Development of production process of useful compounds from cyanide.

该项目的最终目的是建立从腈中生产有用化合物或从氰化物中生产腈的生物转化工艺。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。它们被发现含有从各种氨基酸和氰化物合成腈的酶。从卵状假单胞菌中纯化并鉴定了β -氰丙氨酸合成酶。为了制备合成的寡核苷酸作为聚合酶链反应的引物，测定了卵形虾β -氰丙氨酸合成酶的n端和内肽片段的氨基酸序列。用扩增的700 bp DNA片段作为探针，克隆出编码部分-氰丙氨酸合成酶的3.0 kb BcII片段，用3.0 kb BcII片段中的680 bp EcoRI-BcII作为探针，克隆出编码整个-氰丙氨酸合成酶的2.2 kb BssHII-EcoRI片段。在假定的ATG起始密码子上游序列修饰的β -氰丙氨酸合成酶基因在大肠杆菌中表达约占总可溶性蛋白的40%。卵圆弧菌β -氰丙氨酸合成酶和红红球菌K22硝化酶在大肠杆菌中共表达。无细胞提取物用于KCN生产天冬氨酸。KCN和o -乙酰丝氨酸立即生成-氰丙氨酸，生成的-氰丙氨酸转化为天冬氨酸。另一方面，P.ovalis β -氰丙氨酸合成酶和R.rhodochrous J1腈水合酶在R.rhodochrous ATCC12674中共表达。无细胞提取物用于生产天冬酰胺。-氰丙氨酸立即产生，生成的-氰丙氨酸转化为天冬酰胺。

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.