10 research outputs found
A Modular System for the Rapid Comparison of Different Membrane Anchors for Surface Display on
Comparison of different membrane anchor motifs for the surface display of a protein of interest (passenger) is crucial for achieving the best possible performance. However, generating genetic fusions of the passenger to various membrane anchors is time-consuming. We herein employ a recently developed modular display system, in which the membrane anchor and the passenger are expressed separately and assembled inâ
situ via SpyCatcher and SpyTag interaction, to readily combine a model passenger cytochrome P450 BM3 (BM3) with four different membrane anchors (Lpp-OmpA, PgsA, INP and AIDA-I). This approach has the significant advantage that passengers and membrane anchors can be freely combined in a modular fashion without the need to generate direct genetic fusion constructs in each case. We demonstrate that the membrane anchors impact not only cell growth and membrane integrity, but also the BM3 surface display capacity and whole-cell biocatalytic activity. The previously used Lpp-OmpA as well as PgsA were found to be efficient for the display of BM3 via SpyCatcher/SpyTag interaction. Our strategy can be transferred to other user-defined anchor and passenger combinations and could thus be used for acceleration and improvement of various applications involving cell surface display
3D-Printed Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4-Hydroxystilbene
Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regio- and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agarose-based modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactor-free phenacrylate decarboxylase enzymes suitable for the production of 4-vinylphenol and test their applicability for the encapsulation and direct 3D printing of disk-shaped agarose-based modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetate-catalysed Heck reaction, 4-hydroxystilbene was synthesized from p-coumaric acid with a total yield of 14.7 % on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches
3DâPrinted Phenacrylate Decarboxylase Flow Reactors for the Chemoenzymatic Synthesis of 4âHydroxystilbene
Continuous flow systems for chemical synthesis are becoming a major focus in organic chemistry and there is a growing interest in the integration of biocatalysts due to their high regioâ and stereoselectivity. Methods established for 3D bioprinting enable the fast and simple production of agaroseâbased modules for biocatalytic reactors if thermally stable enzymes are available. We report here on the characterization of four different cofactorâfree phenacrylate decarboxylase enzymes suitable for the production of 4âvinylphenol and test their applicability for the encapsulation and direct 3D printing of diskâshaped agaroseâbased modules that can be used for compartmentalized flow microreactors. Using the most active and stable phenacrylate decarboxylase from Enterobacter spec. in a setup with four parallel reactors and a subsequent palladium(II) acetateâcatalysed Heck reaction, 4âhydroxystilbene was synthesized from pâcoumaric acid with a total yield of 14.7â% on a milligram scale. We believe that, due to the convenient direct immobilization of any thermostable enzyme and straightforward tuning of the reaction sequence by stacking of modules with different catalytic activities, this simple process will facilitate the establishment and use of cascade reactions and will therefore be of great advantage for many research approaches
An Immobilised SiliconâCarbon BondâForming Enzyme for Anaerobic Flow Biocatalysis
The recent development of tailored cytochrome enzymes has enabled ânew-to-natureâ reactivities, such as the biocatalytic formation of carbon-silicon bonds using the cytochrome c from Rhodothermus marinus. To maximise the potential of this remarkable biocatalyst by increasing its turnover numbers (TON) and to enable its reusability in continuous processes, we report the use of the SpyTag/SpyCatcher bioconjugation system to immobilise this enzyme. We successfully modified the enzyme with a SpyTag without significant effects on its catalytic activity. Even after immobilization on microparticles the enzyme retained 60â% activity. When the immobilized enzyme was used in sequential batch or continuous flow to produce an organosilicon, we observed up to 6-fold higher turnover numbers over a total period of 10â
days compared to the free enzyme reaction, however we observed a drop in stereoselectivity under these conditions. This is the first report on the successful immobilisation of a carbon-silicon bond forming enzyme for the continuous, biocatalytic production of organosilicons
Valency engineering of monomeric enzymes for self-assembling biocatalytic hydrogels
All-enzyme hydrogels are efficient reagents for continuous flow biocatalysis. These materials can be obtained by self-assembly of two oligomeric enzymes, modified with the complementary SpyTag and SpyCatcher units. To facilitate access to the large proportion of biocatalytically relevant monomeric enzymes, we demonstrate that the tagging valency of the monomeric (S)-stereoselective ketoreductase Gre2p from Saccharomyces cerevisiae can be designed to assemble stable, active hydrogels with the cofactor-regenerating glucose 1-dehydrogenase GDH from Bacillus subtilis. Mounted in microfluidic reactors, these gels revealed high conversion rates and stereoselectivity in the reduction of prochiral methylketones under continuous flow for more than 8 days. The sequential use as well as parallelization by ânumbering upâ of the flow reactor modules demonstrate that this approach is suitable for syntheses on the semipreparative scale
