• KSBB Journal
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• 제16권2호
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• pp.188-199
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• 2001

10%의 $\alpha-(1\rightarrow3)$가지 결합을 갖고 $\alpha-(1\rightarrow6)$으로 연결된 댁스트란을 합성하는 텍스트란수크라제를 coding하는 유전자 (dsCB)를 Leuconostoc mesenteroides B-742CB로부터 분리하여 염기서열과 아미노산 서열을 결정하였다. dsCB가 포항된 6 6.lkb띄 DNA fragment는 4,536 bp의 염기로 구성된 하나의 open reading 잔ame(ORF)를 가지고 있었다. 추정된 아미노산 서열은 ORF의 698벤째 nucleotide 위치에 있는 start codon (ATG)으로부터 5,223번째 위치에 있는 slop codon(TAA)까지 였다. 구조 유전자의 아마노산은 1,5087H로 구성되고 분자량의 계산값은 168.6 kDa었고 non-denatured SDS-PAGE를 이용하여 활성 band툴 분석한 결과 170 kDa이었다. pDSCB를 까지고 있는 재조합 E. coli는 2 % sucrose배치에서 세포외로 댁스트란수크라제를 생산하였으며, soluble과 insoluble 텍스트 란을 생산하였다. 텍스트란수크라체의 효소 촉매작용에 관여 하는 것으로 얄려져있는 conserved region의 아미노산 중 Asp-492를 Asparagine으로 바꾸고자 point mutation을 시도하였고, 결과로 얻어친 D492N은 돌연변이 이전의 균에 비하여 활성이 1.6배 감소함을 확인하였다.

• Food Science and Biotechnology
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• 제15권1호
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• pp.77-81
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• 2006

The dextransucrase activity of microorganisms which were identified as contributing to the fermentation of jeungpyeon made with yeast was measured. The dextran generated during fermentation was quantified and the viscosity changes were measured. The mechanism of network structure formation was clarified by observing the inside of the network structure over the fermentation periods ranging from 1 to 7 hr using scanning electron microscopy (SEM). The pH of jeungpyeon batter decreased significantly as the fermentation proceeded, whereas the viscosity increased. The identified lactic acid bacteria (LAB) were Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus pentosaceus, Tetragenococcus halophilus, and Leuconostoc mesenteroides subsp. dextranicum. The yeast was identified as Saccharomyces cerevisiae A/Tor. Pretorien. The dextransucrase extracted from those microorganisms showed high activity. On the other hand, the amount of dextran generated from the batter increased significantly beyond 2 hr of fermentation, and the viscosity increment showed a similar trend. The SEM photos showed that the most homogeneous fine network structure was observed in the batter fermented for 2 hr. Therefore, we assumed that the dextran that was generated by microorganisms during fermentation interacted with the components of the batter to increase the stability of the network structure.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.829-835
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• 2009

A dextransucrase (LcDS) gene from Leuconostoc citreum HJ-P4 has been amplified and cloned in E. coli. The LcDS gene consists of 4,431 nucleotides encoding 1,477 amino acid residues sharing 63-98% of amino acid sequence identities with other known dextransucrases from Leuc. mesenteroides. Interestingly, 0.1 mM of IPTG induction at $15^{\circ}C$ remarkably increased the LcDS productivity to 19,187 U/I culture broth, which was over 330-fold higher than that induced at $37^{\circ}C$. Optimal reaction temperature and pH of LcDS were determined as $35^{\circ}C$ and pH 5.5 in 20 mM sodium acetate buffer, respectively. Meanwhile, 0.1 mM $CaCl_2$ increased its activity to the maximum of 686 U/mg, which was 2.1-fold higher than that in the absence of calcium ion. Similar to the native Leuconostoc dextransucrase, recombinant LcDS could successfully produce a series of isomaltooligosaccharides from sucrose and maltose, on the basis of its transglycosylation activity.

