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Kinetic analysis of enhanced thermal stability of an alkaline protease with engineered twin disulfide bridges and calcium‐dependent stability
Author(s) -
Ikegaya Kazuo,
Sugio Shigetoshi,
Murakami Kohji,
Yamanouchi Kouichi
Publication year - 2002
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/bit.10489
Subject(s) - chemistry , calcium , enthalpy , thermal stability , cysteine , protease , enzyme , thermodynamics , biochemistry , organic chemistry , physics
The thermal stability of a cysteine‐free alkaline protease (Alp) secreted by the eukaryote Aspergillus oryzae was improved both by the introduction of engineered twin disulfide bridges (Cys‐69/Cys‐101 and Cys‐169/Cys‐200), newly constructed as part of this study, and by the addition of calcium ions. We performed an extensive kinetic analysis of the increased thermal stability of the mutants as well as the role of calcium dependence. The thermodynamic activation parameters for irreversible thermal inactivation, the activation free energy ( ΔG ‡ ), the activation enthalpy ( ΔH ‡ ), and the activation entropy ( ΔS ‡ ) were determined from absolute reaction rate theory. The values of ΔH ‡ and ΔS ‡ were significantly and concomitantly increased as a result of introducing the twin disulfide bridges, for which the increase in the value of ΔH ‡ outweighed that of ΔS ‡ , resulting in significant increases in the value of ΔG ‡ . The enhancement of the thermal stability obtained by introducing the twin disulfide bridges is an example of the so‐called low‐temperature stabilization of enzymes. The stabilizing effect of calcium ions on wild‐type Alp is similar to the results we obtained by introducing the engineered twin disulfide bridges. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 187–192, 2003.