Self-immobilizing Biocatalysts for fluidic Reaction Cascades
The industrial implementation of whole-cells and enzymes in flow biocatalysis microreactors is essential for the
emergence of a biobased circular economy. Major challenges concern the efficient immobilization of delicate enzymes
inside miniaturized reactors without compromising their catalytic activity. We describe the design and application of four
different immobilization techniques including self-immobilizing whole-cells and purified enzymes on magnetic
microbeads, as well as reactor modules manufactured by 3D printing of bioinks containing thermostable enzymes. To
increase the volumetric activity of our microreactors we furthermore developed and applied self-assembling all-enzyme
hydrogels with cofactor-regenerating capabilities. The resulting reactor formats have excellent operational stability times
of > 14 days and maximum space-time yields of > 450 g product/L-1day-1 paving the way for mild and effective
immobilization techniques of biocatalysts in microfluidic systems
A Magnetosome-Based Platform for Flow Biocatalysis
Biocatalysis in flow reactor systems is of increasing importance for the transformation of the chemical industry. However, the necessary immobilization of biocatalysts remains a challenge. We here demonstrate that biogenic magnetic nanoparticles, so-called magnetosomes, represent an attractive alternative for the development of nanoscale particle formulations to enable high and stable conversion rates in biocatalytic flow processes. In addition to their intriguing material characteristics, such as high crystallinity, stable magnetic moments, and narrow particle size distribution, magnetosomes offer the unbeatable advantage over chemically synthesized nanoparticles that foreign protein âcargoâ can be immobilized on the enveloping membrane via genetic engineering and thus, stably presented on the particle surface. To exploit these advantages, we develop a modular connector system in which abundant magnetosome membrane anchors are genetically fused with SpyCatcher coupling groups, allowing efficient covalent coupling with complementary SpyTag-functionalized proteins. The versatility of this approach is demonstrated by immobilizing a dimeric phenolic acid decarboxylase to SpyCatcher magnetosomes. The functionalized magnetosomes outperform similarly functionalized commercial particles by exhibiting stable substrate conversion during a 60 h period, with an average spaceâtime yield of 49.2 mmol Lâ1 hâ1. Overall, our results demonstrate that SpyCatcher magnetosomes significantly expand the genetic toolbox for particle surface functionalization and increase their application potential as nano-biocatalysts
A Phenolic Acid Decarboxylase-Based All-Enzyme Hydrogel for Flow Reactor Technology
Carrier-free enzyme immobilization techniques are an important development in the field of efficient and streamlined continuous synthetic processes using microreactors. Here, the use of monolithic, self-assembling all-enzyme hydrogels is expanded to phenolic acid decarboxylases. This provides access to the continuous flow production of p-hydroxystyrene from p-coumaric acid for more than 10 h with conversions â„98% and space time yields of 57.7 g·(d·L)â1. Furthermore, modulation of the degree of crosslinking in the hydrogels resulted in a defined variation of the rheological behavior in terms of elasticity and mesh size of the corresponding materials. This work is addressing the demand of sustainable strategies for defunctionalization of renewable feedstocks
MOFâHosted Enzymes for Continuous Flow Catalysis in Aqueous and Organic Solvents
Fully exploiting the potential of enzymes in cell-free biocatalysis requires stabilization of the catalytically active proteins and their integration into efficient reactor systems. Although in recent years initial steps towards the immobilization of such biomolecules in metal-organic frameworks (MOFs) have been taken, these demonstrations have been limited to batch experiments and to aqueous conditions. Here we demonstrate a MOF-based continuous flow enzyme reactor system, with high productivity and stability, which is also suitable for organic solvents. Under aqueous conditions, the stability of the enzyme was increased 30-fold, and the space-time yield exceeded that obtained with other enzyme immobilization strategies by an order of magnitude. Importantly, the infiltration of the proteins into the MOF did not require additional functionalization, thus allowing for time- and cost-efficient fabrication of the biocatalysts using label-free enzymes
A Phenolic Acid Decarboxylase-Based All-Enzyme Hydrogel for Flow Reactor Technology
Carrier-free enzyme immobilization techniques are an important development in the field of efficient and streamlined continuous synthetic processes using microreactors. Here, the use of monolithic, self-assembling all-enzyme hydrogels is expanded to phenolic acid decarboxylases. This provides access to the continuous flow production of p-hydroxystyrene from p-coumaric acid for more than 10 h with conversions ≥98% and space time yields of 57.7 g·(d·L)−1. Furthermore, modulation of the degree of crosslinking in the hydrogels resulted in a defined variation of the rheological behavior in terms of elasticity and mesh size of the corresponding materials. This work is addressing the demand of sustainable strategies for defunctionalization of renewable feedstocks