Accelerated production and characterization of catalytically active inclusion bodies via automated workflows

2020 ◽  
Vol 92 (9) ◽  
pp. 1201-1201
Author(s):  
K. Küsters ◽  
W. Wiechert ◽  
M. Oldiges ◽  
S. Albert
Keyword(s):  
Inclusion Bodies ◽  
Active Inclusion Bodies ◽  
Catalytically Active

Catalytically-active inclusion bodies—Carrier-free protein immobilizates for application in biotechnology and biomedicine

2017 ◽  
Vol 258 ◽  
pp. 136-147 ◽  
Author(s):  
Ulrich Krauss ◽  
Vera D. Jäger ◽  
Martin Diener ◽  
Martina Pohl ◽  
Karl-Erich Jaeger
Keyword(s):  
Inclusion Bodies ◽  
Free Protein ◽  
Active Inclusion Bodies ◽  
Carrier Free ◽  
Catalytically Active

scholarly journals Catalytically-active inclusion bodies for biotechnology—general concepts, optimization, and application

2020 ◽  
Vol 104 (17) ◽  
pp. 7313-7329
Author(s):  
Vera D. Jäger ◽  
Robin Lamm ◽  
Kira Küsters ◽  
Gizem Ölçücü ◽  
Marco Oldiges ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Active Inclusion Bodies ◽  
Catalytically Active

scholarly journals Tailoring the properties of (catalytically)-active inclusion bodies

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
V. D. Jäger ◽  
R. Kloss ◽  
A. Grünberger ◽  
S. Seide ◽  
D. Hahn ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Active Inclusion Bodies ◽  
Catalytically Active

scholarly journals Construction and comprehensive characterization of an EcLDCc-CatIB set—varying linkers and aggregation inducing tags

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Kira Küsters ◽  
Martina Pohl ◽  
Ulrich Krauss ◽  
Gizem Ölçücü ◽  
Sandor Albert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzymatic Activity ◽  
Inclusion Bodies ◽  
Early Stage ◽  
Volumetric Productivity ◽  
Efficient Production ◽  
Wild Type ◽  
Conversion Rates ◽  
Active Inclusion Bodies

Abstract Background In recent years, the production of inclusion bodies that retained substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) were formed by genetic fusion of an aggregation inducing tag to a gene of interest via short linker polypeptides and overproduction of the resulting gene fusion in Escherichia coli. The resulting CatIBs are known for their high stability, easy and cost efficient production, and recyclability and thus provide an interesting alternative to conventionally immobilized enzymes. Results Here, we present the construction and characterization of a CatIB set of the lysine decarboxylase from Escherichia coli (EcLDCc), constructed via Golden Gate Assembly. A total of ten EcLDCc variants consisting of combinations of two linker and five aggregation inducing tag sequences were generated. A flexible Serine/Glycine (SG)- as well as a rigid Proline/Threonine (PT)-Linker were tested in combination with the artificial peptides (18AWT, L6KD and GFIL8) or the coiled-coil domains (TDoT and 3HAMP) as aggregation inducing tags. The linkers were fused to the C-terminus of the EcLDCc to form a linkage between the enzyme and the aggregation inducing tags. Comprehensive morphology and enzymatic activity analyses were performed for the ten EcLDCc-CatIB variants and a wild type EcLDCc control to identify the CatIB variant with the highest activity for the decarboxylation of l-lysine to 1,5-diaminopentane. Interestingly, all of the CatIB variants possessed at least some activity, whilst most of the combinations with the rigid PT-Linker showed the highest conversion rates. EcLDCc-PT-L6KD was identified as the best of all variants allowing a volumetric productivity of 457 g L− 1 d− 1 and a specific volumetric productivity of 256 g L− 1 d− 1 gCatIB−1. Noteworthy, wild type EcLDCc, without specific aggregation inducing tags, also partially formed CatIBs, which, however showed lower activity compared to most of the newly constructed CatIB variants (volumetric productivity: 219 g L− 1 d− 1, specific volumetric activity: 106 g L− 1 d− 1 gCatIB− 1). Furthermore, we demonstrate that microscopic analysis can serve as a tool to find CatIB producing strains and thus allow for prescreening at an early stage to save time and resources. Conclusions Our results clearly show that the choice of linker and aggregation inducing tag has a strong influence on the morphology and the enzymatic activity of the CatIBs. Strikingly, the linker had the most pronounced influence on these characteristics.

scholarly journals Catalytically active inclusion bodies of L-lysine decarboxylase from E. coli for 1,5-diaminopentane production

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Ramona Kloss ◽  
Michael H. Limberg ◽  
Ursula Mackfeld ◽  
Doris Hahn ◽  
Alexander Grünberger ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Lysine Decarboxylase ◽  
E Coli ◽  
Active Inclusion Bodies ◽  
Catalytically Active

scholarly journals Thermostable adenosine 5′-monophosphate phosphorylase from Thermococcus kodakarensis forms catalytically active inclusion bodies

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sarah Kamel ◽  
Miriam C. Walczak ◽  
Felix Kaspar ◽  
Sarah Westarp ◽  
Peter Neubauer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Treatment ◽  
Inclusion Bodies ◽  
Specific Activity ◽  
High Thermal Stability ◽  
Active Protein ◽  
E Coli ◽  
Thermococcus Kodakarensis ◽  
Active Inclusion Bodies ◽  
Catalytically Active

AbstractCatalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5′-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5′-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2–6 folds higher specific activity compared to these heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.

Tailor-made catalytically active inclusion bodies for different applications in biocatalysis

2018 ◽  
Vol 8 (22) ◽  
pp. 5816-5826 ◽  
Author(s):  
Ramona Kloss ◽  
Tobias Karmainski ◽  
Vera D. Jäger ◽  
Doris Hahn ◽  
Alexander Grünberger ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Coiled Coil ◽  
Reaction Systems ◽  
Active Inclusion Bodies ◽  
Catalytically Active

CatIB properties can be tailored to the requirements of different reaction systems using two different coiled-coil domains as fusion tags.

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