Zhen Wang1,2, Xu Huang1,2, Juan Liu1,2, Feiyao Xiao1,2, Miaomiao Tian1,2, Shenghua Ding3,4,5, Yang Shan6,7,8. 1. Longping Branch Graduate School of Hunan University, Changsha, 410125, People's Republic of China. 2. Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha, 410125, People's Republic of China. 3. Longping Branch Graduate School of Hunan University, Changsha, 410125, People's Republic of China. shhding@hotmail.com. 4. Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha, 410125, People's Republic of China. shhding@hotmail.com. 5. Hunan Province International Joint Laboratory On Fruits & Vegetables Processing, Quality and Safety, Changsha, 410125, People's Republic of China. shhding@hotmail.com. 6. Longping Branch Graduate School of Hunan University, Changsha, 410125, People's Republic of China. sy6302@sohu.com. 7. Hunan Agricultural Product Processing Institute, Hunan Academy of Agricultural Sciences, Changsha, 410125, People's Republic of China. sy6302@sohu.com. 8. Hunan Province International Joint Laboratory On Fruits & Vegetables Processing, Quality and Safety, Changsha, 410125, People's Republic of China. sy6302@sohu.com.
Abstract
OBJECTIVES: In this study, 44 flavone synthases (FNS) and flavonol synthases (FLS) from different origins were collected. The instability index and conserved domain of the enzymes were analyzed through bioinformatics analysis, the results of which allowed us to screen suitable enzymes for constructing recombinant Escherichia coli. Defective enzymes were selected as controls. RESULTS: Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were conducted to isolate the heterologously expressed proteins. Liquid chromatography-mass spectrometry, 1H nuclear magnetic resonance, and ultra-performance liquid chromatography were performed to qualitatively and quantitatively analyze the products. The cellular transformation results showed that recombinant E. coli catalyzed the synthesis of diosmetin from hesperetin, and in vitro catalysis showed that heterologously expressed FNS/FLS played a catalytic role in this reaction. AnFNS (from Angelica archangelica) showed the highest substrate conversion (38.80% for cellular transformation, 12.93% for in vitro catalysis). CONCLUSIONS: The catalytic capacity of FNS/FLS from different origins exhibited the expected results, indicating that bioinformatics analysis is useful for screening enzymes. In addition, the catalytic properties of AnFNS and CaFLS (from Camellia sinensis) differed significantly, although these enzymes are structurally similar. Based on this difference, C-2 was predicted as the key site for FNS/FLS catalytic synthesis of diosmetin rather than C-3.
OBJECTIVES: In this study, 44 flavone synthases (FNS) and flavonol synthases (FLS) from different origins were collected. The instability index and conserved domain of the enzymes were analyzed through bioinformatics analysis, the results of which allowed us to screen suitable enzymes for constructing recombinant Escherichia coli. Defective enzymes were selected as controls. RESULTS: Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were conducted to isolate the heterologously expressed proteins. Liquid chromatography-mass spectrometry, 1H nuclear magnetic resonance, and ultra-performance liquid chromatography were performed to qualitatively and quantitatively analyze the products. The cellular transformation results showed that recombinant E. coli catalyzed the synthesis of diosmetin from hesperetin, and in vitro catalysis showed that heterologously expressed FNS/FLS played a catalytic role in this reaction. AnFNS (from Angelica archangelica) showed the highest substrate conversion (38.80% for cellular transformation, 12.93% for in vitro catalysis). CONCLUSIONS: The catalytic capacity of FNS/FLS from different origins exhibited the expected results, indicating that bioinformatics analysis is useful for screening enzymes. In addition, the catalytic properties of AnFNS and CaFLS (from Camellia sinensis) differed significantly, although these enzymes are structurally similar. Based on this difference, C-2 was predicted as the key site for FNS/FLS catalytic synthesis of diosmetin rather than C-3.
Authors: T Akashi; M Fukuchi-Mizutani; T Aoki; Y Ueyama; K Yonekura-Sakakibara; Y Tanaka; T Kusumi; S Ayabe Journal: Plant Cell Physiol Date: 1999-11 Impact factor: 4.927
Authors: Frank Koopman; Jules Beekwilder; Barbara Crimi; Adele van Houwelingen; Robert D Hall; Dirk Bosch; Antonius J A van Maris; Jack T Pronk; Jean-Marc Daran Journal: Microb Cell Fact Date: 2012-12-08 Impact factor: 5.328