Development and Optimization of an In Vitro Multienzyme Synthetic System for Production of Kaempferol from Naringenin

• Published in: Journal of agricultural and food chemistry Vol. 66; no. 31; pp. 8272 - 8279
• Main Authors: Zhang, Zhiping, He, Yanzhi, Huang, Yue, Ding, Li, Chen, Lei, Liu, Yaxian, Nie, Yesen, Zhang, Xinyue
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 08.08.2018
• Subjects: kaempferol; optimization; flavanone 3-hydroxylase; in vitro synthesis; flavonol synthase
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/acs.jafc.8b01299

An in vitro multienzyme synthetic system was developed and optimized to efficiently produce kaempferol in a single reaction tube. Two key genes, Atf3h and Atfls1, in the biosynthetic pathway of kaempferol were cloned into a prokaryotic expression vector and overexpressed in Escherichia coli. The recombinant proteins were purified through affinity chromatography and showed activities of flavanone 3-hydroxylase and flavonol synthase, respectively, followed by development of an in vitro synthetic system for producing kaempferol. The system contains 8.2 mM α-ketoglutaric acid, 0.01 mM ferrous ion, 0.4% sodium ascorbate, 25 μg/mL of each recombinant enzyme, and 10% glycerol in 100 mM Tris-HCl (pH 7.2). When the reaction was carried out at 40 °C for 40–50 min, the yield of kaempferol was 37.55 ± 1.62 mg/L and the conversion rate from NRN to KMF was 55.89% ± 2.74%. Overall, this system provides a promising and efficient approach to produce kaempferol economically.