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Vol 43(2009) N 1 p. 8-15; V.A. Chernukhin1, J. Seggewiss2, Yu.G. Kashirina1, D.A. Gonchar1, S.Kh. Degtyarev1 Purification and properties of recombinant DNA methyltransferase M2. BstSE of the BstSEI nickase-modification system 1SibEnzyme, Novosibirsk, 630117, Russia2SE-BIOTECH, 46325, Borken, Germany Received - 2008-03-11; Accepted - 2008-05-27 The operon for the Bacillus stearothermophilus SE-589 nickase-modification system (NM. BstSEI, recognition site 5\'-GAGTC-3\') includes two DNA methyltransferase (M.) genes, bstSEIM1 and bstSEIM2. The gene encoding M2. BstSEI was cloned in pJW and expressed in Escherichia coli cells. M2. BstSEI was purified by chromatography and displayed maximal activity at 55° C and pH 7.5. The enzyme modified adenine in the nickase recognition site 5\'-GAGTC-3\' and was specific for 5\'-GASTC-3\' substrates. The kinetic parameters of the methylation reaction were determined. The catalytic constant was 2.2 min-1, and the Michaelis constant was 9.8 nM on T7 DNA and 5.8 μM on SAM. DNA methyltransferase nickase, Bacillus stearothermophilus, enzyme kinetics |
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