Fgol, a type II restriction endonuclease from the thermoanaerobe Fervidobacterium gondwanense AB39(T)

Restriction endonuclease activity was detected in 11 out of 13 Fervidobacteriumisolates, including F. islandicum H21T, F. gondwanense AB39T, and nine other Fervidobacterium-like strains isolated from the Great Artesian Basin of Australia. The restriction endonuclease from F. gondwanense AB39T was partially purified and designated FgoI. FgoI recognized a 4 nucleotide sequence 5'-CTAG-3' and cleaved between nucelotides C and T to produce a 2 base 5' overhang (5'-C TAG-3'). As predicted from the enzyme recognition and cleavage specificity, FgoI was found to cleave δ DNA 13 times, phiX174 three times, pBR322 five times, pUC18 four times, and pSK six times. FgoI exhibited a broad temperature optimum range (between 60 to 70°C) and was active at pH6.5 to 8.5, but not at pH9.0. Manganese could replace magnesium as a cofactor for activity, but not cobalt chloride, calcium chloride, cupric chloride, or zinc chloride. The restriction endonuclease was completely inactivated by phenol chloroform extraction and was heat inactivated at 80°C for 60 min or at 100°C for 15 min. FgoI has been identified as a heat stable isoschizomer of the Type II restriction endonucleases, MaeI and BfaI