Purification of penicillin-induced penicillinase of Bacillus cereus NRRL 569: a comparison of its properties with those of a similarly purified penicillinase produced spontaneously by a constitutive mutant strain

Abstract

Penicillin-induced penicillinase from B. cereus 569 grown in casein hydrolysate was purified by absorption on fine-mesh glass powder, elution with 0.5-saturated (NH4SO4, fractionation with 0.60-0.75-saturated (NH4SO4, and crystallization from 30% ethanol. The enzyme appears to be homogeneous as shown by electrophoretic and ultracentrifugal analysis; the molecular weight is 31,000. A constitutive penicillinase, produced without penicillin treatment, from strain (B. cereus 569/H) derived from B. cereus 569 by spontaneous mutation, was purified by the same technique. The constitutive enzyme does not differ significantly from the induced enzyme of its parent strain by any of the characters examined (specific activity, sedimentation and diffusion constants, electrophoretic mobility and salt solubility). This similarity is consistent with the hypothesis that induced and constitutive enzymes are formed by basically the same mechanism. The constitutive penicillinase of B. cereus 569/H is, however, quite distinct physicochemically from the constitutive penicillinase of B. cereus 5/B. Fractionation of the supernatant fluid from an uninduced culture of B. cereus 569 by the specific technique used for isolation of penicillinase yielded only 2.5% of the amount of protein obtained under comparable conditions from an induced culture. It is concluded that induction of penicillinase activity by penicillin corresponds to formation of a specific, physicochemically distinct protein. Diffusion and sedimentation of 4 samples of penicillinase were seen. Sample 5/B differs from all others; samples 569 (induced) and 569/H(2) appear to be identical; samples 569/H(l) and 569/H(2) are different. All samples except 569/H(1) appear homogeneous.