• Journal of Microbiology and Biotechnology
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• 제19권7호
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• pp.709-712
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• 2009

Enzymatic glucosylation with glycosyltransferases can be used to regulate the water solubility of aglycone. The drawback of this process is the demand of UDP-glucose as a sugar donor. We made an in-frame fusion of the flavonoid O-glucosyltransferase (OsUGT-3) and sucrose synthase (AtSUS) genes. The resulting fusion protein, OsUGT3-AtSUS, was expressed in E. coli and purified. When sucrose and UDP were supplied, the fusion protein was able to convert quercetin into quercetin O-glucoside without the addition of UDP-glucose. In addition, UDP-glucose was recycled when sucrose was added to the reaction mixture. This fusion protein is useful for the enzymatic production of flavonoid O-glucosides.

• Journal of Microbiology and Biotechnology
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• 제29권2호
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• pp.268-273
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• 2019

The specificity of a Bacillus licheniformis uridine diphosphate (UDP) glycosyltransferase, YjiC, was increased towards thymidine diphosphate (TDP)-sugar by site-directed mutagenesis. The Arg-282 of YjiC was identified and investigated by substituting with Trp. Conversion rate and kinetic parameters were compared between YjiC and its variants with several acceptor substrates such as 7-hydroxyflavone (7-HF), 4',7-dihydroxyisoflavone, 7,8-dihydroxyflavone and curcumin. Molecular docking of TDP-glucose and 7-HF with YjiC model showed pi-alkyl interaction with Arg-282 and His-14, and pi-pi interaction with $His^{14}$ and thymine ring. YjiC (H14A) variant lost its glucosylation activity with TDP-glucose validating significance of His-14 in binding of TDP-sugars.

• Molecules and Cells
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• 제37권2호
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• pp.172-177
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• 2014

A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-${\alpha}$-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4' and 7 hydroxyl groups, but not at the $5^{th}$ hydroxyl group of the A-ring, resulting in the production of genistein 4'-O-${\beta}$-D-glucoside, genistein 7-O-${\beta}$-D-glucoside (genistin), genistein 4',7-O-${\beta}$-D-diglucoside, biochanin A-7-O-${\beta}$-D-glucoside (sissotrin), daidzein 4'-O-${\beta}$-D-glucoside, daidzein 7-O-${\beta}$-D-glucoside (daidzin), daidzein 4', 7-O-${\beta}$-D-diglucoside, and formononetin 7-O-${\beta}$-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOF-ESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli $BL21(DE3)/{\Delta}pgi{\Delta}zwf{\Delta}ushA$ overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of $200{\mu}M$ of supplemented isoflavonoids, without any additional UDP-${\alpha}$-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.

• 원예과학기술지
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• 제33권4호
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• pp.470-478
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• 2015

본 연구는 국내에서 재배중인 '개량머루', '거봉', '홍이슬' 품종의 anthocyanin 조성을 UPLC-ESI-MS/MS를 이용하여 조사하였다. '개량머루'는 착색에 불량하게 작용하는 기상 조건에서도 착색이 우수한 흑포도 품종이고, '거봉'과 '홍이슬'은 교잡종 포도로 각각 검정색과 분홍색 과피를 갖고 있다. 과피에서 추출된 anthocyanin의 조성은 UPLC-ESI-MS/MS를 이용하여 분석하였다. 각 품종에서 검출된 anthocyanin은 '개량머루'와 '거봉'에서는 각각 25가지, '홍이슬'에서는 21가지였고, 각 품종에서 검출량이 많았던 anthocyanin은 8(개량머루), 15(거봉), 5(홍이슬)가지였다. 세 품종 모두 mono-glucoside가 di-glucoside보다 많았으며, '개량머루'와 '거봉'은 malvidin이 많았던 반면, '홍이슬'은 cyanidin이 가장 많았다. '개량머루'는 acylated anthocyanin(2.0%)은 거의 존재하지 않았고 '거봉'과 '홍이슬'에서는 p-coumaric acid의 acylation이 가장 많았다. Cyanidin feruloyl glucoside가 '홍이슬'에서만 검출되어 품종 지표로 활용이 가능하였다. 결론적으로 anthocyanin의 조성을 anthocyanidin, 결합된 당의 개수, acylation 및 특정 anthocyanin을 기준으로 분류하여 품종의 특징이 확인되었다. 이러한 품종별 anthocyanin 특성을 이용하여 '개량머루'가 Vitis amurensis 혹은 그 교잡종의 가능성과 '홍이슬'에서 품종 구별의 기준으로 활용 가능성이 확인되었다.