Plasmid‐based, D‐aminopeptidase‐catalysed synthesis of (R)‐amino acids
Plasmid‐based, D‐aminopeptidase‐catalysed synthesis of (R)‐amino acids
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基于质粒、D-氨肽酶催化的 (R)-氨基酸合成
DOI：
10.1002/recl.19911100513
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
K. Kondo
中科院分区：
文献类型：
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作者：
Y. Asano;Katsuhiko Kishino;A. Yamada;S. Hanamoto;K. Kondo
来自人苍白杆菌 SCRC Cl-38 的 D-氨肽酶基因被克隆到大肠杆菌 JM109 中。构建了表达质粒pCl38DP(4.5kb)。大肠杆菌 JM109/pCl38DP 无细胞提取物中的酶量提高至 288000 单位/升培养物，大约是 O. anthropi SCRC Cl-38 的 3600 倍。据计算，该酶约占总可提取细胞蛋白的 30%。大肠杆菌转化体的完整细胞用作几种外消旋氨基酰胺盐酸盐的(R)-立体定向水解的催化剂。使用大肠杆菌转化体细胞，5.0M 外消旋丙氨酸酰胺 HCl 在短时间内 (4½ 小时) 即可完全水解 (R)-丙氨酸酰胺。 (R)-丙氨酸的浓度达到220g/l。细胞或无细胞提取物以类似的方式催化从其酰胺合成(R)-2-氨基丁酸、(R)-甲硫氨酸、(R)-正缬氨酸和(R)-正亮氨酸。
The gene for D-aminopeptidase from Ochrobactrum anthropi SCRC Cl-38 was cloned in Escherichia coli JM109. An expression plasmid pCl38DP (4.5 kb) was constructed. The amount of the enzyme in a cell-free extract of E. coli JM109/pCl38DP was elevated up to 288000 units/liter culture, which is about 3600-fold over that of O. anthropi SCRC Cl-38. It was calculated that the enzyme comprised about 30% of the total extractable cellular protein. The intact cells of the E. coli transformant were used as a catalyst for (R)-stereospecific hydrolysis of several racemic amino amides HCl. Complete hydrolysis of (R)-alanine amide was achieved in a short time (4½h) from 5.0M racemic alanine amide HCl using cells of the E. coli transformant. The concentration of (R)-alanine reached 220 g/l. The cells or the cell-free extract catalyzed the synthesis of (R)-2-aminobutyric acid, (R)-methionine, (R)-norvaline and (R)-norleucine from their amides in a similar manner.