876/81749
dc.language.iso en
dc.source.bitstream archive/lib.dr.iastate.edu/rtd/8729/r_8604499.pdf|||Sat Jan 15 02:16:09 UTC 2022
dc.subject.disciplines Biochemistry
dc.subject.keywords Biochemistry and biophysics
dc.subject.keywords Biochemistry
dc.title Purification, surfactant stabilization, molecular weight studies, and divalent metal ion kinetics of dextransucrase from Leuconostoc mesenteroides NRRL B-512F
dc.type article
dc.type.genre dissertation
dspace.entity.type Publication
relation.isOrgUnitOfPublication faf0a6cb-16ca-421c-8f48-9fbbd7bc3747
thesis.degree.level dissertation
thesis.degree.name Doctor of Philosophy
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