title: Rational design of Pseudozyma antarctica lipase B yielding a general esterification catalyst.
abstract: 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.
authors.affiliation: Institute of Technical Biochemistry, Universität Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
authors.forename: Danni
authors.initials: D
authors.lastname: Liu
authors.forename: Peter
authors.initials: P
authors.lastname: Trodler
authors.forename: Sabine
authors.initials: S
authors.lastname: Eiben
authors.forename: Katja
authors.initials: K
authors.lastname: Koschorreck
authors.forename: Monika
authors.initials: M
authors.lastname: Müller
authors.forename: Jürgen
authors.initials: J
authors.lastname: Pleiss
authors.forename: Steffen C
authors.initials: SC
authors.lastname: Maurer
authors.forename: Cecilia
authors.initials: C
authors.lastname: Branneby
authors.forename: Rolf D
authors.initials: RD
authors.lastname: Schmid
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D055162:Biocatalysis; D002175:Candida / Q000201:enzymology*; D002219:Carbamates / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004951:Esterification; D005656:Fungal Proteins; D006868:Hydrolysis; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism
chemical_list: D002219:Carbamates; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D011994:Recombinant Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica