ENZYMATIC PREPARATION OF [1-C-13]D-FRUCTOSE-6-PHOSPHATE FROM [C-13]FORMALDEHYDE AND D-RIBOSE-5-PHOSPHATE USING THE FORMALDEHYDE-FIXING SYSTEM OF METHYLOMONAS-AMINOFACIENS 77A

The formaldehyde-fixing system of a methanol-using bacterium, Methylomonas aminofaciens 77a, was used for the production of C-13-labelled D-fructose-6-phosphate from [C-13]formaldehyde and D-ribose-5-phosphate. Although the cell-free extract of the methanol-grown cells gave a relatively high yield of the sugar phosphate, it was unsuited for the specific production of C-13-labelled sugar phosphate because of contamination due to non-labelled formaldehyde brought in with the extract. The specific and high-yield production of [1-C-13]D-fructose-6-phosphate was performed using an enzyme system that contained partially purified 3-hexulose-6-phosphate synthase and phospho-3-hexuloisomerase from the bacterium, and spinach phosphoriboisomerase. Several reaction conditions, amounts of the enzymes and the substrate concentration, were investigated. A fed-batch reaction, in which 50 mM formaldehyde and 75 mM D-ribose-5-phosphate were added at 15-min intervals, afforded 205 mm (53.3 Mg/Ml) D-fructose-6-phosphate from 300 mm formaldehyde in added total over 90 min. The C-1 position of the sugar phosphate was confirmed to be labelled with C-13 by nuclear magnetic resonance analysis.