Preparation of [1-13C]D-Glucose 6-Phosphate from [13C]Methanol andD-Ribose 5-Phosphate with Methylotrophic Enzymes

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Abstract
[13C]Formaldehyde was selectively incorporated into the C-1 position of D-fructose 6-phosphate by condensation with D-ribulose 5-phosphate catalyzed by a partially purified enzyme system for formaldehyde fixation in Methylomonas aminofaciens 77a. Much of the [1-13C]D-fructose 6-phosphate produced in this reaction was converted to [1-13C]D-glucose 6-phosphate by the addition of glucose-6-phosphate isomerase. A fed-batch reaction with periodic additions of the substrates afforded 56.2 g/liter D-glucose 6-phosphate and 26.8g/liter D-fructose 6-phosphate. When [13C]methanol was used as the C1-donor, the yield of [1-13C]D-glucose 6-phosphate was high when alcohol oxidase was added. The optimum conditions for sugar phosphate production in the fed-batch reaction gave 45.6g/liter [1-13C]D-glucose 6-phosphate and 16.4g/liter [1-13C]D-fructose 6-phosphate in 165min. The molar yield of the total sugar phosphates to methanol added was 95%. The addition of H2O2 and catalase to the reaction system supplied molecular oxygen for methanol oxidation to formaldehyde by alcohol oxidase.

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