title: Purification and simultaneous immobilization of Arabidopsis thaliana hydroxynitrile lyase using a family 2 carbohydrate-binding module.
abstract: Tedious, time- and labor-intensive protein purification and immobilization procedures still represent a major bottleneck limiting the widespread application of enzymes in synthetic chemistry and industry. We here exemplify a simple strategy for the direct site-specific immobilization of proteins from crude cell extracts by fusion of a family 2 carbohydrate-binding module (CBM) derived from the exoglucanase/xylanase Cex from Cellulomonas fimi to a target enzyme. By employing a tripartite fusion protein consisting of the CBM, a flavin-based fluorescent protein (FbFP), and the Arabidopsis thaliana hydroxynitrile lyase (AtHNL), binding to cellulosic carrier materials can easily be monitored via FbFP fluorescence. Adsorption properties (kinetics and quantities) were studied for commercially available Avicel PH-101 and regenerated amorphous cellulose (RAC) derived from Avicel. The resulting immobilizates showed similar activities as the wild-type enzyme but displayed increased stability in the weakly acidic pH range. Finally, Avicel, RAC and cellulose acetate (CA) preparations were used for the synthesis of (R)-mandelonitrile in micro-aqueous methyl tert-butyl ether (MTBE) demonstrating the applicability and stability of the immobilizates for biotransformations in both aqueous and organic reaction systems.
authors.affiliation: Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum Jülich, Jülich, Germany.
authors.forename: Benita
authors.initials: B
authors.lastname: Kopka
authors.forename: Martin
authors.initials: M
authors.lastname: Diener
authors.forename: Astrid
authors.initials: A
authors.lastname: Wirtz
authors.forename: Martina
authors.initials: M
authors.lastname: Pohl
authors.forename: Karl-Erich
authors.initials: KE
authors.lastname: Jaeger
authors.forename: Ulrich
authors.initials: U
authors.lastname: Krauss
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040143:Cellulomonas / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000302:isolation & purification*; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D010940:Plant Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; D000446:Aldehyde-Lyases; C021541:mandelonitrile lyase
keywords: Biocatalysis; Carbohydrate-binding module; Flavin-based fluorescent protein; Hydroxynitrile lyase; Micro-aqueous organic solvent