Tailoring the properties of (catalytically)-active inclusion bodies
Abstract Background Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromato...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2019-02-01
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| Series: | Microbial Cell Factories |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12934-019-1081-5 |
| _version_ | 1819169160977973248 |
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| author | V. D. Jäger R. Kloss A. Grünberger S. Seide D. Hahn T. Karmainski M. Piqueray J. Embruch S. Longerich U. Mackfeld K.-E. Jaeger W. Wiechert M. Pohl U. Krauss |
| author_facet | V. D. Jäger R. Kloss A. Grünberger S. Seide D. Hahn T. Karmainski M. Piqueray J. Embruch S. Longerich U. Mackfeld K.-E. Jaeger W. Wiechert M. Pohl U. Krauss |
| author_sort | V. D. Jäger |
| collection | DOAJ |
| description | Abstract Background Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. Results We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. Conclusion We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry. |
| first_indexed | 2024-12-22T19:15:06Z |
| format | Article |
| id | doaj.art-fd6bd3eb40e84dfabafbcac1ca939e09 |
| institution | Directory Open Access Journal |
| issn | 1475-2859 |
| language | English |
| last_indexed | 2024-12-22T19:15:06Z |
| publishDate | 2019-02-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj.art-fd6bd3eb40e84dfabafbcac1ca939e092022-12-21T18:15:32ZengBMCMicrobial Cell Factories1475-28592019-02-0118112010.1186/s12934-019-1081-5Tailoring the properties of (catalytically)-active inclusion bodiesV. D. Jäger0R. Kloss1A. Grünberger2S. Seide3D. Hahn4T. Karmainski5M. Piqueray6J. Embruch7S. Longerich8U. Mackfeld9K.-E. Jaeger10W. Wiechert11M. Pohl12U. Krauss13Institut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum JülichIBG-1: Biotechnology, Forschungszentrum Jülich GmbHIBG-1: Biotechnology, Forschungszentrum Jülich GmbHIBG-1: Biotechnology, Forschungszentrum Jülich GmbHIBG-1: Biotechnology, Forschungszentrum Jülich GmbHIBG-1: Biotechnology, Forschungszentrum Jülich GmbHInstitut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum JülichIBG-1: Biotechnology, Forschungszentrum Jülich GmbHInstitut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum JülichIBG-1: Biotechnology, Forschungszentrum Jülich GmbHInstitut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum JülichIBG-1: Biotechnology, Forschungszentrum Jülich GmbHIBG-1: Biotechnology, Forschungszentrum Jülich GmbHInstitut für Molekulare Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf, Forschungszentrum JülichAbstract Background Immobilization is an appropriate tool to ease the handling and recycling of enzymes in biocatalytic processes and to increase their stability. Most of the established immobilization methods require case-to-case optimization, which is laborious and time-consuming. Often, (chromatographic) enzyme purification is required and stable immobilization usually includes additional cross-linking or adsorption steps. We have previously shown in a few case studies that the molecular biological fusion of an aggregation-inducing tag to a target protein induces the intracellular formation of protein aggregates, so called inclusion bodies (IBs), which to a certain degree retain their (catalytic) function. This enables the combination of protein production and immobilization in one step. Hence, those biologically-produced immobilizates were named catalytically-active inclusion bodies (CatIBs) or, in case of proteins without catalytic activity, functional IBs (FIBs). While this strategy has been proven successful, the efficiency, the potential for optimization and important CatIB/FIB properties like yield, activity and morphology have not been investigated systematically. Results We here evaluated a CatIB/FIB toolbox of different enzymes and proteins. Different optimization strategies, like linker deletion, C- versus N-terminal fusion and the fusion of alternative aggregation-inducing tags were evaluated. The obtained CatIBs/FIBs varied with respect to formation efficiency, yield, composition and residual activity, which could be correlated to differences in their morphology; as revealed by (electron) microscopy. Last but not least, we demonstrate that the CatIB/FIB formation efficiency appears to be correlated to the solvent-accessible hydrophobic surface area of the target protein, providing a structure-based rationale for our strategy and opening up the possibility to predict its efficiency for any given target protein. Conclusion We here provide evidence for the general applicability, predictability and flexibility of the CatIB/FIB immobilization strategy, highlighting the application potential of CatIB-based enzyme immobilizates for synthetic chemistry, biocatalysis and industry.http://link.springer.com/article/10.1186/s12934-019-1081-5ImmobilizationBiocatalysisInclusion bodiesProtein aggregatesProtein engineeringStructure–function relations |
| spellingShingle | V. D. Jäger R. Kloss A. Grünberger S. Seide D. Hahn T. Karmainski M. Piqueray J. Embruch S. Longerich U. Mackfeld K.-E. Jaeger W. Wiechert M. Pohl U. Krauss Tailoring the properties of (catalytically)-active inclusion bodies Microbial Cell Factories Immobilization Biocatalysis Inclusion bodies Protein aggregates Protein engineering Structure–function relations |
| title | Tailoring the properties of (catalytically)-active inclusion bodies |
| title_full | Tailoring the properties of (catalytically)-active inclusion bodies |
| title_fullStr | Tailoring the properties of (catalytically)-active inclusion bodies |
| title_full_unstemmed | Tailoring the properties of (catalytically)-active inclusion bodies |
| title_short | Tailoring the properties of (catalytically)-active inclusion bodies |
| title_sort | tailoring the properties of catalytically active inclusion bodies |
| topic | Immobilization Biocatalysis Inclusion bodies Protein aggregates Protein engineering Structure–function relations |
| url | http://link.springer.com/article/10.1186/s12934-019-1081-5 |
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