Permeabilization of Microbacterium oxylans shifts the conversion of puerarin from puerarin-7-O-glucoside to puerarin-7-O-fructoside

Abstract

The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 +/- 31.9 microM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 +/- 48.0 microM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, (13)C NMR, (1)H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 +/- 5.4 microM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 +/- 27.7 microM puerarin-7-O-glucoside and 187.8 +/- 29.5 microM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 +/- 24.0 microM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product's composition.

Immobilization of puerarin glycosidase from Microbacterium oxydans CGMCC 1788 increases puerarin transformation efficiency

“…However, the transformation by resting cells is limited by low transformation efficiency and long transformation time because extracellular puerarin diffuses slowly into the intracellular space through the barrier of the cell membrane. When the intracellular puerarin reaches a certain concentration, it is transformed by M. oxydans CGMCC 1788 glycosidase to glycosylated puerarin that is then secreted through the cell membrane to the extracellular solution (Jiang et al, 2008;Yu et al, 2010). In order to increase the transformation efficiency and shorten the transformation time, free glycosidase extracted from disrupted cells of M. oxydans CGMCC 1788 has been used to transform puerarin (Yu et al, 2010).…”

“…When the intracellular puerarin reaches a certain concentration, it is transformed by M. oxydans CGMCC 1788 glycosidase to glycosylated puerarin that is then secreted through the cell membrane to the extracellular solution (Jiang et al, 2008;Yu et al, 2010). In order to increase the transformation efficiency and shorten the transformation time, free glycosidase extracted from disrupted cells of M. oxydans CGMCC 1788 has been used to transform puerarin (Yu et al, 2010). Although the transformation efficiency of free enzyme is increased by its direct reaction with puerarin in solution without the barrier of the cell membrane, it is difficult to isolate free enzyme from products and reuse it for transformation, increasing the production cost (Yu et al, 2010).…”

“…The reaction mixture was centrifuged at 8,000 × g for 10 min and the supernatant was heated to 100 °C for 10 min and centrifuged again at 12,000 × g for 10 min. The final supernatant was applied to an AB-8 macroporous resin adsorption chromatography column for further separation and purification (Yu et al, 2010). Each collected fraction was subjected to HPLC analysis and the fractions of > 95% purity of the transformation product were pooled, concentrated to 40-50 ml in a rotary evaporator, and freeze-dried to obtain the powder.…”

“…However, the pharmacological development of puerarin is limited by its low water solubility and bioavailability (Ren et al, 2006). Thus, significant effort has been made to chemically or biologically modify the structure of puerarin to obtain novel puerarin derivatives with higher water solubility and biological activity (Li et al, 2004;Huang et al, 2008;Jiang et al, 2008;Yu et al, 2010;Ko et al, 2012). Previous studies have shown that transformation of flavonoid substances in the form of aglycon to glycosides improves not only their physical and chemical properties, such as water solubility, taste and sweetness but also their pharmacological activities, including circulation, metabolism, and concentration in body fluids (Lee et al, 1999;Kren et al, 2001;Daines et al, 2004;Blanchard et al, 2006;Hyung et al, 2006;Salas et al, 2007).…”

“…Previous studies have shown that transformation of flavonoid substances in the form of aglycon to glycosides improves not only their physical and chemical properties, such as water solubility, taste and sweetness but also their pharmacological activities, including circulation, metabolism, and concentration in body fluids (Lee et al, 1999;Kren et al, 2001;Daines et al, 2004;Blanchard et al, 2006;Hyung et al, 2006;Salas et al, 2007). Jiang et al (2008) and Yu et al (2010) from our laboratory have previously reported that puerarin is transformed to puerarin-7-O-glucoside and puerarin-7-O-fructoside, respectively, under different condi-…”