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Creating Space for Large Secondary Alcohols by Rational Redesign of Candida antarctica Lipase B
Author(s) -
Magnusson Anders O.,
RotticciMulder Johanna C.,
Santagostino Alberto,
Hult Karl
Publication year - 2005
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200400410
Subject(s) - candida antarctica , stereospecificity , substituent , chemistry , enantiomer , stereochemistry , active site , oxyanion hole , substrate (aquarium) , lipase , kinetic resolution , selectivity , enantiomeric excess , catalysis , organic chemistry , enzyme , enantioselective synthesis , biology , ecology
The active site of Candida antarctica lipase B (CALB) hosts the catalytic triad (Ser‐His‐Asp), an oxyanion hole and a stereospecificity pocket. During catalysis, the fast‐reacting enantiomer of secondary alcohols places its medium‐sized substituent in the stereospecificity pocket and its large substituent towards the active‐site entrance. The largest group to fit comfortably in the stereospecificity pocket is ethyl, and this restricts the number of secondary alcohols that are good substrates for CALB. In order to overcome this limitation, the size of the stereospecificity pocket was redesigned by changing Trp104. The substrate specificity of the Trp104Ala mutant compared to that of the wild‐type lipase increased 270 times towards heptan‐4‐ol and 5500 times towards nonan‐5‐ol; this resulted in the high specificity constants 1100 and 830 s −1 M −1 , respectively. The substrate selectivity changed over 400 000 times for nonan‐5‐ol over propan‐2‐ol with both Trp104Ala and the Trp104Gln mutations.