An extracellular proteinase from Thermus strain Rt41A was immobilized to controlled pore glass (CPG) beads. The properties of the free and CPG-immobilized enzymes were compared using both a large (azocasein) and a small (peptidase) substrate. The specific activity of the immobilized proteinase was 5284 azoU/mg with azocasein and 144 sucU/mg for SucAAPFpNA. The percentage recovery of enzyme activity was unaffected by pore size when it was immobilized at a fixed level of activity/g of beads, whereas it increased with increasing pore size when added at a fixed level/m(2) of support. Saturation of the CPG beads was observed at 540 azoU/m(2) of 105-nm beads. Lower levels (50 azoU/m(2) of 50-nm beads) were used in characterization experiments. The pH optimum of the immobilized Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA, compared with the free proteinase which was 10.5 for both substrates. The immobilized enzyme retained 65% of its maximum activity against azocasein at pH 12, whereas the free proteinase retained less than 10% under the same conditions. Stability at 80 degrees C increased on immobilization at all pH values between 5 and 11, the greatest increase in half-life being approximately 12-fold at pH 7.0. Temperature-activity profiles for both the free and immobilized enzymes were similar for both substrates. The stability of the immobilized proteinase, however, was higher than that of the free enzyme in the absence and presence of CaCl(2). Overall, the results show that low levels of calcium (10 muM) protect against thermal denaturation, but that high calcium or immobilization are required to protect against autolysis. (c) 1994 John Wiley & Sons, Inc.
A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield.
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