2 DIFFERENT ISOSCHIZOMERS OF THE TYPE-II RESTRICTION-ENDONUCLEASE TAQ-I (T CGA) WITHIN THE SAME THERMUS ISOLATE - TSP32-I, AN ENZYME WITH SIMILAR HEAT-STABILITY PROPERTIES TO THE PROTOTYPE ENZYME TAQ-I, AND TSP32-II, A HYPERTHERMOSTABLE ISOSCHIZOMER OF TAQ-I/

We have recently screened 112 separate isolates of the genus Thermus, collected from neutral and alkaline hot water springs on four continen ts, for the presence of the Type-II restriction endonuclease Tag I (T/ CGA). One particular isolate from the Azores (strain 32) was found to contain high levels of a restriction endonuclease with the same recogn ition and cleavage site as Tag I. Initial studies revealed that the pa rtially purified enzyme from strain 32 was considerably more resistant to heat inactivation than the prototype enzyme Tag I, being able to w ithstand temperatures at least 10 degrees C higher than Tag I, before showing evidence of heat inactivation. Subsequently it became clear th at the partially purified extract from strain 32 contains two separate enzymes, both of which are isoschizomers of Taq I. One of the enzymes , Tsp32 I, has similar thermal stability characteristics to Tag I, whe reas the second Tag I isoschizomer, Tsp32 II, found in the same Thermu s isolate as Tsp32 I, is considerably more thermostable than Taq I, re taining full enzyme activity up to a temperature of 85 degrees C. Tsp3 2 I and Tsp32 II were further distinguished by virtue of their differe nt requirements for magnesium ions.