Comparison of methods for thermolysin-catalyzed peptide synthesis including a novel more active catalyst.
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-NH(2) 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.
Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow G1 1 XL, Scotland.rein.ulijn@strath.ac.uk
R V
RV
Ulijn
M
M
Erbeldinger
P J
PJ
Halling
D001671:Biochemistry / Q000379:methods*; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013820:Thermolysin / Q000737:chemistry / Q000378:metabolism*; D014867:Water
D010455:Peptides; D012997:Solvents; D014867:Water; D013820:Thermolysin