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Kinetics of enzymatic solid‐to‐solid peptide synthesis: Synthesis of Z ‐aspartame and control of acid–base conditions by using inorganic salts
Author(s) -
Erbeldinger Markus,
Ni Xiongwei,
Halling Peter J.
Publication year - 2000
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/1097-0290(20010105)72:1<69::aid-bit10>3.0.co;2-p
Subject(s) - aspartame , chemistry , thermolysin , yield (engineering) , base (topology) , reaction rate , peptide synthesis , peptide , catalysis , enzyme , stereochemistry , inorganic chemistry , organic chemistry , biochemistry , mathematical analysis , materials science , trypsin , mathematics , metallurgy
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. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 69–76, 2001.