Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299.
Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.
D002413:Cations, Divalent; D003911:Dextrans / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006146:Guanidines / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D011489:Protein Denaturation; D013393:Sucrase / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D002413:Cations, Divalent; D003911:Dextrans; D006146:Guanidines; D014508:Urea; D004492:Edetic Acid; D013393:Sucrase