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Molecular functional analysis of gene cluster involved in nitrile metabolism

腈代谢相关基因簇的分子功能分析

基本信息

批准号：
06660101
负责人：
KOBAYASHI Michihiko
金额：
$ 1.34万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660101/
关键词：
Nitrile hydratase Promoter Rhodococcus Amide Gene Sequence 誘導クロトンアミド塩基配列転写開始点

项目摘要

Actinomycete Rhodococcus rhodochrous J1 has two NHases depending on an inducer in the presence of cobalt ions ; when the strain is cultured in the medium containing urea and cyclohexanecarboxamide, high-Mr- (H-) and low-Mr- (L-) NHases are selectively induced, respectively.H-NHase gene was expressed as an active form in R.rhodochrous ATCC12674 using a Rhodococcus-E.coli host-vector (pK4) system, whereas it was expressed as an insoluble (inactive) form in E.coli. SDS/PAGE of cell-extracts of R.rhodochrous transformant carrying plasmid pHJK19, in which a 6.5-kb sequence covering 4.6-kb upstream region, 1.3-kb H-NHase gene and 0.6-kb downstream region was inserted into pK4, showed that the amount of alpha-and beta-subunits of H-NHase was about 50% of the total soluble protein. Analysis using the host-vector system revealed that the 4.6kb region was required for the expression of H-NHase gene. Sequence analysis clarified that one of OFRs in the region had significantly similarity to AmiC which was the negative regulatory protein involved in the expression of an amidase gene from Pseudomonas aeruginosa. Northern blot analysis showed that the genes for the alpha-and beta-subunits of H-NHase were cotranscribed as a 1.8-kb mRNA.Determination of H-NHase activity and H-NHase mRNA levels indicated that the expression of H-NHase gene was regulated by urea at transcriptional level, but not by cobalt ions.

放线菌Rhodococcus rhodochrous J1在钴离子存在下有两个依赖于诱导物的NH酶;当该菌株在含有尿素和环己烷甲酰胺的培养基中培养时，分别选择性地诱导高-Mr-（H-）和低-Mr-（L-）NH酶。使用红球菌-大肠杆菌宿主载体（pK 4）系统在玫瑰色红球菌ATCC 12674中表达H-NH酶基因作为活性形式，而它在大肠杆菌中表达为不溶性（无活性）形式。将6.5kb的H-NHase基因插入到pK 4中，其上游4.6kb、下游1.3kb和下游0.6kb的长度分别为6.5kb和6.5kb。SDS/PAGE分析表明，H-NHase α和β亚基的含量约占可溶性蛋白的50%。利用宿主-载体系统分析表明，4.6kb的区域是H-NHase基因表达所必需的。序列分析表明，该区域的一个OFR与铜绿假单胞菌酰胺酶基因表达的负调控蛋白AmiC具有显著的相似性。北方印迹分析表明，H-NHase α-和β-亚基基因共转录为1.8kb的mRNA，H-NHase活性和mRNA水平的测定表明，尿素在转录水平上调控H-NHase基因的表达，而钴离子不调控H-NHase基因的表达。