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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.

L-丝氨酸降解活性可以通过控制甲醇浓度的L-丝氨酸合成使用Hyphomicrobium methylovorum sp.NCIB10099细胞抑制。由100 mg/ml甘氨酸和104 mg/ml甲醇生产53 mg/ml L-丝氨酸。从一株产丝氨酸的甲基营养菌（H.methylovorum GM 2）中分离纯化了不依赖CoASAC的磷酸烯醇式丙酮酸羧化酶（PEPC），并与其它甲基营养菌的PEPC进行了比较。该酶的活性不受CoASAc的影响，CoASAc是一种众所周知的各种异养生物酶的激活剂，NADH和ADP是来自甲胺生长的假单胞菌MA的PEPC的效应物。这表明，H.methylovorum酶应归类为第二组。从嗜甲基菌GM 2中克隆了编码羟基丙酮酸还原酶（HPR）的基因及其侧翼序列。重组质粒的核苷酸序列分析表明，HPR基因编码322个氨基酸的蛋白，计算分子量为35，726 Da。该酶的氨基酸序列与D-异构体特异性2-羟基酸脱氢酶家族的成员相似。将重组质粒导入大肠杆菌HB 101中。从转化的大肠杆菌细胞中纯化的重组酶经免疫学和酶学分析与从H.methylovorum GM 2中分离的酶没有区别。