Specific method for the purification of Streptococcus mutans dextransucrase

Abstract

A convenient and rapid method for the purification of Streptococcus mutans dextransucrase is described. Affinity chromatography, on a column containing insoluble dextran purified from a culture of S. mutans 6715-49, gave an almost 300-fold purification, with 76% recovery of enzyme. Subsequent hydrophobic chromatography on butyl-agarose increased the overall enzyme purification to more than 1,000-fold, with a 65% recovery of activity. Two components of the dextransucrase activity were separated during hydrophobic chromatography. Both synthesized insoluble glucan as their major product and were capable of synthesizing soluble glucan in the presence of exogenous soluble dextran. However, the major enzyme component, which coeluted with a catalytically inert, dextran-binding protein, was greatly stimulated by exogenous soluble dextran, whereas the second enzyme component was not.

Effect of sucrose in culture media on the location of glucosyltransferase of Streptococcus mutans and cell adherence to glass surfaces

“…2) renders definite support to this hypothesis. Several other investigators have reported the binding or adsorption of GTase to insoluble glucans elaborated by extracellular GTase of S. mutans (12,17,22), but without special reference to the adherence ability of S. mutans.…”

“…McCabe and Smith (17) reported that the addition of clinical dextran to an insoluble glucan-crude GTase complex column resulted in desorption of reversibly bound GTase activity. It has also been found that addition of glucan to extracellular GTase resulted in the formation of salt-stable enzyme-glucan complexes, indicating a high affinity of GTase for glucans (4-6).…”

Search for a dextransucrase minimal motif involved in dextran binding

“…DGTIVYFDKK-GHQVFDQYIT YG 4 NGNAYYFDDA-GVMLKSGLATI YG 5 DGHQQYFDQN-GVQVKDKFVIG YG 6 -NQWFYFDGN-GHAVT-GFQTI YG 7 NGKKQYFYND-GHQSK YG 8 -DGDTFYTSATDGRLVT-GVQKI YG 9 NGITYAFDNT-GNLI YG 10 TN-QYYQLAD-GKYMLLDDSGR YG 11 DGVLRYFDQN-GEQVKDAIIVD YG 12 DTNLSYYFNATQGVAV YG 13 -KNDYFEYQ-GNWY YG 14 TD-ANYQLIK-GFKAVDD YG 15 AQGKVYQFDNN-GNAVSA Boldface type indicates conserved amino acids, x corresponds to nonconserved amino acids, and H°to hydrophobic residues. ary and tertiary structure formation.…”

“…Over the past decade several studies have revealed the role of the GBD in dextran binding. For example, the strong affinity for dextran has been used to purify glucansucrases by dextran/polyethyleneglycol phase demixion [5], or affinity chromatography onto Sephadex Ò gels [6][7][8], which consist of crosslinked dextrans. Kaseda et al [9] proposed the use of the 30 kDa GBD of Streptococcus sobrinus GTF-I glucansucrase as an affinity tag for recombinant protein purification.…”

A novel approach for purification of recombinant proteins using the dextran‐binding domain

“…Adsorbed GTF molecules were easily eluted from the beads with water‐soluble dextran in excess. Moreover, GTF from oral bacteria, Streptococcus sobrinus 6715, was purified by binding to insoluble dextrans followed by elution using soluble dextran or denaturing solvents such as guanidine hydrochloride [11,12]. These observations suggest the possibility of collecting recombinant proteins fused to DBD specifically from the cell lysates, and eluting them by addition of water‐soluble dextran.…”