Purification and characterization of two activities of the intracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299.
Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5) activity of Leuconostoc mesenteroides NRRL B-1299 cells has been purified by adsorption on hydroxyapatite, followed by chromatographies on DEAE-cellulose and DEAE-Sephadex. The enzyme activity was readily separated into two principal forms of the enzyme, I and II, by DEAE-cellulose chromatography. Both enzymes I and II were purified to an electrophoretically homogeneous state in which the relative enzyme activities reached 32-and 14-fold of the original specimen, respectively. Molecular weights were 69 000 for the enzyme I and 79 000 for the enzyme II as determined by electrophoresis in sodium dodecyl sulphate-polyacrylamide, and no subunit structure was observed. Enzyme I had an optimum pH at 6.3-6.5 and exhibited a maximal activity at 45 degrees C, while the optimum pH and temperature of enzyme II were pH 5.5-5.9 and 35-40 degrees C. The Km values of enzymes I and II were 10.7 and 250 mM, respectively. The effects of several metal ions, chemical reagents, and addition of various dextrans were also examined. Beside linear alpha-1,6-linkages the polymer synthesized by the enzyme II contained lesser amount of alpha-1,2-and alpha-1,3-linkages, which seems to be a primary characteristic of the B-1299 dextran.
D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides; D013395:Sucrose; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D006146:Guanidines; D008670:Metals; D009844:Oligosaccharides; D011134:Polysaccharides; D013395:Sucrose; D014508:Urea; D005964:Glucosyltransferases