High-level production and purification of a fully active recombinant dextransucrase fromLeuconostoc mesenteroidesNRRL B-512F

Abstract

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.

Search for a dextransucrase minimal motif involved in dextran binding

“…Escherichia coli TOP10 (Invitrogen) was used as host for both cloning and recombinant protein expression and purification. Transformation and cultivation of E. coli transformants were performed as described before [12]. Cells were concentrated to an OD 600 nm of 80 in 50 mM potassium phosphate buffer pH 7.2 supplemented with 0.05 g/l of CaCl 2 and 1 mM PMSF, and then sonicated.…”

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

“…Soluble dextrans have well documented wide applications in industry [16][17][18]39 . The dextransucrase activity of L. mesenteroides was used in acceptor reactions with maltose in order to obtain oligosaccharides with α-1,2 glycosidic bonds 19,40 .…”

“…The induction in Bacillus megaterium, produced very high specifi c (362 Ug -1 ) and volumetric (28,600 Ul -1 ) activity of dextran free dextransucrase 20 . A recent report describes the production of recombinant L. mesenteroides B-512F dextransucrase with 6 His tag at the C-terminal and its expression in E. coli TOP10 cells 16 . The recombinant enzyme was purifi ed by affi nity chromatography using Ni 2+ ion immobilized column.…”

“…A wide variety of techniques have been used for the purifi cation of Leuconostoc mesenteroides dextransucrase [6][7][8][9][10][11][12][13][14][15] . A recent report describes the production of recombinant dextransucrase of L. mesenteroides B-512F with a histidine tag at the C-terminal using E. coli TOP10 cells for expression and purifi cation of the recombinant enzyme by affi nity chromatography using Ni 2+ ion immobilized column 16 .…”

Role of the C-terminal region of dextransucrase from Leuconostoc mesenteroides IBT-PQ in cell anchoring

“…The expression was carried out in E. coli TOP10, after optimization of arabinose concentration. The Thio-DsrS-His was produced using the construction and procedure reported by Moulis et al (2006b), and was kindly provided by Professor Magali Remaud-Simeon, INSA-Toulouse, France.…”