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Rational Design of Pseudozyma antarctica Lipase B Yielding a General Esterification Catalyst
Author(s) -
Liu Danni,
Trodler Peter,
Eiben Sabine,
Koschorreck Katja,
Müller Monika,
Pleiss Jürgen,
Maurer Steffen C.,
Branneby Cecilia,
Schmid Rolf D.,
Hauer Bernhard
Publication year - 2010
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.200900776
Subject(s) - chemistry , stereoselectivity , lipase , candida antarctica , saturated mutagenesis , transesterification , enantiomer , hydrolysis , stereospecificity , alcohol , kinetic resolution , stereochemistry , directed evolution , catalysis , organic chemistry , enzyme , enantioselective synthesis , mutant , biochemistry , gene
Pseudozyma antarctica lipase B (CALB) shows activity in the acrylation of hydroxypropylcarbamate, a racemic mixture of enantiomers of primary and secondary alcohols. However, full conversion is hampered by the slowly reacting S enantiomer of the secondary alcohol. The same is true for a wide range of secondary alcohols, for example, octan‐2‐ and ‐3‐ol. In order to get high conversion in these reactions in a short time, the stereospecificity pocket of CALB was redesigned by using predictions from molecular modeling. Positions 278, 104, and 47 were targeted, and a library for two‐site saturation mutagenesis at positions 104 and 278 was constructed. The library was then screened for hydrolysis of acrylated hydroxypropylcarbamates. The best mutants L278A, L278V, L278A/W104F, and L278A/W104F/S47A showed an increased conversion in hydrolysis and transesterification of more than 30 %. While the wild‐type showed only 73 % conversion in the acrylation of hydroxypropylcarbamate after 6 h, 97 % conversion was achieved by L278A in this time. Besides this, L278A/W104F reached >96 % conversion in the acrylation of octan‐2‐ and ‐3‐ol within 48 h and showed a significant decrease in stereoselectivity, while the wild‐type reached only 68 and 59 % conversion, respectively. Thus the new biocatalysts can be used for efficient transformation of racemic alcohols and esters with high activity when the high stereoselectivity of the wild‐type hampers complete conversion of racemic substrates in a short time.