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Characterization of PAD-fusion proteins and immobilized PAD on different carrier materials. (A) Denaturing 16% SDS-PAGE analysis of 4 μg of each PAD fusion protein after heterologous expression in E. coli and purification via a C-terminal 6×His-tag. The proteins were obtained in purities >95% according to grayscale analysis. Lane 1: PAD (20.4 kDa); Lane 2: PAD-ST (21.8 kDa); Lane 3: PAD-HOB (53.8 kDa); Marker: PageRuler prestained protein ladder (Thermo Scientific). Molecular weight of to the monomer is given. (B) Enzymatic activity in (μmol pCA μmol PAD −1 min −1 ) of the PAD variants by using 0.1 mM p-coumaric acid (pCA) as substrate, determined by an absorbance-based assay in PAD Buffer (25 mM potassium phosphate buffer, pH 6) at 30 °C. (C) Specific activities per milligram of carrier material of PAD-functionalized SCmagnetosomes (PAD-ST@Mag; yellow) in a batch reaction in comparison to the alternative magnetically immobilizable biocatalyst systems. The conversion of pCA to pHS through PAD-ST@Mag, PAD-ST immobilized on SC-Dynabeads (PAD-ST@Dyn; dark blue), or PAD-HOB immobilized on CH-Dynabeads (PAD-HOB@Dyn; light blue) was monitored at different points in time by using HPLC analysis. All experiments were performed in PAD-Buffer at 30 °C and 600 rpm at least in duplicates by using different batches of magnetosomes or particles.
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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 f...
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... potentially detrimental influence of the binding tags on the biocatalyst's activity has to be investigated. However, we found that the fusion of PAD with the tags used in this work could be heterologously expressed in high purity ( Figure 5A) with no significant differences in the substrate conversion rate ( Figure 5B). Furthermore, maximizing the volumetric activity of flow reactors is a crucial parameter for their efficiency and strongly depends on the effective surface area and binding capacity of the support matrix used. ...
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... potentially detrimental influence of the binding tags on the biocatalyst's activity has to be investigated. However, we found that the fusion of PAD with the tags used in this work could be heterologously expressed in high purity ( Figure 5A) with no significant differences in the substrate conversion rate ( Figure 5B). Furthermore, maximizing the volumetric activity of flow reactors is a crucial parameter for their efficiency and strongly depends on the effective surface area and binding capacity of the support matrix used. ...
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... to application in a flow process, the PAD activity per milligram of carrier material was analyzed via the conversion of p-coumaric acid (pCA) to p-hydroxystyrene (pHS) in a batch assay. PAD-ST@Mag showed a superior activity per milligram of carrier material in comparison to PAD-ST@Dyn and PAD-HOB@Dyn ( Figure 5C), which might be due to the higher surface area of the magnetosome nano-biocatalyst in comparison to the Dynabeads. ...
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... slow decline in reactivity of the PAD-ST@Mag could potentially be due to disintegration of the magnetosomes. However, we could not detect obvious changes in magnetosome morphology when comparing particles before and after application in the flow reactor ( Figure S5). On the contrary, the stability of the PAD appears to be improved by immobilization. ...
Citations
... Further expanding the applications of magnetosome-based biosensors, SpyCatcher magnetosomes served as a transformative platform for the development of biocatalytic nanoparticles (Figure 7) [99]. This innovative approach significantly expanded the available genetic toolbox for engineering magnetic nanoparticles with tailored functionalities. ...
Inspired by nature’s remarkable ability to form intricate minerals, researchers have unlocked transformative strategies for creating next-generation biosensors with exceptional sensitivity, selectivity, and biocompatibility. By mimicking how organisms orchestrate mineral growth, biomimetic and bioinspired materials are significantly impacting biosensor design. Engineered bioinspired materials offer distinct advantages over their natural counterparts, boasting superior tunability, precise controllability, and the ability to integrate specific functionalities for enhanced sensing capabilities. This remarkable versatility enables the construction of various biosensing platforms, including optical sensors, electrochemical sensors, magnetic biosensors, and nucleic acid detection platforms, for diverse applications. Additionally, bioinspired materials facilitate the development of smartphone-assisted biosensing platforms, offering user-friendly and portable diagnostic tools for point-of-care applications. This review comprehensively explores the utilization of naturally occurring and engineered biominerals and materials for diverse biosensing applications. We highlight the fabrication and design strategies that tailor their functionalities to address specific biosensing needs. This in-depth exploration underscores the transformative potential of biominerals and materials in revolutionizing biosensing, paving the way for advancements in healthcare, environmental monitoring, and other critical fields.
... These biogenic magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ) crystals display critical properties that distinguish them from abiotically formed counterparts, such as high chemical purity, few crystal defects, and, most importantly, restricted size and shape ranges so as to form stable single-magnetic domains (Amor et al., 2020). Biogenic magnetic nanoparticles are also encapsulated within a lipidic membrane, forming an organelle called the magnetosome, which can now be functionalized for the surface display of fluorescent tags, antibodies or biocatalysts (Mickoleit et al., 2020;Mittmann et al., 2022;Xu et al., 2019). Magnetosomes can easily be isolated from lysed cells, enabling their use in a number of applications, mostly in the biomedical field (e.g. in magnetically targeted drug delivery, pathogen detection, magnetic hypothermia for the treatment of tumours, or as contrast agents in magnetic imaging techniques), but also for toxic metal bioremediation or the detection of pathogens in food -applications that are described in greater details in a number of recent reviews (Basit et al., 2020;Kotakadi et al., 2022;Qin et al., 2020;Vargas et al., 2018). ...
