Stenotrophomonas maltophilia has two types of b-lactamase, L1 and L2. Both b-lactamases are co-inducibly expressed. To characterize the L1 and L2 b-lactamases of S. maltophilia, two parts are included in this study, in respect of quality and quantity. Firstly, to qualitatively characterize these b-lactamases, the zymogram pattern of isoelectric focusing electrophoresis (IEF) is generally referred. The zymogram of twenty S. maltophilia isolates were determined by IEF and two types of zymogram were revealed. In addition to the typical zymogram type of an acid b-lactamase and a basic one, a peculiar IEF zymogram pattern with an acid?涀-lactamase and several basic (ladder-shapped) b-lactamases was identified, like S. maltophilia KH did. The isogenic L2 mutant of strain KH was constructed by gene replacement strategy. From IEF and native-PAGE zymograms of strains KH and KHL2-, we demonstrate that the basic b-lactamases were encoded by the same L2 gene. Secondly, to differentially quantify the induced L1 and L2 b-lactamases in the cell extracts of S. maltophilia, a CENTA-clavulanic acid method is proposed. The L1 and L2 enzymes were characterized, including the enzyme kinetic assay against nitrocefin and CENTA, as well as the inhibitory effect toward clavulanic acid. CENTA was shown to be a susceptible substrate for L1, although it was hardly hydrolyzed by L2 enzyme. The L2 activity was sensitive to 10 mM clavulanic acid. In contrast, the L1 activity was unaffected by clavulanic acid. A differential quantification method for L1 enzyme activity in the induced sonicated extracts was developed based on their discrepant characteristics of L1 and L2 in the aspects of the hydrolysis ability against CENTA and the inhibitory effect by clavulanic acid. The prepared sonicated extracts were pre-incubated with clavulanic acid for 10 min, and then the activity of L1 enzyme was determined by spectrophotometrically monitoring the degradation of substrate CENTA.