• Food Science and Biotechnology
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• 제17권6호
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• pp.1391-1395
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• 2008

Leuconostoc citreum HJ-P4 is a strain isolated for kimchi fermentation with its low temperature-adapted growth feature and its high dextransucrase activity. The detailed characteristics of cell growth and dextran sucrase activities were investigated at various environmental conditions such as temperatures, pHs, salts, and raw ingredients. This strain showed almost 2-fold higher maximal cell concentration ($X_{max}$) than that of the type culture Leuconostoc mesenteroides B-512F at $10^{\circ}C$. The $X_{max}$ of the strain was maximum at pH 7 and the cell growth was inhibited by salts in a dose-dependent mode up to 7%. Addition of pepper (<6%), garlic (<10%), and ginger (<2%) in kimchi gave no inhibition effect on the growth of HJ-P4. Dextransucrase synthesized by this strain retained over 80% of its maximum activity at $10^{\circ}C$ showing a comparable cold-adapted feature to its host microbe. This culture can be used as a starter culture in the industrial kimchi production giving desirable functions and predominance at low temperature.

• Journal of Microbiology and Biotechnology
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• 제22권4호
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• pp.510-515
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• 2012

A recombinant putative dextransucrase (DexT) was produced from Leuconostoc citreum KM20 as a 160 kDa protein, but its productivity was very low (264 U/l). For optimization, we examined enzyme activity in 7 Escherichia coli strains with inducer molecules such as lactose or IPTG. E. coli BL21-CodonPlus(DE3)-RIL exhibited the highest enzyme activity with lactose. Finally, DexT activity was remarkably increased by 12-fold under the optimized culture conditions of a cell density to start induction ($OD_{600}$) of 0.95, a lactose concentration of 7.5 mM, and an induction temperature of $17^{\circ}C$. These results may effectively apply to the heterologous expression of other large DexT genes.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1075-1080
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• 2004

Dextransucrase (DSRB742) from Leuconostoc mesenteroides NRRL B-742CB is a glucosyltransferase that catalyzes the synthesis of dextran using sucrose, or the synthesis of oligosaccharides when acceptor molecules, like maltose, are present. The DSRB742 gene (dsrB742) was cloned and the properties were characterized. In order to identify critical amino acid residues, the DSRB742 amino acid sequence was aligned with glucosyltransferase sequences, and three amino acid residues reported as sucrose binding amino acids in Streptococcus glucosyltransferases were selected for site-directed mutagenesis experiments. Asp-533, Asp-536, and His-643 were independently replaced with Ala or Asn. D533A and D536A dextransucrases showed reduced dextran synthesis activities, 2.3% and 40.8% of DSRB742 dextransucrase, respectively, and D533N, D536N, H643A, end H643N dextransucrases showed complete suppression of dextran synthesis activities altogether. Additionally, D536N dextransucrase showed complete suppression of oligosaccharide synthesis activities. However, modifications at Asp-533 or at His-643 retained acceptor reaction activities in the range of 8.4% to 21.3% of DSRB742 acceptor reaction activity. Thus at least two carboxyl groups of Asp-533 and Asp-536, and His-643 as a proton donor, are essential for the catalysis process.

• Journal of Microbiology and Biotechnology
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• 제10권4호
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• pp.559-563
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• 2000

A dextransucrase gene (dsrB742) that expresses a dextransucrase to synthesize mostly ${\alpha}-1{\rightarrow}6$ linked dextran with a low amount (3-5%) of ${\alpha}-1{\rightarrow}3$ branching was cloned and sequenced from Leuconostoc mesenteroides B-742CB. The 6.1-kb PstI fragments were ligated with pGEM-3Zf(-) and transformed into E. coli $DH5{\alpha}$. The recombinant clone (pDSRB742) synthesized dextran on an agar plate containing 2% (w/v) sucrose. The dextran synthesized was hydrolyzed with Penicillium endo-dextranase. The hydrolyzate was composed of glucose, isomaltose, isomaltotriose, and branced pentasaccharide. The nucleotide sequence of dsrB742 showed one open reading frame (ORF) composed of 4,524 bp encoding dextrasnsucrase. The deduced amino acid sequence revealed a calculated molecular mass of 168.6 kDa. It also showed an activity band of 184 kKa on a non-denaturing SDS-PAGE (10%). The amino acid sequence of DSRB742 exhibited a 50% similarity with DSRA from L. mesenteroides B-1299, a 70% similarity with DSRS from L. mesenteroides B-512 (F, FMCM) and a 45-56% similarity with Streptococcal GTFs.

