Biocatalytic Systems Research Articles

Cofactor immobilization for efficient dehydrogenase driven upgrading of xylose

In this study, the coupled immobilization on nanosilica of xylose dehydrogenase and alcohol dehydrogenase was accomplished with high efficiency, which was 90 %. Moreover, immobilization of the cofactors oxidized and reduced nicotinamide adenine dinucleotide, i.e., NAD+ and NADH, on silica material was examined and the impact on the effectiveness of the process was determined. The highest efficiency of NAD+ immobilization was found to be 56 %, which was obtained after 24 h of immobilization at 30 °C, pH 7 For NADH, the best immobilization efficiency was 53 % which was achieved after 24 h at 25 °C, pH 7. The KM and Vmax values were determined for various configurations of the biocatalytic systems showing, as expected, that immobilization of the enzymes decreased the catalytic rate (Vmax) and slightly increased the KM, but verifying that the immobilization of the cofactors did not significantly affect the kinetics, but would enable high conversion, and potentially continued enzymatic reaction. The use of the system configuration with co-immobilized enzymes, immobilized NAD+ and immobilized NADH thus allowed for obtaining over 90 % efficiency of xylose conversion in one batch, which was significantly higher than the systems with single free or only one immobilized cofactor. Using UV-Vis measurements, it was confirmed that effective cofactor regeneration occurred in the systems with immobilized components thus allowing for sustained enzyme catalyzed upgrading of xylose to xylonic acid.

Long-Term Stability of Nicotinamide Cofactors in Common Aqueous Buffers: Implications for Cell-Free Biocatalysis.

The use of nicotinamide cofactors in cell-free biocatalytic systems is necessitated by the high specificity that these enzymes show for their natural redox mediators. Unfortunately, isolation and use of natural cofactors is costly, which suggests that enhancing their stability is key to enabling their use in industrial processes. This study details NAD+ and NADH stability in three buffer systems (sodium phosphate, HEPES, and Tris) at 19 °C and 25 °C and for up to 43 d. In Tris, both NADH and NAD+ were found to be highly stable. NADH degradation rates of 4 μM/d (19 °C) and 11 μM/d (25 °C) were observed in Tris buffer, corresponding to >90% and 75% remaining after 43 d, respectively. Higher degradation rates (up to 34 μM/d) were observed when sodium phosphate or HEPES buffers were used. The effect of a mild increase in temperature was determined to be significant for long-term stability, and it was shown that degradation under these conditions can be easily monitored via UV-Vis, because the degradation proceeds via the oxidation/de-aromatization of the dihydropyridine ring. Overall, this work emphasizes that the choice of buffer system is consequential for bioreactor systems employing natural nicotinamide cofactors for extended periods of time.

Enzymatic and Bio-Inspired Enantioselective Oxidation of Non-Activated C(sp 3)–H Bonds

AbstractThe enantioselective oxidation of C–H bonds relies on two different approaches: the use of enzymes or bio-inspired transition metal catalysts. Both are powerful tools, as they transform ubiquitous C(sp3)–H bonds into valuable oxygenated building blocks. However, the reaction remains a challenge in synthetic chemistry, continuously demanding efficient catalytic systems to improve substrate scopes. Optimization of site- and enantioselectivities in bio-catalytic systems is underpinned by protein engineering, while ligand design and medium effects play crucial roles in bio-inspired synthetic complexes. In this Short Review, recent advances in the field are described, focusing on reactions that target strong, non-activated C–H bonds.1 Introduction1.1 Enantioselective Catalytic C–H Oxidation in Nature and Bio-Inspired Systems1.2 Biological C–H Oxidation Mechanism and Challenges for the Implementation of Chirality with Synthetic Catalysts1.3 Bio-Catalytic C–H Oxidation Systems: From Microorganism to Engineered Enzymes1.4 Mimicking Nature: The Bio-Inspired C–H Oxidation Approach1.5 Origin of Enantioselectivity2 Enantioselective C–H Oxidation of Non-Activated C–H Bonds2.1 Hydroxylation at Non-Activated C–H Bonds by Bio-Catalytic Systems2.2 Enantioselective C–H Lactonization with Enzymatic Systems2.3 Oxidation at Non-Activated C–H Bonds by Synthetic Catalysts2.4 Enantioselective Lactonization with Small-Molecule Catalysts3 Conclusions

