Purification and properties of an extracellular exonuclease from Thermus thermophilus HB8.
An extracellular exonuclease has been found and purified about 10,000-fold from the culture broth of an extreme thermophile, Thermus thermophilus HB8. The enzyme had an isoelectric point at pH 5.1 and seems to be of a multimolecular type, with molecular weights estimated to be ca. 530,000 (Peak I) and around 330,000 (Peak II) by gel filtration. The properties of the most highly purified enzyme fraction, Peak I were investigated. The enzyme requires divalent cations (Mg2+ greater than Sn2+ greater than Ca2+, Mn2+) and is inactive in the presence of EDTA. The pH optimum is 9.4-9.5 in glycine-NaOH buffer and the optimum temperature is 85 degrees C. The rate of hydrolysis increases in the order heat-denatured DNA greater than native DNA greater than RNA. The enzyme hydrolyzes deoxyoligonucleotides bearing 5'-monophosphate to liberate 5'-mononucleotides in an exonucleolytic manner. However, oligonucleotides lacking a 5'-phosphoryl group, irrespective of the presence or absence of phosphate at the 3'-termini, give both 5'-mononucleotides and dinucleoside monophosphates derived from the 5'-termini. It was also found that dinucleotides terminated with a 5'-phosphoryl group were cleaved to 5'-mononucleotides, but dinucleoside monophosphates were resistant to this enzyme. This exonuclease should be useful in the direct determination of sequence at the 5'-terminus and the penultimate position of oligonucleotides by the use of high-performance liquid chromatography.
D003851:Deoxyribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D005092:Exonucleases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013824:Thermus / Q000201:enzymology*
D046911:Macromolecular Substances; D003851:Deoxyribonucleases; D005092:Exonucleases; D012260:Ribonucleases