• Journal of Microbiology and Biotechnology
• /
• 제26권3호
• /
• pp.493-497
• /
• 2016

Glucosyl rubusosides were synthesized by two dextransucrases. LcDexT was obtained from Leuconosotoc citreum, that LlDexT was obtained from Leuconostoc lactis. LcDexT and LlDexT regioselectively transferred a glucosyl residue to the 13-O-glucosyl moiety of rubusoside with high yield of 59-66% as analyzed by TLC and HPLC. Evaluation of the sweetness of these glucosyl rubusosides showed that their quality of taste, in particular, was superior to that of rubusoside. These results indicate that transglucosylation at the 13-O-glucosyl moiety of rubusoside by different regioselective dextransucrases can be applicable for increasing its sweetness and quality of taste.

• 한국미생물·생명공학회지
• /
• 제27권4호
• /
• pp.304-310
• /
• 1999

We have developed a new process for the production of new structure oligosaccharides using the mixed-culture fementation of Lipomyces starkeyi KSM22 and leuconostoc mesenteroides B-512FMCM.L.starkeyi KSM22 produces a novel DXAMase(an enzyme containing both dextranase and amylase activities). It hydrolyzes the soluble starch and dextran. The hydrolyzates were used as acceptors for dextransucrase of L.mesenteroides to synthesize the new oligosaccharides(NOS). In fermentation, as the concentration of sucrose was increased from 9%(w/v) to 15%(w/v), the yields of dextran(sum of dextran I, MW=66kD, and dextran II, MW=21kD) was increased from 12.7% to 42.5%, and NOS was increased from 3.9% to 5.2% of the theoretical, respectively. The NOS of dp(degree of polymerization) 5 and over was increased from 33.1% to 58.3% of the total NOS. The NOS showed heat resistant up to 12$0^{\circ}C$ and was stable at pHs ranged from 2 to 6. The NOS decreased the pH changes in the culture of S. mutans, and also showed inhibitory effects on the growth of S. aureus or S. typhimurium.

• 한국미생물생명공학회:학술대회논문집
• /
• 한국미생물생명공학회 2004년도 Annual Meeting BioExibition International Symposium
• /
• pp.215-218
• /
• 2004

Dextrans make up a class of polysaccharides that are D-glucans of various structures with contiguous $\alpha$-1longrightarrow6 ~6 glycosidic linkages in the main chains and $\alpha$-1longrightarrow2, $\alpha$-1longrightarrow3, or $\alpha$-1longrightarrow4 branch glycosidic linkages, depending on the specificity of the particular dextransucrase. Glucansucrases that catalyze glucans synthesis from sucrose. When other carbohydrates, in addition to sucrose, are present in the enzyme digest, the enzyme transfers glucose to the carbohydrate acceptors in the secondary reaction that diverts some of the glucose from incorporation into glucan. Many carbohydrate acceptors have been recognized and the products that result are dependent on the particular enzyme and the structure of the particular acceptor. Because of these unique catalytic characteristics, various dextransucrases have many important industrial and medical uses. To improve the understanding of their action mode and extend their applications, this study describes mechanism of glucan synthesis and potential industrial uses of dextransucrases, and our recent findings on the structural, functional organization and directed evolution of the glucansucrases to offer for designing glucansucrases with improved properties.

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