Microbial Fuel Cell Based on Ensifer meliloti

The world’s growing energy crisis demands renewable energy sources. This issue can be solved using microbial fuel cells (MFCs). MFCs are biocatalytic systems which convert chemical energy into electrical energy, thereby reducing pollution from hazardous chemical compounds. However, during the development of MFCs, one of the most significant challenges is finding and assessment of microorganisms that generate sufficient redox potential through metabolic and catalytic processes. In this research, we have used Ensifer meliloti (E. meliloti) bacteria to design MFCs based on consecutive action of two redox mediators (9,10 - phenanthrenequinone (PQ) and potassium ferricyanide), which transferred charge between E. meliloti bacteria and graphite rod electrode. A viability study of E. meliloti culture showed that PQ significantly inhibits the growth of bacteria at 0.036 mM. Cyclic voltammograms were registered in the presence of 20 mM of potassium ferricyanide and different concentrations (0.036 and 0.071 mM, 0.11 mM, 0.14 mM, 0.172 mM, 0.32 mM) of PQ. Four days of lasting assessment of the microbial fuel cells in two-electrode systems showed that the maximal open circuit potential during the experiment raised from 174.9 to 234.6 mV. Power increased from 0.392 to 0.741 mW m−2.

Ionic liquid supported hydrogel–lipase biocatalytic systems in asymmetric synthesis of enantiomerically pure S-ibuprofen

Novel hydrogel biocatalysts with immobilized lipase, stabilized by ionic liquids (ILs) of different hydrophobicity, were synthesized and evaluated. Variations of the time of immobilization and ratio of substrates during hydrogel synthesis were considered to obtain the most stable biocatalyst with the highest activity. Physicochemical characterization proved the success of the hydrogel synthesis and enzyme deposition on the surface of the support. Nevertheless, the key objective was to produce a biocatalyst for further application in ibuprofen methyl ester resolution, with the aim of obtaining an enantiomerically pure product. The hydrogel biocatalysts obtained in the presence of 5 wt% ILs after 8 h of immobilization achieved the highest activity recovery of 62 %. After 10 reaction cycles, enzymatic activity was still above 60 %, and the negative effect of pH and temperature on the activity of immobilized lipase was much lower than in the case of the free enzyme. After application of the catalyst in the resolution of ibuprofen methyl ester, the enantiomeric excess and conversion rate of the process were obtained for the dynamic kinetic resolution in isooctane. A conversion rate of 95 % was achieved due to the stabilization of the biocatalyst with IL and its resulting high catalytic activity. The study thus provides the pharmaceutical industry with a new potential approach with a strong scientific foundation.

Choline Oxidase and Choline Ionic Liquids in Biocatalytic Heme Peroxidase Cascades

