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

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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.