Kinetics of enzymatic solid-to-solid peptide synthesis: synthesis of Z-aspartame and control of acid-base conditions by using inorganic salts.
Enzymatic peptide synthesis can be carried out efficiently in solid-to-solid reaction mixtures with 10% (w/w) water added to a mixture of substrates. The final reaction mass contains >/=80% (by weight) of product. This article deals with acid-base effects in such reaction mixtures and the consequences for the enzyme. In the Thermoase-catalyzed synthesis of Z-Asp-Phe-OMe, the reaction rate is strongly dependent on the amount of basic salts added to the system. The rate increases 20 times, as the KHCO(3) or K(2)CO(3) added is raised 2.25-fold from an amount equimolar to the Phe-OMe. HCL starting material. With further increases in KHCO(3) addition, the initial rate remains at the maximum, but with K(2)CO(3) it drops sharply. Addition of NaHCO(3) is less effective, but rates are faster if more water is used. With >1.5 equivalents of basic salt, the final yield of the reaction decreases. Similar effects are observed when thermolysin catalyzes the same reaction, or Z-Gln-Leu-NH(2) synthesis. These effects can be rationalized using a model estimating the pH of these systems, taking into account the possible formation of up to ten different solid phases.
Erbeldinger
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Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, UK.
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Halling
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D001218:Aspartame / Q000138:chemical synthesis* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D001709:Biotechnology / Q000379:methods; D010450:Endopeptidases; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007700:Kinetics; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism; D012492:Salts
D007202:Indicators and Reagents; D010455:Peptides; D012492:Salts; D010450:Endopeptidases; D001218:Aspartame