Rational design of thermostable lactate oxidase by analyzing quaternary structure and prevention of deamidation.

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.

journal_name

Biotechnol Lett

journal_title

Biotechnology letters

authors

Kaneko H,Minagawa H,Shimada J

doi

10.1007/s10529-005-3555-2

keywords:

subject

Has Abstract

pub_date

2005-11-01 00:00:00

pages

1777-84

issue

22

eissn

0141-5492

issn

1573-6776

journal_volume

27

pub_type

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