Large-scale preparation of highly purified dextransucrase from a high-producing constitutive mutant of Leuconostoc mesenteroides B-512FMC

Conditions for the cultivation of Leuconostoc mesenteroides B-512FMC-16 (formerly L. mesenteroides B-512FMC/6HG8), a high-producing constitutive mutant of dextransucrase, has been studied in a jar fermentor by varying the amount of nitrogen (Bactopeptone and yeast extract) and the amount of carbon (glucose) with pH control. The optimum conditions were Sound to be 3.76 g l(-1) Bactopeptone, 3.76 g l(-1) yeast extract, 33.8 g l(-1) glucose with the pH controlled at 6.0 for 23 h of fermentation. The mutant produced 20-25 IU ml(-1) of the enzyme under the optimum conditions. The enzyme was concentrated using polysulfone ultrafiltration hollow fiber cartridges in the presence of 0.1% Tween 80 and I mM CaCl2. It can be concentrated not only by a hollow fiber cartridge with molecular weight cut-off of 100,000, but also by one with a particle cut-off size of 0.1 mu m. The enzyme that was concentrated using the Inter hollow fiber column gave a pure protein band with a molecular mass of 190 kDa on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the highest specific activity (183 IU mg(-1)) ever reported for B-512F dextransucrase. The role of Tween 80 and CaCl2 in the concentration procedure was studied and it was found that Tween 80 dissociates inactive enzyme aggregates and produces active enzyme molecules. CaCl2 produces aggregation; in the presence of both Tween 80 and CaCl2, active enzyme aggregates are formed that do not pass through the hollow fiber membrane, permitting concentration and purification. Using the methods described, B-512FMC dextransucrase call be purified on a 10 g or larger scale using only three operation steps. (C) 1998 Elsevier Science Inc.