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Cloning of a gene for heat-stable tryptophanase produced by an obligatorily symbiotic and thermophilic bacterium.

克隆由强制共生嗜热细菌产生的热稳定色氨酸酶基因。

基本信息

批准号：
61560113
负责人：
UOZUMI Takeshi
金额：
$ 1.54万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-61560113/
关键词：
tryptophanase gene cloning thermophile heat-stable enzyme symbiosis tryptophan 発現耐熱性

项目摘要

An obligatorily symbiotic bacterium, strain T, associating with a specific strain S of a thermophilic Bacillus sp. produces heat-stable tryptophanase. We purified the tryptophanase and determined the NH_2-terminal amino acid sequence. Two corresponding oligonucleotide sequences were synthesized as the probes for cloning of this enzyme gene. A Bam HI fragment of about 5.4-kb hybridized with these probes was isolated and E.coli harboring the subcloned 2.0-kb fragment on expression vector pUC19 produced approximately ten-fold higher level of the tryptophanase than that in strain T after induction with IPTG. The determination of nucleotide sequence of this fragment is now in progress. Since the sequence corresponding to NH_2-terminal sequence is already found, the fragment must contain an entire gene for the tryptophanase.A molecular weight of the purified enzyme estimated by gel filtration column chromatography was 208 kd, and the molecular weight of subunit was determined by SDS-PAGE to be 46 kd, indicating that the native enzyme is composed of four homologous subunits. The optimum temperature for the enzyme activity was 70 C and the enzyme was stable at 65 C. These temperatures are higher than those of the tryptophanase from E. coli, which are 55 C and 40 C, respectively. So this heat-stable tryptophanase will be available for industrial production of tryptophan using reverse reaction of this enzyme activity.Growth of strain T required co-culture with Bacillus strain S which was not replaced by other strains of thermophilic Bacillus species. No independent growth of strain T was observed in any of the medium so far tested. Further studies are required for elucidating the mechanism of symbiosis of the tryptophanase-producing microorganism strain T.

一种专性共生细菌，菌株T，与嗜热芽孢杆菌属的特定菌株S结合，产生热稳定的葡聚糖酶。对该酶进行了纯化和氨基端氨基酸序列测定。合成了两个相应的寡核苷酸序列作为探针，用于克隆该酶基因。分离出与这些探针杂交的约5.4-kb的BamH Ⅰ片段，在表达载体pUC 19上含有亚克隆的2.0-kb片段的大肠杆菌在用IPTG诱导后产生比菌株T中高约10倍的酶水平。该片段的核苷酸序列测定正在进行中。由于已经找到了与NH_2端序列相对应的序列，因此该片段一定含有完整的色氨酸酶基因。凝胶过滤柱层析法估计纯化酶的分子量为208 kd，SDS-PAGE测定亚基的分子量为46 kd，表明天然酶由四个同源亚基组成。该酶的最适反应温度为70 ℃，在65 ℃时酶活性稳定.这些温度高于E.大肠杆菌的最适发酵温度分别为55 ℃和40 ℃。因此，该酶可用于工业化生产色氨酸。菌株T的生长需要与芽孢杆菌菌株S共培养，而芽孢杆菌菌株S没有被其他嗜热芽孢杆菌菌株所取代。在迄今为止测试的任何培养基中均未观察到菌株T的独立生长。该菌株的共生机制有待于进一步研究。