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

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

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

• Food Science and Biotechnology
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• v.14 no.3
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• pp.317-322
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• 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
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• v.8 no.3
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• pp.287-290
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• 1998

Acarbose effectively inhibited the synthesis of dextran, and the inhibition pattern was a noncompetitive type with a $K_i$ value of 1.35 mM. It also inhibited the disproportionation reaction of dextransucrase with isomaltotriose and decreased the efficiency of the maltose acceptor reaction. Increased concentration of dextransucrase or maltose in reaction digests, however, decreased the degree of inhibition by acarbose.

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

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.545-549
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• 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
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• v.26 no.2
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• pp.179-186
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• 1998

Dextransucrase hyper-producing Leuconostoc mesenteroides B-512FMCM and dextransucrase constitutive mutants B-742CB and B-1355C catalyzed the transfer of glucose from sucrose to other carbohydrates which were present or were added to the reaction digests. When the acceptor was a maltose, gentiobiose, lactose or raffinose, there was produced a series of oligosaccharide acceptor products or single product based on the kinds of enzymes and reaction conditions. To obtain the quantitative information about the yield and the distribution of acceptor products and dextran two experimental parameters were studied: a) the ratio of acceptor to sucrose and b) the amount of enzyme at constant carbohydrate concentration (100 mM). As the amount of enzyme increased, the synthesis of acceptor products (of maltose or gentiobiose) increased, and the formation of dextran decreased. As the ratio of acceptor to sucrose increased, the amount of dextran and the number of acceptor-products decreased and the amount of acceptor-products increased. When maltose or gentiobiose was an acceptor, the glucose from sucrose was transferred to the C-6 hydroxyl group of the nonreducing-end glucose residue of accepters to give a homologous series of isomaltosyl dextrins. In case of lactose or raffinose, there was produced only one acceptor product from B-512FMCM dextransucrase reaction. In the lactose acceptor reaction, the glucose from sucrose was transferred to the C-2 hydroxyl of the reducing end glucose residue of lactose. To get a series of oligosaccharides from lactose or raffinose acceptor reaction we used B-742CB dextransucrase or B-1355C alternansucrase with 500 mM sucrose in reaction digest.

• KSBB Journal
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• v.9 no.1
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• pp.1-7
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• 1994

A qualitative study was made on the mechanism of dextran synthesis by dextransucrase. Enzymatic synthesis of dextran was experimentally studied with initial sucrose concentration from 50g/$\ell$ to 150g/$\ell$. The molecular weight distribution of synthesized dextran was measured by using on-line gel Permeation chromatographic system Sucrose was observed not to work as a primer within the range of concentration tested. At the initial sucrose concentration of 50g/$\ell$, dextran with molecular weight of medium range ($10^4-2{\times}10^6$) was synthesized due to the mass transfer limitation of sucrose. The amount of the dextran of medium range decreased with the initial sucrose concentration. Dextran was likely to be synthesized by radical chain polymerization mechanism since the dextran of medium range was not produced at higher sucrose concentrations.

• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.235-238
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• 2002

Leuconostoc mesenteroides B-512FMCM, 742CB3, 1299C의 dextransucrase들의 glycosyl기 전이 특성을 수용체 반응과 transglycosylation반응을 통해 확인하였다. 수용체 반응의 경우 10% sucrose에 수용체로 4% maltose를 첨가하여 반응시켰고 transglycosylation반응은 다른 크기, 다른 농도 그리고 다른 종류의 가지 결합의 dextran 을 합성하는 효소들을 이용하여 수행하였다. 각각의 효소들은 maltose를 이용한 수용체 반응에서 유사한 종류의 수용체 산물들을 합성한 것에 비해 세 dextransucrase들 (512FMCM, 742CB3, 1299C) 을 일정 비율로 혼합하여 maltose를 이용한 수용체 반응 결과 512FMCM 효소의 활성 비율을 줄이고 742CB3, 1299C 효소의 활성 비율을 증가시켰을 경우에는 ${\alpha}-1{\rightarrow}$3 의 가지결합이 많은 dextran 을 합성하였다. 또한, 세 가지 다른 구조의 dextran(T40, 742CB, B1299)에 100mM maltose을 수용체로 첨가해 각각의 dextransucrase(512FMCM, 742CB3, 1299C)와 transgly cosylation을 수행한 결과 1299C 효소가 세 종류의 dextran(T40, 742CB, B1299) 에 모두 가지 결합이 많은 dextran을 합성함을 확인하였다. 또한 ${\alpha}-1{\rightarrow}$6 결합으로 주로 이루어진 2%, 5% dextran(T10, T40, T7O, T500, T2000)에 dextransucrase(512FMCM, 742CB3, 1299C)를 반응시켜 기존의 dextran 보다 가지 결합이 더 많이 형성된 transglycosylation 산물을 합성하였다. 이때 maltose를 첨가했을 경우 이 수용 체에 의해 많은 ${\alpha}-1{\rightarrow}$6 가지 결합의 dextran 을 합성함을 확인하였다.