Rational Design of Thermostable Lactate Oxidase by Analyzing Quaternary Structure and Prevention of Deamidation |
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Authors: | Email author" target="_blank">Hiroki?KanekoEmail author Hirotaka?Minagawa Jiro?Shimada |
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Institution: | (1) Department of Integrated Sciences in Physics and Biology, College of Humanities and Sciences, Nihon University, 156-8550 Tokyo, Setagaya, Japan;(2) Fundamental and Environmental Research Laboratories, NEC Corporation, 305-8501 Ibaraki, Tsukuba, Japan |
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Abstract: | Our current knowledge of protein unfolding is overwhelmingly related to reversible denaturation. However, to engineer thermostable
enzymes for industrial applications and medical diagnostics, it is necessary to consider irreversible denaturation processes
and/or the entire quaternary structure. In this study we have used lactate oxidase (LOD), which is employed in lactic acid
sensors, as a model example to design thermostable variants by rational design. Twelve mutant proteins were tested and one
of them displayed a markedly greater thermostability than all the mutants we had previously obtained by random mutagenesis.
This mutant was designed so as to strengthen the interaction between the subunits and stabilize the quaternary structure.
Since LOD is difficult to crystallize, its three-dimensional structure remains unknown. This study shows that it is possible
to carry out rational design to improve thermostability using a computer-aided quaternary structure model based on the known
tertiary structure of a related protein. Critical factors required for increasing the thermal stability of proteins by rational
design, where the 3-D structure is not available, are discussed.
Revisions requested 18 August 2005; Revisoins received 6 September 2005 |
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Keywords: | biosensor computer-aided design lactate oxidase protein engineering thermostability |
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