Purification of beta-lactamase from Streptomyces cellulosae by affinity chromatography on Blue Sepharose.
A beta-lactamase from culture supernatant of Streptomyces 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 microM. The beta-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 x 10(3) M-1. The beta-lactamase decreases its enzymatic activity against benzylpenicillin in the presence of NADP+. From these results, it is suggested that this beta-lactamase has a dinucleotide binding fold.
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000096:biosynthesis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D008722:Methods; D009711:Nucleotides / Q000494:pharmacology; D012685:Sepharose; D013302:Streptomyces / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000302:isolation & purification*
D000596:Amino Acids; D001426:Bacterial Proteins; D009711:Nucleotides; D065093:beta-Lactamase Inhibitors; D012685:Sepharose; D001618:beta-Lactamases