ORIGINAL ARTICLE Molecular cloning, overexpression and biochemical characterization of hypothetical b-lactamases of Mycobacterium tuberculosis H37Rv K.M. Nampoothiri, R. Rubex*, A.K. Patel*, S.S. Narayanan, S. Krishna, S.M. Das and A. Pandey Biotechnology Division, National Institute for Interdisciplinary Science and Technology (Formerly Regional Research Laboratory), CSIR, Trivandrum, Kerala, India Introduction The increasing resistance of mycobacterial strains to antimycobacterial drugs and the resurgence of tubercu- losis in the recent past underline the urgent need for new therapeutic regime for the treatment of tuberculo- sis. b-lactam antibiotics that inhibit transpeptidase reac- tions and prevent cell wall assembly in bacteria are the most widely used antimicrobial agents. However, most of the mycobacteria are naturally resistant to b-lactams, presumably because of their extremely hydrophobic cell wall, presence of periplasmic penicillin-binding proteins (PBS) and most importantly the presence of an active b-lactamase catalysing the hydrolysis of b-lactam anti- biotics (Massova and Mobashery 1998; Nampoothiri 2003; Wang et al. 2006). Most of the isolates of M. tuberculosis produce b-lactamase and opinions differ in elucidating the enzyme structure, classification and whether it is secreted cytoplasmically or bound to the cell membrane and as to whether its production is inducible or constitutive (Kasik 1979). Most of the kinetic studies of mycobacterial b-lactamase are with impure preparations of enzyme or have been inferred indirectly via the result of susceptibility tests involving b-lactams and b-lactamase inhibitor combinations (Segura et al. 1998). Systematic study of M. tuberculosis b-lactamases and that of related species and their correlation with b-lactam and their inhibitor suscepti- bility profile are useful in developing new antibiotic regime. Keywords b-lactamases, b-lactam antibiotics, Mycobacterium tuberculosis, nitrocefin. Correspondence Kesavan Madhavan Nampoothiri, Biotechnology Division, National Institute for Interdisciplinary Science and technology (NIIST), Formerly as Regional Research Laboratory, CSIR, Thiruvananthapuram – 695 019, Kerala, India. E-mail: madhavan85@hotmail.com *Both authors contributed equally to this work. 2007 ⁄ 1420: received 3 September 2007, revised and accepted 5 December 2007 doi:10.1111/j.1365-2672.2007.03721.x Abstract Aim: Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypo- thetical b-lactamases activity. Methods and Results: Analysis of the M. tuberculosis H37Rv genome revealed that Rv2068c, Rv0406c and Rv3677c gene products were predicted to exhibit b-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have b-lactam- ase activity by the hydrolysis of nitrocefin and other b-lactams. Catalytic para- meters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of b-lactam antibiotics. Conclusion: The study revealed the possibility of more than one gene in M. tuberculosis, encoding proteins having b-lactamase or b-lactamase-like activity, giving wide spectrum of resistance against b-lactams. Significance and Impact of the Study: Systematic study of hypothetical b-lacta- mases of M. tuberculosis and related species and their correlation with b-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains. Journal of Applied Microbiology ISSN 1364-5072 ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 105 (2008) 59–67 59