Choline oxidase from Alcaligenes sp. (ChOx) is used to generate hydrogen peroxide in situ from choline‐based ionic liquids (ILs) to fuel peroxidase‐mediated biocatalysis, while mitigating oxidative degradation of the heme‐dependent enzymes. Horseradish peroxidase (HRP) and chloroperoxidase from Caldariomyces fumago (CPO), in combination with the ChOx, are evaluated in enzymatic cascades for the ability of the biocatalytic systems to withstand elevated concentrations of different choline additives in oxidative and halogenative enzymatic assays. The findings are applied in various synthetic scenarios to produce important oxygen‐ and nitrogen‐containing heterocycles, using choline ILs in a dual‐purpose fashion, as a substrate‐solubilizing component in the reaction medium as well as the source for hydrogen peroxide. The ChOx/HRP couple is used to induce intramolecular cyclizations of hydroxamic acids and hydroxycarbamates in a nitroso‐ene‐type pathway with choline dihydrogen phosphate as IL additive. The ChOx/CPO cascade successfully mediates brominative cyclizations of α‐allenic alcohols, while amphiphilic surfactants are employed to turn the aqueous choline propionate IL media into a colloidal suspension. ChOx/CPO partnering is also evaluated in an oxygenative rearrangement of 1‐furylethanol with choline acetate IL. The results show the wide potential of choline oxidase for hydrogen peroxide‐driven biocatalysis with both aqueous and micellar choline ionic liquid solutions.

Coupling of the catalytic reactions from formate dehydrogenase and hydrogenase in solution: Insights from computations and in situ IR spectroscopy

Sophisticated enzymatic systems have evolved in nature to efficiently couple distinct biochemical reactions in form of cascades. As such they serve as reference models to understand the indirect interactions of catalytic centers. Herein, we studied, in solution, the coupling of the reactions from membrane‐bound [NiFe] hydrogenase (MBH) from Cupriavidus necator (reversible H2 splitting into H+ and e‐) and the molybdenum‐dependent formate dehydrogenase from Rhodobacter capsulatus (reversible formate to CO2 interconversion). To follow their interplay via the characteristic absorptions from the MBH's active site or the respective substrate and product bands of FDH, we utilized in situ IR spectrocopy and GC(‐MS), in the absence or presence of soluble redox mediators. Coarse grained molecular dynamics (cgMD) computations revealed the lack of productive enzyme complexes for direct electron transfer (ET). Thus, the observed minor amounts of H2 or formate were produced from transient interactions between the two enzymes. On the contrary, the significantly increased product formation in the presence of methylene viologen can be related to the putative multiple interaction sites of the redox mediator with FDH identified by cgMD. Our study represents a proof‐of‐concept approach that can be used in future to develop novel coupled biocatalytic systems by identifying potential ET pathways.

Biocatalytic pathways, cascades, cells and systems: general discussion.

Biocatalytic pathways, cascades, cells and systems: general discussion.

Improving the Selectivity and Efficiency of Synthetic Organic Electrosynthesis

Although electrochemistry is a current focus area in improving the sustainability and safety of chemical manufacturing, electrochemical techniques for synthesis rarely have sufficient selectivity and frequently have low faradaic, product, and energy efficiencies. This paper will discuss the use of biocatalytic systems to improve the selectivity of electrosynthesis followed by a discussion of materials, reactor, and technique strategies to improve the faradaic, product, and energy efficiencies in these systems. Examples will be shown that range from commodity chemical synthesis to fine chemical synthesis to pharmaceutical synthesis with a comparison of the concerns and advantages of electrosynthesis for each chemical industry.

A synthetic cell-free pathway for biocatalytic upgrading of one-carbon substrates.

Biotechnological processes hold tremendous potential for the efficient and sustainable conversion of one-carbon (C1) substrates into complex multi-carbon products. However, the development of robust and versatile biocatalytic systems for this purpose remains a significant challenge. In this study, we report a hybrid electrochemical-biochemical cell-free system for the conversion of C1 substrates into the universal biological building block acetyl-CoA. The synthetic reductive formate pathway (ReForm) consists of five core enzymes catalyzing non-natural reactions that were established through a cell-free enzyme engineering platform. We demonstrate that ReForm works in a plug-and-play manner to accept diverse C1 substrates including CO2 equivalents. We anticipate that ReForm will facilitate efforts to build and improve synthetic C1 utilization pathways for a formate-based bioeconomy.

