Abstract
BackgroundWhole 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.ResultsThis 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.ConclusionIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0371-9) contains supplementary material, which is available to authorized users.
Highlights
Whole cell biocatalysts and isolated enzymes are considered as state of the art in biocatalytic prepara‐ tions for industrial applications
Production of cellular envelopes with immobilized β‐galactosidase In order to evaluate the new system, the β-galactosidase from E. coli K12 was fused with the C-terminal hydrophobic sequence of cytochrome b5 from rabbit liver, resulting in the β-gal-cyt b5-fusion protein [18]
A new one-step expression and immobilization system was established for the production of cellular envelopes with immobilized enzymes as new biocatalytic preparations
Summary
Whole cell biocatalysts and isolated enzymes are considered as state of the art in biocatalytic prepara‐ tions for industrial applications. Whole cell biocatalysts are cheap in production and have internal cofactor regeneration [1] They can produce undesired byproducts due to the cellular metabolism, which complicate downstream processing [2]. Immobilization of enzymes after purification increases preparation costs for biocatalysts, but allows for the efficient reuse of the Sührer et al Microb Cell Fact (2015) 14:180 enzymes in the biocatalytic process [5, 8]. The enzymes are attached to the outer membrane by fusing them to outer membrane proteins or so called autotransporters, to ensure sufficient translocation of the enzymes to the outer membrane [9,10,11] Such whole cell biocatalysts displaying enzymes on their surface retain their complete metabolism, which can again cause the formation of undesired byproducts. A new approach of one-step expression and immobilization was aimed for
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