title: HaloTag™: Evaluation of a covalent one-step immobilization for biocatalysis.
abstract: Easy, fast and gentle immobilization for the efficient reuse of important biocatalysts is highly demanded. We used the commercially available HaloTag™ technology (Promega), so far relatively unknown in the context of biocatalysis, to immobilize the benzaldehyde lyase from P. fluorescence (PfBAL). Immobilization mediated by this fusion tag proceeds rapidly within minutes from crude extracts yielding covalently attached enzymes in high purity, making expensive and laborious previous chromatographic purification steps obsolete, which strongly reduces the costs for biocatalyst immobilization. Further, we introduce a novel design of HaloTag fusions and demonstrate the positive effect of the tag on soluble expression and activity of PfBAL. The immobilized biocatalyst was stable at 4°C for months and was successfully reused in several repetitive batches for the carboligation of aggressive aldehydes.
authors.affiliation: IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Döbber
authors.affiliation: IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany. Electronic address: ma.pohl@fz-juelich.de.
authors.forename: Martina
authors.initials: M
authors.lastname: Pohl
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000378:metabolism; D005818:Genetic Engineering; D011551:Pseudomonas fluorescens / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D012240:Rhodococcus
chemical_list: D001426:Bacterial Proteins; D004800:Enzymes, Immobilized; D011994:Recombinant Proteins; D000446:Aldehyde-Lyases; C059416:benzaldehyde lyase
keywords: Benzaldehyde lyase; CC bond formation; Covalent enzyme immobilization; Economizing production process; Enzyme fusion; Repetitive batch