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Rational and combinatorial engineering of the glucan synthesizing enzyme amylosucrase

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Rational engineering of amylosucrase required detailed investigations of the molecular basis of catalysis. Biochemical characterization of the enzyme coupled to structural analyses enabled the polymerization mechanism to be elucidated. This provided key information for successfully changing amylosucrase to a hydrolase or an improved polymerase using site-directed mutagenesis. In parallel, a combinatorial approach was developed to further improve the catalyst. The method, based on random mutagenesis and recombination of parent sequences, has generated libraries of variants. These were searched for improved polymer formation using a first step of selection followed by a screening test including a double detection method. Several mutants with desired properties have been isolated, their sequences revealed that they could not have been designed following a rational approach.
OriginalsprogEngelsk
TidsskriftBiocatalysis and Biotransformation
Vol/bind21
Udgave nummer4-5
Sider (fra-til)271-277
Antal sider7
ISSN1024-2422
DOI
StatusUdgivet - 1 aug. 2003

ID: 45670807