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

一种强制性共生细菌，菌株T，与嗜热芽孢杆菌的特定菌株S相结合，产生热稳定色氨酸酶。我们纯化了色氨酸酶并测定了其nh_2末端氨基酸序列。合成了两个相应的寡核苷酸序列作为该酶基因克隆的探针。与这些探针杂交的约5.4 kb的Bam HI片段被分离出来，在表达载体pUC19上含有亚克隆的2.0 kb片段的大肠杆菌在IPTG诱导后产生的色氨酸酶水平比菌株T高约10倍。目前正在对该片段的核苷酸序列进行测定。由于已经找到了与nh_2末端序列相对应的序列，因此该片段必须包含一个完整的色氨酸酶基因。经凝胶过滤柱层析得到的纯化酶分子量为208 kd， SDS-PAGE测定的亚基分子量为46 kd，表明该酶由4个同源亚基组成。最适温度为70℃，最稳定温度为65℃，高于大肠杆菌色氨酸酶的55℃和40℃。因此，利用该酶活性的逆反应，这种热稳定色氨酸酶可用于工业生产色氨酸。菌株T的生长需要与芽孢杆菌菌株S共培养，而S不被其他嗜热芽孢杆菌菌株所取代。到目前为止，没有观察到菌株T在任何培养基中独立生长。色氨酸酶产生菌T的共生机制有待进一步研究。

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.