Koirala Niranjan, Pandey Ramesh Prasad, Parajuli Prakash, Jung Hye Jin, Sohng Jae Kyung
Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Asansi, Chungnam 336-708, Republic of Korea.
Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, Asansi, Chungnam 336-708, Republic of Korea.
J Biotechnol. 2014 Aug 20;184:128-37. doi: 10.1016/j.jbiotec.2014.05.005. Epub 2014 May 22.
An O-methyltransferase SpOMT2884, originating from Streptomyces peucetius ATCC 27952, was cloned, expressed, and applied for the production of target metabolite from Escherichia coli. Biochemical characterization of the 25kDa recombinant protein by in vitro and in vivo experiments showed that SpOMT2884 was an S-adenosyl-l-methionine-dependent O-methyltransferase. SpOMT2884 catalyzed O-methylation of different classes of flavonoids such as flavones (7,8-dihydroxyflavone (7,8-DHF), luteolin), flavonols (quercetin, rutin), flavanone (naringenin), and isoflavonoids (daidzein, formononetin). Biotransformation of 7,8-DHF, a preferred substrate of SpOMT2884, in a grown-induced culture of E. coli BL21 (DE3) harboring the recombinant pET-28a-SpOMT2884 stoichiometrically converted 7,8-DHF into 7-hydroxy-8-methoxyflavone, which was confirmed by liquid chromatography, mass spectrometry and various nuclear magnetic resonance (NMR) spectroscopy analyses. In order to improve the biotransformation substrate, time and media parameters were optimized and the production was scaled up using a 3-L fermentor. The maximum yield of 7-hydroxy-8-methoxyflavone was 192μM (52.57mg/L), representing almost 96% bioconversion within 12h, when 200μM of 7,8-DHF was supplemented in the culture. Further, the 7-hydroxy-8-methoxyflavone was purified in large scale and was used as a substrate separately for in vitro glycosylation to produce glucose, galactose and 2-deoxyglucose conjugated at 7th hydroxyl position of 7-hydroxy-8-methoxyflavone. Biological activity showed that 7-hydroxy-8-methoxyflavone had long term cytoprotective and antioxidant effects compared to 7,8-DHF suggesting that methylation enhances the stability of substrate and glycosylation has proved to increase the water solubility.
克隆、表达了一种源自变铅青链霉菌ATCC 27952的O-甲基转移酶SpOMT2884，并将其应用于从大肠杆菌中生产目标代谢物。通过体外和体内实验对25kDa重组蛋白进行的生化特性分析表明，SpOMT2884是一种依赖S-腺苷-L-甲硫氨酸的O-甲基转移酶。SpOMT2884催化不同类别的黄酮类化合物的O-甲基化反应，如黄酮（7,8-二羟基黄酮（7,8-DHF）、木犀草素）、黄酮醇（槲皮素、芦丁）、黄烷酮（柚皮素）和异黄酮（大豆苷元、刺芒柄花素）。在含有重组质粒pET-28a-SpOMT2884的大肠杆菌BL21（DE3）的生长诱导培养物中，SpOMT2884的首选底物7,8-DHF的生物转化反应将7,8-DHF化学计量地转化为7-羟基-8-甲氧基黄酮，这通过液相色谱、质谱和各种核磁共振（NMR）光谱分析得到了证实。为了优化生物转化的底物、时间和培养基参数，并使用3-L发酵罐扩大生产规模。当在培养物中添加200μM的7,8-DHF时，7-羟基-8-甲氧基黄酮的最大产量为192μM（52.57mg/L），在12小时内的生物转化率几乎达到96%。此外，大规模纯化了7-羟基-8-甲氧基黄酮，并将其分别用作体外糖基化反应的底物，以生成在7-羟基-8-甲氧基黄酮的第7位羟基上共轭的葡萄糖、半乳糖和2-脱氧葡萄糖。生物活性表明，与7,8-DHF相比，7-羟基-8-甲氧基黄酮具有长期的细胞保护和抗氧化作用，这表明甲基化增强了底物的稳定性，并且糖基化已被证明可增加水溶性。
