Immobilization and characterization of benzoylformate decarboxylase from Pseudomonas putida on spherical silica carrier

Abstract

If an adequate biocatalyst is identified for a specific reaction, immobilization is one possibility to further improve its properties. The immobilization allows easy recycling, improves the enzyme performance, and it often enhances the stability of the enzyme. In this work, the immobilization of the benzoylformate decarboxylase (BFD) variant, BFD A460I-F464I, from Pseudomonas putida was accomplished on spherical silica. Silicagel is characterized by its high mechanical stability, which allows its application in different reactor types without restrictions. The covalently bound enzyme was characterized in terms of its activity, stability, and kinetics for the formation of chiral 2-hydroxypropiophenone (2-HPP) from benzaldehyde and acetaldehyde. Moreover, temperature as well as pressure dependency of immobilized BFD A460I-F464I activity and enantioselectivity were analyzed. The used wide-pore silicagel shows a good accessibility of the immobilized enzyme. The activity of the immobilized BFD A460I-F464I variant was determined to be 70% related to the activity of the free enzyme. Thereby, the enantioselectivity of the enzyme was not influenced by the immobilization. In addition, a pressure-induced change in stereoselectivity was found both for the free and for the immobilized enzyme. With increasing pressure, the enantiomeric excess (ee) of (R)-2-HPP can be increased from 44% (0.1 MPa) to 76% (200 MPa) for the free enzyme and from 43% (0.1 MPa) to 66% (200 MPa) for the immobilized enzyme.

Heterofunctional Magnetic Metal‐Chelate‐Epoxy Supports for the Purification and Covalent Immobilization of Benzoylformate Decarboxylase From Pseudomonas Putida and Its Carboligation Reactivity

“…These synthons are important intermediates in the production of antifungals . as well as of pharmaceuticals like Wellbutrin (treatment of depression) and Zyban (smoking cessation) …”

“…30,31 BFD accepts a broad range of aldehydes as substrates to catalyze the enantioselective synthesis of 2-hydroxyketones, which are useful as synthons in organic chemistry. [32][33][34][35][36] These synthons are important intermediates in the production of antifungals. 36,37 as well as of pharmaceuticals like Wellbutrin (treatment of depression) and Zyban (smoking cessation).…”

“…[32][33][34][35][36] These synthons are important intermediates in the production of antifungals. 36,37 as well as of pharmaceuticals like Wellbutrin (treatment of depression) and Zyban (smoking cessation). 36,38 In this work, the synthesis of (R)-benzoin starting from benzaldehyde and (S)-2-hydroxy-1-phenylpropanone [(S)-2-HPP] starting from benzaldehyde and acetaldehyde was catalyzed by covalently immobilized BFD on M-chelate-epoxy supports.…”

“…36,37 as well as of pharmaceuticals like Wellbutrin (treatment of depression) and Zyban (smoking cessation). 36,38 In this work, the synthesis of (R)-benzoin starting from benzaldehyde and (S)-2-hydroxy-1-phenylpropanone [(S)-2-HPP] starting from benzaldehyde and acetaldehyde was catalyzed by covalently immobilized BFD on M-chelate-epoxy supports. The M-chelate-epoxy supports were optimized to achieve the one-step purification, covalent immobilization, and stable 6xHis-tagged BFD via multipoint covalent attachment.…”

“…ThDP and is mainly engaged in a variety of carbon–carbon bond‐forming reactions, in which each unit has a special role in enzymatic catalysis . BFD accepts a broad range of aldehydes as substrates to catalyze the enantioselective synthesis of 2‐hydroxyketones, which are useful as synthons in organic chemistry . These synthons are important intermediates in the production of antifungals .…”