PURIFICATION OF BETA-LACTAMASE FROM STREPTOMYCES-CELLULOSAE BY AFFINITY CHROMATOGRAPHY ON BLUE SEPHAROSE

A β-lactamase from culture supernatant of Streptomyees cellulosae was purified about 1,450-fold to apparent homogeneity in polyacrylamide gel electrophoresis and isoelectric focusing on polyacrylamide gel sheet. The methods used were ammonium sulfate precipitation, CM-52 cellulose ion-exchange chromatography and affinity chromatography on Blue Sepharose CL-6B. The molecular weight was determined to be approximately 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This value was in good agreement with the previous value determined by gel filtration on Sephadex G-75. The isoelectric point was pH 9.5. The enzyme behaved primarily as penicillinase and apparent Km value for benzylpenicillin was 500 µ M. The β-lactamase of S. cellulosae interacted strongly with blue dextran and NADP+-agarose but not with Sepharose. In addition, the presence of NADP+ but not NAD+ and ATP diminished sharply the intrinsic fluorescence intensity of the enzyme and the apparent association constant was calculated to be 1.4 χ103 M-1. The β-lactamase decreases its enzymatic activity against benzylpenicillin in the presence of NADP+. From these results, it is suggested that this β-lactamase has a dinucleotide binding fold. © 1979, JAPAN ANTIBIOTICS RESEARCH ASSOCIATION. All rights reserved.