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

用甲基营养菌的C_1-固定系统酶法制备^ 13 C-标记的化合物

• 批准号: 04660118
• 负责人: KITA Keiko
• 金额: $ 1.28万
• 依托单位: Tottori Uniersity
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04660118/
• 关键词: ^<13>C-Labelled sugar phosphate; Methanl; Formaldehyde; Methyomonas aminofaciens; C_1-fixing system; Alcohol oxidase; Catalase; 安定同位体標識化合物; ^<13>C-グルコース; ^<13>C-フルクトース; メチロトローフ; リブロースモノリン酸経路

[^<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.

[^<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。

项目成果

H.Yanase et al.: "Preparation of [1-^<13>C]D-glucse 6-phosphate from [^<13>C]methanol and D-ribose 5-phosphate with methylotrophic enzymes" Bopsci.Biotech.Biochem. 57. 308-312 (1993)

H.Yanase等人：“用甲基营养酶从[^ 13 C]甲醇和D-核糖5-磷酸制备[1-^ 13 C]D-葡萄糖6-磷酸”Bopsci.Biotech.Biochem

H.Yanase: "Production of D-[1-^<13>C]fructose 6-phosphate by a formaldehyde fixing system in Methylomonas aminofaciens 77a" Biosci.Biotechnol.Biochem.56. 541-542 (1992)

H.Yanase：“通过氨基单胞菌77a中的甲醛固定系统生产D-[1-^ 13 C]果糖6-磷酸”Biosci.Biotechnol.Biochem.56。

H.Yanase et al.: "Enzymatic preparation of [1-^<13>C]D-fructose-6-phosphate from [^<13>C]formaldehyde and D-ribose-5-phsphate using the formaldehyde-fixing system of Methhyomonas amnofaciens 77a" Appl.Microbiol.Biotechnol. 37. 301-304 (1992)

H.Yanase 等人：“使用甲醛固定系统从 [^ 13 C] 甲醛和 D-核糖-5-磷酸酯酶法制备 [1-^ 13 C]D-果糖-6-磷酸酯”

H.Yanase: "Preparation of[1-^<13>C]D-glucose 6-phosphate from[^<13>C]methanol and D-ribose 5-phosphate with Methylotrophic enzymes" Biosci.Biotech.Biochem.57. 308-312 (1993)

H.Yanase：“用甲基营养酶从[^ 13 C]甲醇和D-核糖5-磷酸制备[1-^ 13 C]D-葡萄糖6-磷酸”Biosci.Biotech.Biochem.57。

H.Yanase: "Enzymatic preparation of〔1-^<13>C〕D-fructose-6-phosphate from〔^<13>C〕formaldehyde and D-ribose-5-phosphate using the formaldehyde-fixing system of Methlomonas aminofaciens 77a" Appl.Microbiol.Biotechnol.37. 301-304 (1992)

H.Yanase：“使用氨基单胞菌的甲醛固定系统，从[^<13>C]甲醛和D-核糖-5-磷酸酶法制备[1-^<13>C]D-果糖-6-磷酸” 77a”应用微生物学生物技术37. 301-304 (1992)