Characterization of BflI – A Thermostable, Co++-Requiring Isoschizomer of BsiYI from Anoxybacillus Flavithermus

A thermophile, isolated from geothermal areas in the northern Himalayan region of India, was identified by partial 16S rDNA sequence (GenBank accession # AF482430) analysis as Anoxybacillus flavithermus. The isolate produced BflI (REBASE # 4910), a Type II restriction endonuclease, which recognized the sequence 5′-CCNNNNN/NNGG-3′ and was the isoschizomer of BsiYI. The enzyme was purified to homogeneity by passing through Cibacron Blue F3GA agarose, DEAE-cellulose, heparin-agarose and MonoQ FPLC. The purified enzyme (MW 36 kDa) worked best at 60 °C in Promega's buffer C and preferentially required Co++(0.4 mM) as cofactor followed by Mg++(10 mM) and Mn++(1 mM). The enzyme showed high specific activity and worked in the presence of high concentrations of β-mercaptoethanol (200 mM), Triton-X-100 (25%), urea (30%), formamide (6%) and guanidine (40 mM) and showed no star activity in the presence of 40% glycerol. In the absence of any stabilizing agent, BflI retained t 1/2 for at least 96 h at 37 °C, 6 h at 60 °C and 6 months at 4 °C. N-terminal sequencing showed that its first 10 amino acid residues were DFHEDKTIAR.