beta-Lactamase

From Bacillus cereus E.C. 3.5.2.6 Lyophilized solid Activity: Each vial contains 500 units β-Lactamase I and 50 units β-Lactamase II Unit Definition: One unit is defined as the amount of enzyme which will hydrolyze 1.0 micromole of benzyl penicillin and 1.0 micromole of cephalosporin C per minute at 25°C in the presence of EDTA pH 7.0


Source: Bacillus cereus

E.C. # 3.5.2.6

Synonyms: Penicillinase; Penicillin amido-b-lactamhydrolase; Cephalosporinase

Activity: Each vial contains approximately 500 units beta-lactamase I and 50 units of beta-lactamase II

Unit Definition: One unit is defined as the amount of enzyme which will hydrolyze 1.0 micromole of benzyl penicillin and 1.0 micromole of cephalosporin C per minute at 25°C in the presence of EDTA, pH 7.0 and Zn²⁺. (one unit is approximately equal to 600 Levy or 75 Pollock units).

Description: b-Lactamase is typically used to destroy b-lactam antibiotics.

Inhibitors: Clavulanic acid, sulbactam, tazobactam. These inhibitors bind to the enzyme more effectively than the b-lactam antibiotic.

b-Lactam Antibiotic Effectiveness: Effective against benzylpenicillin, ampicillin, amoxycillin, carbenicillin, methicillin, cloxacillin, flucloxacillin.

Cephalosporin Antibiotic Effectiveness: Effective against cephaloridine, cephradine (above 100 ug/ml requires a 30 minute incubation to ensure complete inactivation), cephalothin, cephalexin, cefuroxime (requires a 24 hour incubation with an elevated concentration of enzyme), cephazolin, cefoxitin (requires a 24 hour incubation with an elevated concentration of enzyme). It is recommended that a control solution of the cephalosporin antibiotic is prepared and incubated under the same conditions as the sample. After incubation, the control should be tested for complete inactivation of the antibiotic.

Typical Assay Method:

beta-Lactamase I catalyzes the following reaction:



Reagents:

A. Benzyl Penicillin Solution: contains 4.5 uM benzyl penicillin, 0.35% gelatin and 5 mM EDTA prepared in double distilled, deionized water.
B. 0.05 M NaOH solution
C. Enzyme Solution: Prepare enzyme in 0.1 M Tris-HCl, pH 7.0 with 1% gelatin.

Method:

Use 2.0 ml benzyl penicillin solution (Reagent A). Equilibrate and titrate to pH 7.0 with 0.05 M NaOH at 25°C. Add 0.02 ml enzyme solution. Determine titration rate at 25°C using 0.05 M NaOH.

Calculation:



beta-lactamase II catalyzes the following reaction:



Reagents:

A. Cephalospo