Abstract
To study enzymological properties and usefulness of continuous enzymic reaction of the insoluble derivatives of semi-alkaline proteinase (SAP) from Aspergillus melleus, the purified SAP was transformed into insoluble forms by coupling its native form to insoluble carriers, using CM-cellulose (CMC), copolymer of ethylene and maleic anhydride, (EMA) and Sephadex. The optimum temperature and thermostability increased by the insolublization. The optimum pH of EMA-SAP and CMC-SAP was at pH 7-10 and 10-11, respectively, against soluble SAP and Sephadex-SAP at pH 8.0. The effect of protein denaturation reagents, metal ions, and surface-active reagents on insoluble SAP was also examined. Insoluble SAP preparations were little inhibited by sodium dioctylsulfosuccinate whereas soluble SAP lost most of the activity. The activity remaining in the insoluble SAP stored in a buffer of pH 6.0 for several months was much higher than that of soluble SAP and a column of Sephadex-SAP through which casein solution was continuously passed for 20 days retained about 40% of initial activity. Little difference between the soluble and insoluble SAP with respect to Km values and the energy of activation suggested that the conformation of SAP does not change by insolublization. From the analysis of amino acid composition of Sephadex-SAP, the basic amino groups of SAP useful for coupling with activated Sephadex were lysyl and arginyl residues.
