Dextran biosynthesis and dextransucrase production by continuous culture of Leuconostoc mesenteroides

Abstract

Leuconostoc mesenteroides NRRL B-512(F) was grown in continuous culture under conditions of energy-limited growth. The extracellular enzyme dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-glucosyltransferase EC 2.4.1.5), was not detected in glucose- or maltose-limited cultures. Under conditions of sucrose-limited growth, the enzyme activity of the cell-free culture supernatant increased with increasing dilution rate only after the critical concentration of enzyme inducer (sucrose) in the chemostat had been achieved. The appearance of fructose in the effluent of the sucrose-limited chemostat at higher dilution rates indicated that sucrose was being diverted to dextran biosynthesis. The competition between bacteria and extracellular enzyme for the common substrate sucrose represents an inefficiency in the system of enzyme production. Dextransucrase was isolated from the cell-free culture supernatant by ammonium sulfate precipitation and DEAE-cellulose chromatography. The enzyme preparation exhibited both dextran biosynthetic activity and an invertase-like activity. The biosynthetic efficiency was increased by decreasing the temperature from 30 to 10 degrees C. The enzyme was irreversibly denatured by prolonged incubation in the absence of Ca2+.

An overview of purification methods of glycoside hydrolase family 70 dextransucrase

“…Therefore, the low yield, the dilution of the dextransucrase activity and the presence of contaminating enzymes restricted the use of hydroxapatite 61 . The precipitation of enzyme from culture supernatant of L. mesenteroides B-512F was done using ammonium sulfate with egg albumin as co-precipitant 62 . The crude preparation was desalted by gel fi ltration with Sephadex G-25 and applied to DEAE-cellulose column.…”

“…The crude preparation was desalted by gel fi ltration with Sephadex G-25 and applied to DEAE-cellulose column. The enzyme was eluted with a linear gradient of NaCl 62 . When the crude extract of enzyme was applied to DEAEcellulose column, the dextransucrase activity could not be eluted from the column, even at very high ionic strengths 6 .…”

“…The enzyme dextransucrase produced by L. mesenteroides mutants in the presence of various sugars was studied for adsorbtion of enzyme to the sephadex column 62 . Partial purifi cation of enzyme was done by ammonium sulfate precipitation and passing through a column of hydoxyapatite 62 .…”

“…Partial purifi cation of enzyme was done by ammonium sulfate precipitation and passing through a column of hydoxyapatite 62 . The partially purifi ed enzyme was run through sephadex column.…”

“…The adsorption by sephadex decreased when the glucose-produced enzyme was pre-incubated with dextrans of molecular size greater than 10 kDa, as the dextran gets associated with the enzyme and reduces the affi nity of sephadex for enzyme 62 . The culture supernatant of constitutive mutant SH 3002 of L. mesenteroides B-512F grown on fructose was, put on DEAE-Sephadex A-50 column and eluted with NaCl gradient 68 .…”