Purification and Some Properties of β‐Lactamase from Mycobacterium smegmatis

Abstract

Mycobacteria which are usually penicillin resistant are known to produce f3-lactamases (I, 2). Kasik and his coworkers (3, 5) investigated some properties of f3-lactamases produced by Mycobacterium smegmatis, M. phlei, and M.fortuitum by using a crude enzyme from bacterial cells. The f3-lactamases from these rapid-growing mycobacteria showed broad substrate specificity. However, the nature of the purified enzyme from mycobacterial cells has not been studied. This paper describes the purification and some of the properties of an enzyme from M. smegmatis cells.M. smegmatis ATCC 607 was grown for three days at 37 C in a Sauton medium which contained (per liter): K 2HP04, 0.5 g; MgS0 4· 7H20, 0.5 g; L-asparagine, 4.0 g; sodium citrate, 3.0 g; ferric ammonium citrate, 0.05 g; and glycerol, 50.0 g (final pH 7.0). The cells were harvested on filter paper in a funnel, washed with water, and then suspended in ice-cold 0.07 M phosphate buffer, pH 6.6, at a concentration of 0.2 to 0.3 g (wet weight) per ml. The cell suspensions were disrupted by sonic treatment at 20,000 Hz for 10 min in an ice-water bath. The suspensions were centrifuged at 20,000 xg for 30 min at 0 C, and the supernatants were retained as the cell-free enzyme preparation. The enzyme preparation was brought to 15% saturation by the addition of solid (NH4)2S04. After 30 min the precipitate formed was removed by centrifugation and solid (NH 4hS04 was again added to the supernatant to 30% saturation. The resulting precipitate was collected by centrifugation and dissolved in 0.07 M phosphate buffer (pH 6.6). After dialysis against distilled water at 2 C, the enzyme solution was applied to a Sephadex G-75 column (3.2 X 50 em) previously equilibrated with 0

Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector

“…1) consists of the CAT reporter cassette from pKK232-8, the origin of replication from pAL5000, and the kanamycin resistance gene along with the pl5A origin of replication derived from pACYC177 (5). The replacement of the ampicillin resistance gene in pSD2 with the kanamycin resistance gene (as in pSD7) was necessary because ampicillin is an unselectable marker in mycobacteria (16). Screening for promoters in the presence of chloramphenicol (20 ,g/ml) is based on the activation of the silent CAT gene in pSD7, resulting in resistance to chloramphenicol.…”

Genetic analysis of the β-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatis and susceptibility to β-lactam antibiotics

“…The major b-lactamase in M. smegmatis mc 2 155 has been biochemically described and is similar to BlaF, the wellstudied molecular class A b-lactamase from M. fortuitum (Kaneda & Yabu, 1983;Quinting et al, 1997). A recent report identified a gene, designated blaA, encoding the major b-lactamase in M. smegmatis (Li et al, 2004), which is the same gene we previously designated blaS and describe in this work (A.R.…”

Beta-lactamase of Mycobacterium fortuitum: kinetics of production and relationship with resistance to beta-lactam antibiotics

“…As in other bacteria, the high resistance of M. fortuitum to most beta-lactam antibiotics may result from different, possibly interacting mechanisms such as poor cell penetration, drug inactivation by ,B-lactamases, or low affinity for penicillin-binding proteins. For most mycobacterial species, 13-lactamases have been described as noninducible, intracellular enzymes with a broad spectrum of activity (6,12,13). Purified P-lactamase of M. fortuitum hydrolyzes both penicillins and cephalosporins (1).…”