Daniel, Roy M.Peek, KeithWilson, Shelley-Ann2026-07-072026-07-071991https://hdl.handle.net/10289/18430A proteinase isolated from Thermus strain Rt41A was purified to apparent homogeneity using ion exchange and FPLC chromatography and had a final specific activity of approximately 9000 azocasein units/mg. The substrate specificity of the proteinase against various peptide p-nitroanilide substrates was investigated and compared with the results of Peek (unpublished results) and Cowan et al. (1987b) Rt41A proteinase has a preference for aromatic amino acids in the P₁ site but, will also cleave the pNA group from peptides with leucine, arginine, alanine and lysine in this position. When utilising p-nitroanilide substrates the peptide must have a minimum of 3 amino acids for activity to be observed. Rt41A proteinase was immobilised to controlled pore glass (CPG) and Polymer Carrier Va-Epoxy Biosynth resin. The former of which was compared to the free proteinase. Both large (azocasein) and small (SucAAPFpNA) molecular weight substrates were utilised to measure the activity of the immobilised proteinase. Pore size of the CPG beads had little effect on % recovery of enzyme activity when it was added at a fixed level of activity/g of beads, whereas, it increased with increasing pore size when added at a fixed level/m² of support. Saturation of the CPG beads, with 105 nm pore size, was observed at 540 azocasein units/m². Lower levels (50 azocasein units/m² of 50 nm beads) were used in characterisation experiments. The specific activity of the immobilised proteinase was 5284 Au/mg with azocasein and 144 U/mg for SucAAPFpNA. The pH optimum of the immobilised Rt41A proteinase was 8.0 for azocasein and 9.5 for SucAAPFpNA compared to 10.5 for both substrates for the free enzyme. The immobilised enzyme retained approximately 65% of its maximum activity, against azocasein, at pH 12 whereas the free proteinase retained less than 10%. Stability towards pH increased on immobilisation, the greatest increase in half-life being approximately 12 fold at pH 7.0 at and 80 °C. Temperature activity profiles for both the free and immobilised enzymes were very similar when using either substrate. The stability of the immobilised proteinase however, was higher than that of the free enzyme in the presence and absence of CaCl₂ at various temperatures. Overall the results show that low levels of calcium (10 μM) protect against thermal denaturation, but that a high calcium concentration or immobilisation are required to protect against autolysis. Immobilisation of the proteinase also increased its stability towards two organic solvents, DMF and heptanol. Both the free and immobilised proteinase were utilised for peptide synthesis and the observed maximum yields were 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 and Tyr-NH₂, Ala-NH₂, Trp-NH₂, Phe-NH₂, Leu-pNA and Val-pNA were all reactive nucleophiles. The substrate specificity for peptide synthesis was therefore very similar to that found for hydrolysis reactions. The synthesis of Bz-Ala-Tyr-NH2 was optimised by studying the effect of pH, temperature, solvent concentration, ionic strength and nucleophile and acyl donor concentration on the partition constant and the maximum yield. The initial conditions used were 25 mM Bz-Ala-OMe, 25 mM Tyr-NH₂, 70 °C, pH 8.0, and 10% v/v DMF. The optimised conditions were 80 mM Bz-Ala-OMe, 615 mM Tyr-NH₂, 40 °C, pH 10.0 and 90% v/v DMF; these conditions increased the maximum yield from 0.75% to 26% (% of original ester concentration). A number of cosolvents were also compared; the best peptide yields were observed with acetonitrile and ethyl acetate. Under optimised conditions acetonitrile gave similar yields (20%) as those observed in DMF except the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100 mM respectively. The effect of different nucleophiles was also investigated with respect to peptide yield. Tyr- βNA and Tyr-pNA were found to be better nucleophiles than Tyr-NH₂. The yields observed were 48%, 61% and 8% respectively (90% v/v DMF, 40 °C, 25 mM Bz-Ala-OMe, 100 mM Tyr-X). No peptide was produced with Tyr-OBu as the nucleophile under the same conditions. With Ala in the P₁ position, and Tyr-pNA as the nucleophile, changing the blocking group or leaving group of the acyl donor had no effect on the peptide yield. Extending the length of the peptide of the acyl donor also had no effect. Peptide yields with Bz-Ala-OMe and Tyr-βNA in both DMF and acetonitrile was compared under optimised conditions. Quantitative yields were observed in DMF whereas only 26% yield was found in acetonitrile. Therefore acetonitrile, compared to DMF, increased peptide yields with the Tyr-NH₂ nucleophile, decreased yields with the Tyr-βNA nucleophile and it was also shown to have no affect on yields with the Tyr-pNA nucleophile.enAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.Thesis