• Journal of Microbiology and Biotechnology
• /
• v.11 no.4
• /
• pp.628-635
• /
• 2001

Recombinant E. coli DH5$\alpha$ harboring a dextransucrase gene (dsrB742) produced an extracellular dextransucrase in a 2% sucrose medium. The enzyme was purified by DEAE-Sepharose and Phenyl-Sepharose column chromatographies upto a 142.97-fold purification with a 11.11% recovery to near homogeneity. The enzyme had a calculated molecular mass of 168.6 kDa, which was in good agreement with the activity band of 170 kDa on a nondenaturing SDS-PAGE. An expression plasmid was constructed by inserting the dsrB742 into a pRSET expression vector. The activity after expression in E. coli BL21(DE3)pLysS increased about 6.7-fold compared to the extracellular dextransucrase from L. mesenteroides B-742CB. The expressed and purified enzyme from the clone showed similar biochemical properties (acceptor reaction, size of active dextransucrase, optimum pH, and temperature) to B-742CB dextransucrase, however, the ability to synthesize ${\alpha}$-(1$\rightarrow$3) branching decreased in comparison to that of L. mesenteroides B-742CB dextransucrase.

• Food Science and Biotechnology
• /
• v.14 no.3
• /
• pp.317-322
• /
• 2005

A simple and convenient method of immobilizing dextransucrase via an affinity interaction is described, along with the use of this system to synthesize leucrose. Dextransucrase was produced in sucrose-free medium by fermenting a constitutive mutant of Leuconostoc mesenteroides NRRL B-512F and was separated using an ultrafiltration membrane. The purified enzyme was free of dextran polymer, which previously was always found with the sucrose-induced enzyme. Therefore, it was possible to immobilize the enzyme on dextran-based resins using an affinity interaction. Sephadex G-200 was the best resin for immobilizing the dextransucrase and gave a fast flow rate through the packed column. The immobilized dextransucrase retained more than 80% of its specific activity after immobilization ($K_m\;=\;18.1\;mM$ and $k_{cat}\;=\;450\;sec^{-1}$ vs. 13.1 mM and $640\;sec^{-1}$, respectively, for the free enzyme). The immobilized dextransucrase showed improved stability over a pH range of 4.0 to 6.5 and at moderately high temperatures over $40^{\circ}C$. When immobilized dextransucrase was used to synthesize leucrose via the transfer reaction with sucrose and fructose, about 74% of the sucrose was converted into leucrose after one day, and the half-life of the enzyme activity was 15 days. Regeneration of the resin by supplementation with dextransucrase enabled the recovery of the initial activity of the system, but both the reaction and the flow rate were lower, probably owing to the accumulation of dextran inside the resin.

• Journal of Microbiology and Biotechnology
• /
• v.9 no.2
• /
• pp.219-222
• /
• 1999

A simple sequence of membrane concentration and DEAE-Cellulose chromatography has been optimized to give a purified dextransucrase from Leuconostoc mesenteroides B-512FMCM with the highest specific activity (248.8 IU/mg protein) ever reported in high yield (overall 88.7%) for dextransucrase. When there was no sucrose in the dextransucrase and the dextran reaction digest, the dextransucrase hydrolyzed glucose from dextran. The glucose was transferred to the other glucoses from dextran and formed isomaltose and isomaltodextrin. The transglycosylation efficiency of glucose from dextran was much higher with acceptors. The dextransucrase can be used for the production of various kinds (or structures) of oligosaccharides using dextran and various acceptors with almost 100% theoretical yield.

• 한국생물공학회:학술대회논문집
• /
• 2003.04a
• /
• pp.545-549
• /
• 2003

After irradiation of a cloned dextransucrase gene (dsrB742) with ultrasoft X-ray, an E. coli transformant (pDSRB742CK) was first developed for the expression of an extracellular dextransucrase, having increased activity and the synthesis of a highly branched dextran. Seven nucleotides of the parent gene (dsrB742) were changed in the nucleotide sequences of dsrB742ck. Among them, four nucleotides were changed at the ORF of dsrB742, resulting in a 30 amino acids deletion in the N-terminal of DSRB742 dextransucrase. The activity of DSRB742CK dextransucrase in culture supernatant was approximately 2.6 times higher (0.035 IU/ml) than that of the DSRB742 clone. The pDSRB742CK clone produced DSRB742CK dextransucrase when grown both on a sucrose medium (inducibly) and on a glucose medium (constitutively). The DSRB742 clone did not produce dextran constitutively on a glucose medium. DSRB742CK dextran had 15.6% branching and 2.7-times higher resistance to dextranase hydrolysis compared to DSRB742 dextran. $^{13}C-NMR$ showed that DSRB742CK dextran contained ${\alpha}-(1{\rightarrow}3)$ branch linkages that were not present in DSRB742 dextran.

• Microbiology and Biotechnology Letters
• /
• v.27 no.1
• /
• pp.86-89
• /
• 1999

The liquid of ripened Kimchi was spread on phenylethylalcohol sucrose medium and incubated at 20$^{\circ}C$ for 2 days in order to isolate Leuconostoc sp. strains. Twenty isolated colonies were identified as Leuconostoc sp. strains from sugar fermentation test. Dextransucrase activities of the isolated strains were determined and the strain J-2 showed highest activity. The morphological, cultural and physiological studies on these 5 strains showed that gram(+), spores(-), motility(-) and produced gas from glucose, acid in Whittenbury C. Only Y-1 strain produced ammonia from arginine.

