NITRILE HYDRATASE GENE FROM RHODOCOCCUS SP N-774 REQUIREMENT FOR ITS DOWNSTREAM REGION FOR EFFICIENT EXPRESSION
NITRILE HYDRATASE GENE FROM RHODOCOCCUS SP N-774 REQUIREMENT FOR ITS DOWNSTREAM REGION FOR EFFICIENT EXPRESSION
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DOI：
10.1271/bbb.58.1859
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发表时间：
1994-10-01
影响因子：
1.6
通讯作者：
BEPPU, T
中科院分区：
文献类型：
--
作者：
HASHIMOTO, Y;NISHIYAMA, M;BEPPU, T
为提高红球菌产腈水合酶(NHase)的效率。用重组DNA技术构建了N-774株NHaseα、β亚基编码基因的上游或下游缺失的表达载体。重组罗氏酵母和大肠杆菌细胞的酶分析表明，氨酶基因的下游区域是两个细胞产生活性氨酶所必需的，但产生活性酰胺酶除了酰胺酶结构基因外，不需要其他基因。其下游核苷酸序列含有一个开放阅读框(Orf1188)，由396个氨基酸组成。Orf1188的氨基酸序列与绿色假单胞菌B23氨酶基因下游的开放阅读框P47K相似，与可能参与脱氮假单胞菌钴胺生物合成的CobW基因产物相似。因为NHaseβ亚基的TGA终止密码子和Orf1188的ATG密码子之间的距离只有98bp，而且Orf1188和NHase的产生都依赖于酰胺酶基因上游的启动子，所以这些基因似乎以多顺反子的方式共转录，形成操纵子。通过对转氨酶、氨酶和Orf1188基因的转化子pKRNH2的培养条件进行优化，结果表明，转化子的氨酶活性比出发菌株提高了6倍。N-774。
For improvement of the production of nitrile hydratase (NHase) from Rhodococcus sp. N-774 by recombinant DNA techniques, several plasmids, each of which had a deletion of the upstream or downstream region of the genes encoding the alpha and beta subunits of NHase, were constructed. Enzyme assays of recombinant R. rhodochrous and Escherichia coli cells showed that a downstream region of the NHase genes was indispensable for the production of active NHase in both cells, but for the production of the active amidase, no genes other than the amidase structural gene were required. The nucleotide sequence of the downstream region contained a single open reading frame (Orf1188) with 396 amino acids. Orf1188 showed similarity in amino acid sequence to P47K, an open reading frame found downstream of the NHase genes from Pseudomonas chlororaphis B23, and also to the cobW gene product, which may be involved in cobalamin biosynthesis in Pseudomonas denitrificans. Because the distance between the TGA stop codon for the NHase beta-subunit and the ATG codon for Orf1188 is only 98bp, and because production of both Orf1188 and NHase is dependent on a promoter upstream of the amidase gene, these genes appear to be co-transcribed in a polycistronic manner, forming an operon. By optimization of the culture conditions of R. rhodochrous carrying pKRNH2, which contained the amidase, NHase, and Orf1188 genes, the transformant showed the NHase activity 6-fold higher than that of the original strain, Rhodococcus sp. N-774.