Two 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 continents, for the presence of the Type-II restriction endonuclease Taq I (T/CGA). One particular isolate from the Azores (strain 32) was found to contain high levels of a restriction endonuclease with the same recognition and cleavage site as Taq I. Initial studies revealed that the partially purified enzyme from strain 32 was considerably more resistant to heat inactivation than the prototype enzyme Taq I, being able to withstand temperatures at least 10 degrees C higher than Taq I, before showing evidence of heat inactivation. Subsequently it became clear that 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 Taq I, whereas the second Taq I isoschizomer, Tsp32 II, found in the same Thermus isolate as Tsp32 I, is considerably more thermostable than Taq I, retaining full enzyme activity up to a temperature of 85 degrees C. Tsp32 I and Tsp32 II were further distinguished by virtue of their different requirements for magnesium ions.
Department of Biochemistry, Faculty of Basic Medical Sciences, Queen Mary and Westfield College, University of London, U.K.
S G
SG
Welch
R A
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Williams
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000302:isolation & purification / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D019914:Taq Polymerase; D013816:Thermodynamics; D013824:Thermus / Q000201:enzymology*
D007527:Isoenzymes; D011994:Recombinant Proteins; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes; D008274:Magnesium