High-level production and purification of a fully active recombinant dextransucrase from Leuconostoc mesenteroides NRRL B-512F.
Recombinant expression of the dextransucrase dsrS gene by Escherichia coli was optimized to produce 5850 U L(-1) (culture) of DSR-S, corresponding to a 30-fold increase compared with previous studies. Rational deletions of the signal peptide, the beginning of the variable region and the last four repeats of the C-terminal end caused no loss of activity. This new variant successfully purified was remarkably stable. With a k(cat) of 584 s(-1), it is the most efficient recombinant glucansucrase described to date. The synthesized polymer possesses more than 95% of alpha-1,6 links, like the dextran produced by the native enzyme, and innovative gel properties were obtained.
Moulis
Claire
C
Laboratoire de Biotechnologies - Bioprocédés, UMR CNRS 5504, UMR INRA 792, INSA, Toulouse, France.
Arcache
Audrey
A
Escalier
Pierre-Claude
PC
Rinaudo
Marguerite
M
Monsan
Pierre
P
Remaud-Simeon
Magali
M
Potocki-Veronese
Gabrielle
G
D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002846:Chromatography, Affinity; D003911:Dextrans / Q000096:biosynthesis* / Q000737:chemistry; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D014783:Viscosity
D003911:Dextrans; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C020025:dextransucrase