Thermostabilization of cellulosomal endoglucanase EngB from Clostridium cellulovorans by in vitro DNA recombination with non-cellulosomal endoglucanase EngD.

Abstract:

:Enhancement of enzyme thermostability by protein engineering gives us information about the thermostabilization mechanism as well as advantages for industrial use of enzymes. In this study, we enhanced the thermostability of endoglucanase EngB, one component of the cellulase complex (cellulosome) from Clostridium cellulovorans, by the directed evolution technique. The library was constructed by in vitro recombination of the genes for EngB and non-cellulosomal cellulase EngD, based on the fact that the catalytic domains of both cellulases were highly homologous. To obtain thermostable clones without loss of activity, the library was screened by a combination of activity and thermostability screening. We obtained three mutants out of 8000 selected clones that showed significantly higher thermostability than those of EngB and EngD without compromising their endoglucanase activities. One of the mutants possessed a sevenfold higher thermostability than EngB. The possible mechanisms of thermostabilization are discussed.

journal_name

Mol Microbiol

journal_title

Molecular microbiology

authors

Murashima K,Kosugi A,Doi RH

doi

10.1046/j.1365-2958.2002.03049.x

subject

Has Abstract

pub_date

2002-08-01 00:00:00

pages

617-26

issue

3

eissn

0950-382X

issn

1365-2958

pii

3049

journal_volume

45

pub_type

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