• 대한치과의사협회지
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• 제9권12호
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• pp.807-811
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• 1971

This report was concerned with the isolation and identification of bacterial flora in the human dental plaque and the dextransucrase activity of isolated species. The results were obtained as follows: 1. The bacterial flora, isolated from the human dental plaque, was identified as 3 species of resembling streptococci, Streptococcus salivarius strain SD-1, Streptococcus bacilli, Lactobacillus brevis strain SD-3, Lactobacillus acidophilus strain SD-2 and SD-7, resembling Staphylococcus sp, and one species of resembling Leuconostoc mesenteroides strain SD-6. 2. The dextransucrase activites of resembling Streptococcus mitis strain SD-9 and Streptococcus salivarius strain SD-1 were exhibited the highest among the isolated species of human dental plaque.

• 한국식품과학회지
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• 제38권3호
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• pp.400-407
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• 2006

26균주의 젖산균을 증편 반죽에서 분리하여 dextransucrase 활성을 측정할 결과 T. halophilus 1-12가 36.95 DSU/mg으로 가장 높았다. 이 중 Leu. mesenteroides subsp mesenteroides 2-9, T. halophilus 1-12, Leu. mesenteroides subsp dextranicum 5-13을 선별하여 starter로 이용하였다. 증편의 점도변화를 관찰한 결과 Leu. mesenteroides subsp mesenteroides 2-9를 첨가한 증편은 발효 적기인 2시간째에 첨가량이 많을수록 점도가 높아졌고 T. halophilus 1-12를 첨가한 증편의 경우는 첨가량에 따라 차이 없이 증편의 점도가 증가하였다. Leu. mesenteroides subsp dextranicum 5-13를 첨가한 증편 반죽의 경우에는 첨가량에 차이 없이 starter 균을 첨가하지 않은 증편 반죽의 발효패턴과 같은 점도 변화를 보였다. 증편반죽의 pH는 Leu. mesenteroides subsp mesenteroides 2-9 와 T. halophilus 1-12를 첨가한 경우 첨가량이 증가할수록 초기 pH도 낮았으며 발효가 진행되면서 계속 감소되는 경향을 보였다. 그러나 Leu. mesenteroides subsp dextranicum 5-13의 경우에는 첨가량에 따른 유의적인 차이가 없이 발효가 진행되면서 감소하는 패턴을 보였다. 증편의 비체적은 Leu. mesenteroides subsp dextranicum 5-13 1.0% 첨가한 군이 2.00으로 가장 높았고, T. halophilus 1-12를 1.0% 첨가한 군이 0.33으로 가장 낮았다. 증편의 단면을 관찰한 결과 Leu. mesenteroides subsp mesenteroides 2-9를 0.5 % 첨가한 것이 균일하였고, T. halophilus 1-12는 첨가량이 많을수록 기공이 거칠고 불규칙하였다. 반면 Leu. mesenteroides subsp dextranicum 5-13를 첨가한 군은 첨가량이 많을수록 기공이 균일하고 부피가 증가한 것을 볼 수 있었다. Texture는 Leu. mesenteroides subsp dextranicum 5-13를 0.5%, 1.0% 첨가한 증편이 hardnesubsp과 gumminesubsp, chewinesubsp가 대조군에 비해 매우 유의적으로 감소하였다. 관능검사를 실시한 결과 Leu. mesenteroides subsp dextranicum 5-13 0.5%를 첨가한 시료가 전체적인 기호도에서 8.500을 받아 매우 우수한 것으로 나타났다.

• Journal of Microbiology and Biotechnology
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• 제33권9호
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• pp.1228-1237
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• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.