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

在甲基单胞菌77a中，在部分纯化的固定甲醛的酶系统催化下，通过与D-核糖5-磷酸的缩合反应，将甲醛选择性地结合到D-果糖6-磷酸的C-1位。该反应生成的[1-^&lt；13&gt；C]D-果糖6-磷酸通过添加葡萄糖-6-磷酸异构酶转化为[1-^&lt；13&gt；C]D-葡萄糖6-磷酸。间歇加料反应可得到56.2g/L的D-葡萄糖6-磷酸和26.8g/升的D-果糖6-磷酸。当以甲醇为C_1给体时，加入乙醇氧化酶时，D-葡萄糖6-磷酸的产率较高。补料分批反应生产磷酸糖的最佳工艺条件为：反应时间165min，磷酸葡萄糖45.6g/L，6-磷酸果糖16.4g/L。反应体系中的过氧化氢酶为乙醇氧化酶氧化甲醇生成甲醛提供了分子氧。

[^<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.