title: 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.
authors.affiliation: Institute of Thermodynamics, Helmut-Schmidt-University Hamburg, Holstenhofweg 85, 22043, Hamburg, Germany, peper@hsu-hh.de.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Peper
authors.forename: Selin
authors.initials: S
authors.lastname: Kara
authors.forename: Wei Sing
authors.initials: WS
authors.lastname: Long
authors.forename: Andreas
authors.initials: A
authors.lastname: Liese
authors.forename: Bernd
authors.initials: B
authors.lastname: Niemeyer
mesh_terms: D000079:Acetaldehyde / Q000737:chemistry; D001547:Benzaldehydes / Q000737:chemistry; D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000378:metabolism*; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006910:Hydroxypropiophenone / Q000138:chemical synthesis*; D007700:Kinetics; D011312:Pressure; D016958:Pseudomonas putida / Q000201:enzymology*; D012822:Silicon Dioxide / Q000737:chemistry; D013237:Stereoisomerism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001547:Benzaldehydes; D004800:Enzymes, Immobilized; D006910:Hydroxypropiophenone; D012822:Silicon Dioxide; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase; D000079:Acetaldehyde; C032175:benzaldehyde