Enzymatic preparation of ^<13>C-labelled compounds with C_1-fix ing system of a methylotroph

[^<13>C]甲醛通过与D-核糖5-磷酸缩合而选择性地掺入到D-果糖6-磷酸的C-1位，所述缩合是在部分纯化的用于甲醛固定的Methylomonassminofaciens 77 a中的酶催化下进行的。通过加入葡萄糖-6-磷酸异构酶，该反应中产生的大部分[1-<13>^C] D-果糖6-磷酸转化为[1-^<13>C] D-葡萄糖6-磷酸。定期加入底物的补料分批反应得到56.2 g/升D-葡萄糖6-磷酸和26.8 g/升D-果糖6-磷酸。当以[^<13>C]甲醇为C_1-供体时，加入醇氧化酶，[1-^<13>C] D-葡萄糖6-磷酸的产率较高。在补料分批反应中生产磷酸糖的最佳条件在165 min内得到45.6 g/L [1-^<13>C] D-葡萄糖6-磷酸和16.4 g/L [1-^<13>C] D-果糖6-磷酸。反应体系的过氧化氢酶为醇氧化酶将甲醇氧化为甲醛提供分子氧。

[^<13>C]Formaldehyde was selectively incorporated into the C-1 position of D-fructose 6-phosphate by condensation with D-ribose 5-phosphate catalyzed by a partially purified enzymesystem for formaldehyde fixation in Methylomonas sminofaciens 77a. Much of the [1-^<13>C]D-fructose 6-phosphate production in this reaction was converted to [1-^<13>C]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 26.8 g/iter D-fructose 6-phosphate. When [^<13>C]methanol was used as the C_1-donor, the yield of [1-^<13>C]D-glucose 6-phosphate was high when alcohol oxidase was added. The optimum conditions for sugar phosphate production in the fed-batch reaction gave 45.6 g/liter [1-^<13>C]D-glucose 6-phosphate and 16.4 g/liter [1-^<13>C]D-fructose 6-phosphate in 165 min. The catalase to the reaction system supplied molecular oxygen for methanol oxidation to formaldehyde by alcohol oxidase.