Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst

Ulijn, R.V. and Erbeldinger, M. and Halling, P.J. (2000) Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst. Biotechnology and Bioengineering, 69 (6). pp. 633-638. ISSN 0006-3592 (http://dx.doi.org/10.1002/1097-0290(20000920)69:6<...)

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Abstract

This is a comparative study of the performance of thermolysin for enzymatic peptide synthesis by reversed hydrolysis in several different reaction systems. Z-Gln-Leu-NH2 was synthesized in acetonitrile containing 5% water (with various catalyst preparation methods) as well as by the solid-to-solid and frozen aqueous methods. Reaction rates (values in nanomoles per minute per milligram) in acetonitrile depended significantly on the method of addition of enzyme: (a) direct suspension in the reaction mixture as freeze-dried powders gave 60 to 95; (b) addition as an aqueous solution, so that enzyme precipitates on mixing with acetonitrile, gave 230; (c) addition as an aqueous suspension gave a remarkable increase in reaction rates (up to 780); (d) immobilized enzymes (adsorbed at saturating loading on celite, silica, Amberlite XAD-7, or polypropylene, then dried by propanol rinsing) all gave <230. It is postulated that, starting with the enzyme already in the form of solid particles in aqueous buffer, there is a minimum chance of alteration of its optimal conformation during transfer to the organic medium. For solid-to-solid synthesis with 10% water content we found initial rates of 670 under optimized conditions. In frozen aqueous synthesis, rates were <10. Equilibrium yields were always around 60% in low water organic solvent, whereas they were found to >80% in the aqueous systems studied

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