UNUSUAL CHROMATOGRAPHIC BEHAVIOR AND ONE-STEP PURIFICATION OF A NOVELMEMBRANE PROTEINASE FROM BACILLUS-CEREUS

Cell envelopes of Bacillus cereus contain a casein-cleaving membrane p roteinase (CCMP) and an insulin-cleaving membrane proteinase (ICMP), w hich differ in their substrate and inhibitor specificity from all Baci llus proteinases described previously. They remained localized in the cytoplasmic membrane after treatment with lysozyme and mutanolysin and they are strongly attached to the membrane compared with other known membrane proteinases. Only high a concentration of the zwitterionic de tergent sulfobetain SB-12 enabled an effective solubilization of both membrane proteinases. The usual conventional purification methods, suc h as chromatofocusing, ion-exchange chromatography and hydrophobic int eraction chromatography in the presence of detergent concentrations be yond their critical micelle concentration, could not be applied to the purification, because the solubilized membrane proteinases bound stro ngly and irreversibly to the chromatographic matrix. In the search for other purification methods, we used a tentacle ion-exchanger (EMD tri methylaminoethyl-Fractogel) to reduce the hydrophobic interactions bet ween the proteinases and the matrix. All contaminating proteins could be removed by a first gradient of sodium chloride without elution of C CMP; a second gradient with isopropanol and a decreasing salt concentr ation resulted in an efficiently purified CCMP. The ICMP was irreversi bly denaturated. Purified CCMP is a member of the metalloproteinase fa mily with a pH optimum in the neutral range and a temperature optimum of 40 degrees C, whose properties differ from the serine-type membrane proteinase of Bacillus subtilis described by Shimizu et al. [Agric. B iol. Chem., 47 (1983) 1775]. It consists of two subunits in sodiumdode cyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reduci ng conditions (M(r) 53 000 and 65 000); however, the molecular mass of the purified enzyme could not be determined by size exclusion or SDS- PAGE, because the purified enzyme aggregated at the top of the gel mat rix. CCMP solubilized before the purification process, could be eluted in the presence of 0.1% octylphenol-poly(ethyleneglycol ether)(9-10) (Triton X-100) in two peaks of M(r) 56 000 and 128 000, respectively. We discuss this special chromatographic behaviour of the CCMP from Bac illus cereus, with regard to the strong hydrophobic interactions of th e enzyme with the chromatographic matrix and additional self-aggregati on, which could only be dissolved by solvents such as isopropanol.