Enzymatic and Molecular Genetic Studies of Serine Production by C_1-Microorganisms

C_1-微生物产生丝氨酸的酶学和分子遗传学研究

基本信息

批准号：
06650917
负责人：
IZUMI Yoshikazu
金额：
$ 1.28万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

L-Serine-degrading activity could be suppressed by controlling the methanol concentration in L-serine synthesis using Hyphomicrobium methylovorum sp. NCIB10099 cells. Fifty-three mg/ml of L-serine were produced from 100 mg/ml of glycine and 104 mg/ml of methanol. It is believed that L-serine-degrading activity contributes to the reverse serine hydroxymethyltransferase reaction.CoASAc-independent phosphoenolpyruvate carboxylase (PEPC) was purified from a serine-producing methylotroph, H.methylovorum GM2, and compared with those from other methylotrophs. The activity of this enzyme was not affected by CoASAc, a well-known activator for enzymes from various heterotrophs, NADH and ADP,effectors for PEPC from methylamine-grown Pseudomonas MA.This suggested that the H.methylovorum enzyme should be classified into the second group. and different from that of Pseudomonas MA.The gene encoding hydroxypyruvate reductase (HPR) and its flanking regions were isolated from H.methylovorum GM2. Nucleotide sequence of the recombinant plasmids revealed that HPR gene codes the 322-amino-acid protein with calculated molecular mass 35,726Da. The amino acid sequence of the enzyme showed similarity to members of the D-isomer-specific 2-hydroxyacid dehydrogenase family. The recombinant plasmid was introduced into Escherichia coli HB101. The recombinant enzyme purified from the transformed E.coli cells was indistinguishable from the enzyme isolated from H.methylovorum GM2 by immnunological and enzymological analysis.

通过使用甲基化菌群SP来控制L链合成中的甲醇浓度，可以抑制L链降解活性。 NCIB10099细胞。从100 mg/ml的甘氨酸和104 mg/ml甲醇产生了53毫克/毫升L丝氨酸。人们认为，L-塞氨酸降解活性有助于丝氨酸羟基甲基转移酶反应。COASAC非依赖性的磷酸烯醇丙酮酸羧化酶（PEPC）是从产生丝氨酸的甲基动物，h.methyloproph，h.methylovorum gm2和与其他甲基甲基球体相比的。该酶的活性不受COASAC的影响，CoAsac是一种众所周知的活化剂，用于来自各种异育酶，NADH和ADP，是来自甲胺生长的假单胞菌的PEPC的效应子，这表明H.Methylovorum酶的酶应分为第二组。并且与编码羟基丙酮酸还原酶（HPR）及其侧翼区域的基因不同的基因不同，并从H.methylovorum gm2中分离出来。重组质粒的核苷酸序列表明，HPR基因用计算的分子质量为35,726Da代码322-氨基酸蛋白。酶的氨基酸序列与D-异构体特异性2-羟基酸脱氢酶家族的成员相似。将重组质粒引入大肠杆菌HB101中。从转化的大肠杆菌细胞中纯化的重组酶与通过Immnologologice和酶学分析从H.methylovorum GM2分离的酶没有区别。