Enzymatic production of trans-4-hydroxy-L-proline by regio- and stereospecific hydroxylation of L-proline
Enzymatic production of trans-4-hydroxy-L-proline by regio- and stereospecific hydroxylation of L-proline
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DOI：
10.1271/bbb.64.746
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发表时间：
2000-04-01
影响因子：
1.6
通讯作者：
Ozaki, A
中科院分区：
文献类型：
--
作者：
Shibasaki, T;Mori, H;Ozaki, A
从Daclendosporangium sp.RH1中克隆了脯氨酸4-羟化酶基因，并对该基因5'端密码子进行了优化后，将该基因在大肠杆菌W1485中以色氨酸串联启动子进行了高效表达。脯氨酸4-羟化酶活性比Daclamposporangium sp，RH 1高1600倍。重组E.大肠杆菌在含有L-脯氨酸和葡萄糖的培养基中培养。2-酮戊二酸是脯氨酸4-羟化酶对L-脯氨酸进行羟基化所必需的，显然通过细胞代谢途径由葡萄糖提供。不能降解L-脯氨酸的W1485的putA突变体允许L-脯氨酸定量转化为Hyp。未观察到羟脯氨酸其他异构体的形成。Hyp的生产力在大规模培养中几乎相同。建立了以L-脯氨酸为原料制备羟脯氨酸的方法。
A proline 4-hydroxylase gene, which was cloned from Dactylosporangium sp. RH1, was overexpressed in Escherichia coli W1485 on a plasmid under a tryptophan tandem promoter after the codon usage of the 5' end of the gene was optimized. The proline 4-hydroxylase activity was 1600-fold higher than that in Dactylosporangium sp, RH1. trans-4-Hydroxy-L-proline(Hyp) was produced and accumulated to 41 g/L (87% yield from L-proline) in 100 h when the recombinant E. coli was cultivated in a medium containing L-proline and glucose. 2-Oxoglutarate, which is necessary for the hydroxylation of L-proline by proline 4-hydroxylase, was apparently supplied from glucose through the cellular metabolic pathway. The putA mutant of W1485, which is not able to degrade L-proline, has allowed the quantitative conversion of L-proline to Hyp. The formation of other isomers of hydroxyproline was not observed. Productivity of Hyp was almost the same in a larger-scale culture. The method of manufacturing Hyp from L-proline was established.