Development of high-functional production processes using microbial cyclic-amide metabolism

利用微生物环酰胺代谢开发高功能生产工艺

基本信息

批准号：
13660089
负责人：
OGAWA Jun
金额：
$ 2.3万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Novel processes for the production of useful compounds using microbial cyclic-amide metabolism were developed. A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using the cells cultivated in the medium containing 2% (w/v) fumarate as the catalysts, 286 mM pyruvate was produced from 500 mM fumarate in 27 h. Bromopyruvate-resistant mutants were derived from P. putida s52, and their pyruvate production was examined. One of the mutants, strain No. 15160 showed much higher pyruvate production than the parent strain. Using the mutant cells cultivated in the medium containing 2% (w/v) fumarate as the catalysts, 770 mM pyruvate was produced from 1000 mM fumrate in 72 h. Brevibacterium linens C-l was found to produce a novel cyclic amidohydrolase which stereoselectively hydrolyzes 5-substituted thiazolidinedione and wasuseful for the production of optically active α-mercapto acids. The gene encoding the enzyme was cloned from the chromosomal DNA of B. lines C-1 and sequenced. It contains an open reading frame consisting of 936 nucleotides corresponding to 312 amino acid residues. The deduced amino acid sequence exhibited no apparent homology with other protein sequences in the protein databases. The enzyme was expressed in recombinant E. coli cells. The E. coli transformant, E. coli BL21/pETBTH, was a good catalyst for stereo selective production of .S-3-phenyl-2-mercaptopropionic (S-PMPA) from RS-5-Benzyl-thiazolidinedione. The optical purity of S-PMPA produced was approximately 80% e.e.

开发了利用微生物环酰胺代谢生产有用化合物的新工艺，使用在含有 2% 的培养基中培养的细胞，发现恶臭假单胞菌 s52 是有效的丙酮酸生产者。 w/v) 富马酸盐作为催化剂，500 mM 富马酸盐在 27 小时内生成 286 mM 丙酮酸盐h. 溴代丙酮酸抗性突变体源自恶臭假单胞菌 s52，并且使用在含有 2 的培养基中培养的突变体细胞检查了其中一种突变体，菌株 No. 15160，其丙酮酸生产量高得多。％（w/v）富马酸盐作为催化剂，在 72 小时内从 1000 mM 富马酸盐产生 770 mM 丙酮酸盐。发现亚麻短杆菌 C-1 产生一种新型环状酰胺水解酶，该酶立体选择性地水解 5-取代的噻唑烷二酮，并可用于生产光学活性 α-巯基酸。编码该酶的基因从 B. lines C-1 和 C-1 的染色体 DNA 中克隆。它包含一个开放阅读框，由对应于 312 个氨基酸残基的 936 个核苷酸组成。推导的氨基酸序列与蛋白质数据库中的其他蛋白质序列没有明显的同源性。该酶在重组大肠杆菌细胞中表达，大肠杆菌转化体大肠杆菌BL21/pETBTH是立体选择性生产的良好催化剂。来自RS-5-苄基-噻唑烷二酮的S-3-苯基-2-巯基丙酸(S-PMPA) 所产生的S-PMPA的光学纯度约为80% 有效量