• Journal of Microbiology and Biotechnology
• /
• v.1 no.3
• /
• pp.176-181
• /
• 1991

Studies on the optimum conditions for dextran production and the properties of dextransucrase (DS) were performed with Leuconostoc mesenteroides from Sikhae and Leuconostoc mesenteroides NRRL B-512(F). Dextransucrases were partially purified by lyophilization of the culture supernatant and subsequent gel chromatography on Bio-Gel A-5(m). The storage stabilities of Sikhae DS and B-512(F) DS were decreaed by the addition of dextranase. The optimum conditions for the enzyme stability were pH 5 and below $30^{\circ}C$. The B-512(F) DS lost the activity at pH 4, while Sikhae DS had 30% of the activity at the same pH. The activity of DS was decreased by EDTA. confirming the metalloprotein character of the enzymes, and was restored by the addition of calcium ions. Concanavalin A completely removed the activity of DSs, confirming the glycoprotein character of the enzymes.

• KSBB Journal
• /
• v.14 no.6
• /
• pp.707-712
• /
• 1999

We have produced new-structured oligosaccharides using mixed-enzyme reactor of dextransucrase from Leuconostoc mesenterides B-512FMCM and ${\alpha}$-amylase. When the concentrations of sucrose and starch were 10%(w/v) and 5%(w/v), respectively, the maximum yield of oligosaccharides with both dextransucrase(100U) and ${\alpha}$-amylase(1000U) was 66.4%. The activity of dextransucrase in mixed-enzyme reactor was increased about 2.5 times by acceptor reaction with starch hydrolyzates. As the activities of dextransucrase:${\alpha}$-amylase were increased from 20U:200U to 500U:5000U, the amount of polymer was increased and the yield of oligosaccharides was decreased. By the addition of sucrose into mixed-enzyme reactor following the prehydrolysis of starch with ${\alpha}$-amylase, the yield was increased up to 12% compared with that of mixed-enzyme reactor without the addition of starch hydrolyzate. New structured-oligosaccharides showed heat resistance up to 140$^{\circ}C$ and was stable in acidic condition at pH 3~6.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.12
• /
• pp.1644-1649
• /
• 2009

This study investigates the buffering effects of calcium salts in kimchi on the total acidity, microbial population, and dextransucrase activity. Calcium chloride or calcium carbonate was added to dongchimi-kimchi, a watery radish kimchi, and the effects on various biochemical attributes were analyzed. The addition of 0.1% calcium chloride produced a milder decrease in the pH after 24 days of incubation, which allowed the lactic acid bacteria to survive longer than in the control. In particular, the heterofermentative Leuconostoc genus population was 10-fold higher than that in the control. When sucrose and maltose were also added along with the calcium salts, the dextransucrase activity in the kimchi was elevated and a higher concentration of isomaltooligosaccharides was synthesized when compared with the control. Calcium chloride was determined as a better activator compound of dextransucrase than calcium carbonate, probably because of its higher solubility. Therefore, the results of this study confirm the ability of the proposed approach to modulate the kimchi fermentation process and possibly enhance the quality of kimchi based on the addition of dietary calcium salts.

• Korean Journal of Food Science and Technology
• /
• v.34 no.6
• /
• pp.1085-1090
• /
• 2002

Leuconostoc mesenteroides, the major bacterium in the initial phase of lactate-fermentation in kimchi, produces lactic acid, acetic acid, mannitol, and $CO_2$. It also secrets dextransucrase, which catalyzes the transfer reaction of glucose from sucrose to maltose, synthesizing mainly panose ($6^_2-{\alpha}-D-glucopyranosylmaltose)$, a probiotic oligosaccharide. To use the strain as a starer culture to produce high amount of panose during kimchi fermentation, we screened psychrotrophic strains showing fast growth rate at low temperature among the isolates of Leuconostoc sp. and selected two strains showing high dextransucrase activity. The strains were identified as Leuconostoc mesenteroides, which can be used as function added-starters for lactate-fermented foods.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.6
• /
• pp.1050-1058
• /
• 2008

A gene encoding a dextransucrase (dsrBCB4) that synthesizes only ${\alpha}$-1,6-linked dextran was cloned from Leuconostoc mesenteroides B-1299CB4. The coding region consisted of an open reading frame (ORF) of 4,395 bp that coded a 1,465-amino-acids protein with a molecular mass of 163,581 Da. The expressed recombinant DSRBCB4 (rDSRBCB4) synthesized oligosaccharides in the presence of maltose or isomaltose as an acceptor, plus the products included ${\alpha}$-1,6-linked glucosyl residues in addition to the maltosyl or isomaltosyl residue. Alignments of the amino acid sequence of DSRBCB4 with glucansucrases from Streptococcus and Leuconostoc identified conserved amino acid residues in the catalytic core that are critical for enzyme activity. The mutants D530N, E568Q, and D641N displayed a 98- to 10,000-fold reduction of total enzyme activity.