ABSTRACT
A general combined purification and immobilization method to facilitate biocatalytic process development is presented. The support material, EziG™, is based on controlled porosity glass (CPG) or polymer-coated versions thereof (HybCPG) and binds protein affinity tags. Biocatalytic reactions in aqueous and organic media with seven enzymes of biocatalytic interest are shown.
Subject(s)
Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Biocatalysis , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Glass , Lipase/chemistry , Lipase/metabolism , Oxygenases/chemistry , Oxygenases/metabolism , Polymers/chemistry , Porosity , Sterol Esterase/chemistry , Sterol Esterase/metabolism , Transaminases/chemistry , Transaminases/metabolismABSTRACT
The major limitation in the synthetic application of two-component Baeyer-Villiger monooxygenases was addressed by identifying the 28-kDa flavin-reductase Fre from Escherichia coli as a suitable supplier of reduced FMN for these enzymes. Coexpression of Fre with either 2,5- or 3,6-diketocamphane monooxygenase from Pseudomonas putida NCIMB 10007 significantly enhanced the conversion of camphor and norcamphor serving as representative ketones. With purified enzymes, full conversion was achieved, while only slight amounts of product were formed in the absence of this flavin reductase. Fusion of the genes of Fre and DKCMOs into single open reading frame constructs resulted in unstable proteins exhibiting flavin reducing, but poor oxygenating activity, which led to overall decreased conversion of camphor.
Subject(s)
Camphor/metabolism , Coenzymes/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , FMN Reductase/metabolism , Flavin Mononucleotide/metabolism , Mixed Function Oxygenases/metabolism , Pseudomonas putida/enzymology , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , FMN Reductase/genetics , Gene Expression , Mixed Function Oxygenases/genetics , Pseudomonas putida/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
We herein report the first directed evolution of Candida antarctica lipase A (CalA), employing a combinatorial active-site saturation test (CAST). Wild-type CalA has a modest E-value of 5.1 in kinetic resolution of 4-nitrophenyl 2-methylheptanoate. Enzyme variants were expressed in Pichia pastoris by using the episomal vector pBGP1 which allowed efficient secretory expression of the lipase. Iterative rounds of CASTing yielded variants with good selectivity toward both the (S)- and the (R)-enantiomer. The best obtained enzyme variants had E-values of 52 (S) and 27 (R).
Subject(s)
Candida/enzymology , Lipase/genetics , Pichia/genetics , Amino Acid Sequence , Catalytic Domain , Combinatorial Chemistry Techniques , Directed Molecular Evolution , Molecular Sequence Data , Peptide Library , PlasmidsABSTRACT
[formula: see text] Silylcupration of allenes with a lower order silylcuprate reagent followed by an electrophilic trapping reaction with allylic diphenyl phosphates has been studied. This reaction provides a new route to 1,4-diene systems having an allylsilane moiety.
ABSTRACT
[formula: see text] Enzymatic resolution in combination with ruthenium-catalyzed racemization of the substrate led to dynamic kinetic resolution of alpha-hydroxy esters in good yields and excellent ee's. Studies of different parameters showed that the best results were obtained using Pseudomonas cepacia lipase, ruthenium catalyst 3, and 4-chlorophenyl acetate as acyl donor in cyclohexane.
ABSTRACT
Enantiomerically pure cis and trans isomers of 4-acetoxy-[eta3(1,2,3)-cyclohexenyl]palladium chloride dimers (cis-1 and trans-1) were prepared from enantiomerically pure trans-1-acetoxy-4-chloro-2-cyclohexene. X-ray analyses of these complexes show that in the trans complex (trans-1) the six-membered ring prefers a chair conformation, whereas in the cis complex (cis-1) the cyclohexenyl ring has a boat conformation. According to the X-ray structure of trans-1 the Pd-C3 bond is shorter than the other allylic terminal palladium-carbon bond (Pd-C1). On the other hand, in cis-1 the Pd-C3 and Pd-C1 bond lengths are identical within the experimental error. The calculated structures (B3PW91/LANL2DZ + P) of trans-1 and cis-1 also display differences in the allylpalladium bonding. The asymmetric allylpalladium bonding in trans-1 is explained on the basis of pi-sigma electronic interactions between the 4-acetoxy substituent and the allyl-metal moiety.
