Erbeldinger M, Ni X, Halling P J
Department of Bioscience and Biotechnology, University of Strathclyde, Glasgow, UK.
Biotechnol Bioeng. 2001 Jan 5;72(1):69-76.
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.
酶促肽合成可以在固-固反应混合物中高效进行，向底物混合物中添加10%（w/w）的水。最终反应产物中产物含量≥80%（重量）。本文探讨了此类反应混合物中的酸碱效应及其对酶的影响。在嗜热酶催化合成Z-天冬酰胺-苯丙氨酸甲酯的过程中，反应速率强烈依赖于添加到体系中的碱性盐的量。当添加的KHCO₃或K₂CO₃从与苯丙氨酸甲酯起始原料等摩尔的量增加2.25倍时，反应速率提高20倍。随着KHCO₃添加量的进一步增加，初始速率保持在最大值，但添加K₂CO₃时反应速率急剧下降。添加NaHCO₃的效果较差，但如果使用更多的水，反应速率会更快。当碱性盐添加量超过1.5当量时，反应的最终产率会降低。当嗜热菌蛋白酶催化相同反应或Z-谷氨酰胺-亮氨酸-氨基合成时，也观察到了类似的效应。考虑到可能形成多达十种不同的固相，使用一个估算这些体系pH值的模型可以解释这些效应。
