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Development of production process of useful compounds from cyanide.

开发从氰化物生产有用化合物的工艺。

基本信息

批准号：
09556018
负责人：
OGAWA Jun
金额：
$ 5.57万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09556018/
关键词：
Cyanide C1 compound nitrile hydratase beta-cyanoalanine synthase nitrilase beta-oyanoalanine Cl化合物 Pseudomonas ovalis PLP

项目摘要

The final purpose of this project is the establishment of bioconversion process for the production of useful compounds from nitriles or that of nitriles from cyanide. Various types of microorganisms that metabolize cyanide were obtained from soil by an enrichment culture technique. They have been found to contain enzymes that synthesize nitriles from various amino acids and cyanide. beta-Cyanoalanine synthases were purified and characterized from Pseudomonas ovalis. The amino acid sequences of N-terminal and internal peptide fragments of the beta-cyanoalanine synthase from P.ovalis were determined in order to prepare synthetic oligonucleotides as primers of the polymerase chain reaction. A 700-bp DNA fragment thus amplified was used as the probe to clone a 3.0-kb BcII fragment coding for the partial beta-cyanoalanine synthase, and the 680-bp EcoRI-BcII from 3.0-kb BcII fragment was used as the probe to clone a 2.2-kb BssHII-EcoRI fragment coding for the whole beta-cyanoalanine synthase. The beta-cyanoalanine synthase gene modified in the sequence upstream of the presumed ATG start codon was expressed to about 40% of the total soluble protein in E, coli. The P.ovalis beta-cyanoalanine synthase and Rhodococcus rhodochrous K22 nitrilase were co-expressed in E.coli. The cell-free extracts were used for the production of aspartic acid from KCN.beta-cyanoalanine was produced from KCN and O-acetyl-serine immediately, and the resultant beta-cyanoalanine was transformed into aspartic acid. On the other hand, P.ovalis beta-cyanoalanine synthase and R.rhodochrous J1 nitrile hydratase were co-expressed in R.rhodochrous ATCC12674. The cell-free extracts were used in the production of asparagine. beta-Cyanoalanine was produced immediately, and the resultant beta-cyanoalanine was converted into asparagine.

本项目的最终目的是建立腈类化合物或氰化物转化腈类化合物的生物转化工艺。通过富集培养技术从土壤中获得了多种代谢氰化物的微生物。已发现它们含有从各种氨基酸和氰化物合成腈的酶。从卵形假单胞菌中纯化并表征β-氰基丙氨酸脱氢酶。测定了卵圆单胞菌β-氰基丙氨酸合酶N-末端和内部肽片段的氨基酸序列，以制备合成的寡核苷酸作为聚合酶链反应的引物。将这样扩增的700-bp DNA片段用作探针，克隆编码部分β-氰基丙氨酸合酶的3.0-kb BcII片段，并将来自3.0-kb BcII片段的680-bp EcoRI-BcII用作探针，克隆编码整个β-氰基丙氨酸合酶的2.2-kb BssHII-EcoRI片段。在假定的ATG起始密码子的上游序列中修饰的β-氰基丙氨酸合酶基因在大肠杆菌中表达至总可溶性蛋白的约40%。在大肠杆菌中共表达卵圆藻β-氰基丙氨酸合酶和紫红红球菌K22腈水解酶。无细胞提取物用于从KCN生产天冬氨酸。由KCN和O-乙酰丝氨酸立即产生β-氰基丙氨酸，并将所得的β-氰基丙氨酸转化为天冬氨酸。另一方面，卵圆假单胞菌β-氰基丙氨酸合酶和玫瑰色红球菌J1腈水合酶在玫瑰色红球菌ATCC 12674中共表达。将无细胞提取物用于天冬酰胺的生产。立即产生β-氰基丙氨酸，并将所得的β-氰基丙氨酸转化为天冬酰胺。