[Back to Issue 12 ToC] [Back to Journal Contents] [Back to Biochemistry (Moscow) Home page]

Recombinant DNA-Methyltransferase M1.BspACI from Bacillus psychrodurans AC: Purification and Properties

M. V. Tarasova1,2*, V. V. Kuznetsov3, N. A. Netesova3, D. A. Gonchar1, and S. Kh. Degtyarev1

1SibEnzyme, ul. Akademika Timakova 2/12, 630117 Novosibirsk, Russia; fax: (383) 333-6853; E-mail: tarasovamv@sibenzyme.ru

2Novosibirsk State University, ul. Pirogova 2, 630090 Novosibirsk, Russia

3State Research Center of Virology and Biotechnology “Vector”, 630559 Kol’tsovo, Novosibirsk Region, Russia

* To whom correspondence should be addressed.

Received May 20, 2010; Revision received June 22, 2010
A restriction–modification system from Bacillus psychrodurans AC (recognition sequence 5′-CCGC-3′) comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature optimum of 30ºC and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence 5′-CCGC-3′. The kinetic parameters of M1.BspACI DNA methylation are as follows: Km for phage λ DNA is 0.053 µM and Km for S-adenosyl-L-methionine is 5.1 µM. The catalytic constant (kcat) is 0.095 min–1.
KEY WORDS: DNA methyltransferase, Bacillus psychrodurans, enzyme kinetics

DOI: 10.1134/S0006297910120096