title: A novel one-step expression and immobilization method for the production of biocatalytic preparations.
abstract: BACKGROUND
  Whole cell biocatalysts and isolated enzymes are considered as state of the art in biocatalytic preparations for industrial applications. Whole cells as biocatalysts are disadvantageous if substrate or products are toxic to the cells or undesired byproducts are formed due to the cellular metabolism. The use of isolated enzymes in comparison is more expensive due to the required downstream processing. Immobilization of enzymes after purification increases preparation costs for biocatalysts significantly, but allows for the efficient reuse of the enzymes in the biocatalytic process. For a more rapid processing one-step expression and immobilization is desirable.
  
  
  RESULTS
  This study focused on the development of a new one-step expression and immobilization technique for enzymes on the example of the β-galactosidase from Escherichia coli K12. The enzyme was expressed in E. coli with a C-terminal membrane anchor originating from cytochrome b5 from rabbit liver and was thus in situ immobilized to the inner surface of the cytosolic membrane. Then, the expression of a lytic phage protein (gene E from PhiX174) caused the formation of a pore in the cell wall of E. coli, which resulted in release of the cytosol. The cellular envelopes with immobilized enzymes were retained. Batch and fed-batch processes were developed for efficient production of these biocatalysts. It was possible to obtain cellular envelopes with up to 27,200 ± 10,460 immobilized enzyme molecules per cellular envelope (753 ± 190 U/gdry weight). A thorough characterization of the effects of membrane immobilization was performed. Comparison to whole cells showed that mass transfer limitation was reduced in cellular envelopes due to the pore formation.
  
  
  CONCLUSIONS
  In this study the feasibility of a new one-step expression and immobilization technique for the generation of biocatalytic preparations was demonstrated. The technique could be a useful tool especially for enzyme systems, which are not suitable for whole-cell biocatalysts due to severe mass transfer limitations or undesired side reactions mediated by cytosolic enzymes.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748, Garching, Germany. i.suehrer@lrz.tum.de.
authors.forename: Ilka
authors.initials: I
authors.lastname: Sührer
authors.affiliation: BIRD-C GmbH & Co KG, Erne-Seder-Gasse 4/2, 1030, Vienna, Austria. timo.langemann@bird-c.at.
authors.forename: Timo
authors.initials: T
authors.lastname: Langemann
authors.affiliation: BIRD-C GmbH & Co KG, Erne-Seder-Gasse 4/2, 1030, Vienna, Austria. werner.lubitz@bird-c.at.
authors.forename: Werner
authors.initials: W
authors.lastname: Lubitz
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748, Garching, Germany. d.weuster-botz@lrz.tum.de.
authors.forename: Dirk
authors.initials: D
authors.lastname: Weuster-Botz
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748, Garching, Germany. k.castiglione@lrz.tum.de.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Castiglione
mesh_terms: D000818:Animals; D001435:Bacteriophages / Q000378:metabolism; D055162:Biocatalysis; D019149:Bioreactors; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D011817:Rabbits; D014764:Viral Proteins / Q000378:metabolism; D001616:beta-Galactosidase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004800:Enzymes, Immobilized; D014764:Viral Proteins; D001616:beta-Galactosidase