Production, purification, and properties of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.
The production of dextransucrase from Leuconostoc mesenteroides NRRL B-512F was stimulated 2-fold by the addition of 0.005% of calcium chloride to the medium; levansucrase levels were unaffected. Dextransucrase was purified by concentration and dialysis of the culture supernatant with a Bio-Fiber 80 miniplant, and by treatment with dextranase followed by chromatography on Bio-Gel A-Fm. A 240-fold purification, with a specific activity of 53 U/mg, was obtained. Contaminating enzyme activities of levansucrase, invertase, dextranase, glucosidase, and sucrose phosphorylase were decreased to non-detectable levels. Poly(acrylamide)-gel electrophoresis of the purified enzyme showed only two protein bands, both of which had dextransucrase activity. These bands also gave a carbohydrate stain, indicating that the dextransucrase could be a glycoprotein. Acid hydrolysis, followed by paper chromatography, of the purified enzyme showed that the major carbohydrate was mannose. Concanavalin A completely removed dextransucrase activity from solution, confirming the mannoglycoprotein character of the enzyme. Dextransucrase activity was not altered by the addition of 0.008-4 mg/ml of dextran, but its storage stability was increased by the addition of 4 mg/ml of dextran. As previously shown by others, the activity of dextransucrase was decreased by EDTA, and was restored by the addition of calcium ions. Zinc, cadmium, lead, mercury, and copper ions were inhibitory to various degrees.
D002122:Calcium Chloride / Q000494:pharmacology; D003911:Dextrans; D004492:Edetic Acid / Q000494:pharmacology; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D007934:Leuconostoc / Q000187:drug effects / Q000201:enzymology*; D008722:Methods; D013395:Sucrose
D003911:Dextrans; D013395:Sucrose; D004492:Edetic Acid; D005964:Glucosyltransferases; C020025:dextransucrase; D002122:Calcium Chloride