Biomineralization, the capacity to form minerals, has evolved in a great diversity of bacterial lineages as an adaptation to different environmental conditions and biological functions. Microbial biominerals often display original properties (morphology, composition, structure, association with organics) that significantly differ from those of abiotically formed counterparts, altogether defining the ‘mineral phenotype’. In principle, it should be possible to take advantage of microbial biomineralization processes to design and biomanufacture advanced mineral materials for a range of technological applications. In practice, this has rarely been done so far and only for a very limited number of biomineral types. This is mainly due to our poor understanding of the underlying molecular mechanisms controlling microbial biomineralization pathways, preventing us from developing bioengineering strategies aiming at improving biomineral properties for different applications. Another important challenge is the difficulty to upscale microbial biomineralization from the lab to industrial production. Addressing these challenges will require combining expertise from environmental microbiologists and geomicrobiologists, who have historically been working at the forefront of research on microbe–mineral interactions, alongside bioengineers and material scientists. Such interdisciplinary efforts may in the future allow the emergence of a mineral biomanufacturing industry, a critical tool towards the development more sustainable and circular bioeconomies.
Exploring the potential of microfluidic systems, this study presents a groundbreaking approach harnessing energy in microfluidic flows within a purpose‐built microreactor, enabling precise deposition of functional biomaterials. Upon optimizing reactor dimensions and integrating it into a microfluidic system, sequentially flow‐induced deposition of DNA hydrogels and transformation into DNA‐protein hybrid materials with SpyTag/SpyCatcher technology is investigated. However, limited functionalization rates restrict its viability for targeted biocatalytic processes. Therefore, the direct deposition of a phenolic acid decarboxylase is investigated, which is efficiently deposited but shows limited biocatalytic performance due to shear‐induced denaturation. This challenge is overcome by a two‐step immobilization process, resulting in microfluidic bioreactors demonstrating initial high space‐time yields of up to 7000 g L⁻¹ d⁻¹, but whose process stability proves unsatisfactory. However, by exploiting the principle of flow‐induced deposition to immobilize recombinant E. coli cells as functional living materials overexpressing biocatalytically relevant enzymes, bioreactors are produced that show equally high space‐time yields in continuous whole‐cell catalysis which remain constant over periods of up to 10 days. The insights gained offer optimization strategies for advanced functional materials and innovative reactor systems holding promise for applications in fundamental materials science, biosensing, and scalable production of microreactors for biocatalysis and bioremediation.
Glycans are the most abundant biopolymers on earth and are constituents of glycoproteins, glycolipids, and proteoglycans with multiple biological functions. The availability of different complex glycan structures is of major interest in biotechnology and basic research of biological systems. High complexity, establishment of general and ubiquitous synthesis techniques, as well as sophisticated analytics, are major challenges in the development of glycan synthesis strategies. Enzymatic glycan synthesis with Leloir-glycosyltransferases is an attractive alternative to chemical synthesis as it can achieve quantitative regio- and stereoselective glycosylation in a single step. Various strategies for synthesis of a wide variety of different glycan structures has already be established and will exemplarily be discussed in the scope of this review. However, the application of enzymatic glycan synthesis in an automated system has high demands on the equipment, techniques, and methods. Different automation approaches have already been shown. However, while these techniques have been applied for several glycans, only a few strategies are able to conserve the full potential of enzymatic glycan synthesis during the process - economical and enzyme technological recycling of enzymes is still rare. In this review, we show the major challenges towards Automated Enzymatic Glycan Synthesis (AEGS). First, we discuss examples for immobilization of glycans or glycosyltransferases as an important prerequisite for the embedment and implementation in an enzyme reactor. Next, improvement of bioreactors towards automation will be described. Finally, analysis and monitoring of the synthesis process are discussed. Furthermore, automation processes and cycle design are highlighted. Accordingly, the transition of recent approaches towards a universal automated glycan synthesis platform will be projected. To this end, this review aims to describe essential key features for AEGS, evaluate the current state-of-the-art and give thought- encouraging impulses towards future full automated enzymatic glycan synthesis.
The recent development of tailored cytochrome enzymes has enabled “new‐to‐nature” 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 60 % 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 10 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.
For the development of efficient and green industrial processes, the combination of biocatalysis and flow chemistry holds great promises. Flow chemical utilization of biocatalysts, essentially made possible by the immobilization (or retention) of enzymes in flow reactors, has attracted increased academic attention during recent years. In the present review we present an overview of immobilization strategies suitable for flow chemistry, particularly focusing on recently developed carrier‐free immobilization methods, highlighting advances in the field and presenting future trends. The combination of biocatalysis and flow chemistry is a promising and rapidly growing field of research. In the present contribution, we therefore present an overview of selected immobilization strategies suitable for the flow chemical use of enzymes, particularly focusing on recently developed carrier‐free immobilization methods.
Hybrid biological robots (biobots) prepared from living cells are at the forefront of micro-/nanomotor research due to their biocompatibility and versatility toward multiple applications. However, their precise maneuverability is essential for practical applications. Magnetotactic bacteria are hybrid biobots that produce magnetosome magnetite crystals, which are more stable than synthesized magnetite and can orient along the direction of earth's magnetic field. Herein, we used Magnetospirillum magneticum strain AMB-1 (M. magneticum AMB-1) for the effective removal of chlorpyrifos (an organophosphate pesticide) in various aqueous solutions by naturally binding with organic matter. Precision control of M. magneticum AMB-1 was achieved by applying a magnetic field. Under a programed clockwise magnetic field, M. magneticum AMB-1 exhibit swarm behavior and move in a circular direction. Consequently, we foresee that M. magneticum AMB-1 can be applied in various environments to remove and retrieve pollutants by directional control magnetic actuation.