Construction of a high-efficiency GjCCD4a mutant and its application for de novo biosynthesis of five crocins in Escherichia coli

Crocins are bioactive natural products that rarely exist in plants. High costs and resource shortage severely limit its development and application. Synthetic biology studies on crocins are of considerable global interest. However, the lack of high-efficiency genetic tools and complex cascade biocatalytic systems have substantially hindered progress in crocin biosynthesis-related research. Based on mutagenesis, a high-efficiency GjCCD4a mutant (N212m) was constructed with a catalytic efficiency that was 25.08-fold higher than that of the wild-type. Solubilized GjCCD4a was expressed via fusion with an MBP tag. Moreover, N212m and ten other genes were introduced into Escherichia coli for the de novo biosynthesis of five crocins. The engineered E57 strain produced crocins III and V with a total yield of 11.50 mg/L, and the E579 strain produced crocins I-V with a total output of 8.43 mg/L at shake-flask level. This study identified a marvelous genetic element (N212m) for crocin biosynthesis and achieved its de novo biosynthesis in E. coli using glucose. This study provides a reference for the large-scale production of five crocins using E. coli cell factories.

In-situ honeycomb spheres for enhanced enzyme immobilization and stability

This paper presents a promising heterogeneous biocatalyst-immobilized cellulase system, which can facilitate the targeted catalytic conversion of biomass to reducing sugars. Magnetic Zeolite Imidazole Framework-8/chitosan (Fe3O4/ZIF-8/CS, FZC) hybrid honeycomb microspheres were synthesized to immobilize cellulase using an in-situ growth strategy. The enhancement of immobilization efficiency induced by each constituent of the composite carrier was explored. This exploration was grounded in varying combinations of components within the honeycomb composite spheres, revealing the indispensability of ZIF-8. Through in-situ assembly, the composite material presents a microsphere with an internal honeycomb structure and multilevel pores. Effective immobilization, temperature-responsive conformational change, and cyclic stability were achieved due to the scaling effect of the channel, extensive specific surface area, and abundant amino group interactions enable immobilized enzymes to maintain excellent performance under challenging operating conditions, demonstrating an enhanced structure-performance relationship. With the obtained immobilized cellulase, we achieved a high load (461.67 mg/g) of cellulase and a significant reducing sugar yield (328.39 mg/g) from corncob waste. In addition, we investigated the enhancement effect of thermal activation on the immobilized enzyme activity of composite carrier. The results showed that the appropriate heat treatment was conducive to the activation of FZC immobilized enzyme with a reducing sugar yield 1.61 times higher than that of the control. The strategy provided in this study promotes the development of immobilized biocatalysts and provides a useful reference for the further design of biocatalytic systems.

Modeling Enzymatic Cascade Reactions Immobilized in Plug‐Flow Reactors for Flow Biocatalysis

AbstractFlow biocatalysis has emerged as a promising technology for the sustainable production of chemicals. Multiple immobilized enzymes are often used as biocatalysts to perform enzymatic cascade reactions in flow biocatalytic systems. When designing packed‐bed reactors with multiple immobilized enzymes, the enzymes are distributed in porous particles, and the porous particles are distributed in catalyst zones. A recently developed methodology is extended for selecting spatial immobilization distributions in batch reactors to packed‐bed reactors. Mechanistic models are used to compare the three most basic designs for a packed‐bed reactor, i.e., mixed individually immobilized enzymes, separated individually immobilized enzymes, and mixed co‐immobilized enzymes. For first‐order microkinetics, the design with co‐immobilized enzymes was found to always outperform the other two. Separating individually immobilized enzymes in two zones was proven to outperform mixing them in one zone for any positive order reaction kinetic.

Kinetic Model for the Heterogeneous Biocatalytic Reactions Using Tethered Cofactors.

Understanding the mechanism of interfacial enzyme kinetics is critical to the development of synthetic biological systems for the production of value-added chemicals. Here, the interfacial kinetics of the catalysis of β-nicotinamide adenine dinucleotide (NAD+)-dependent enzymes acting on NAD+ tethered to the surface of silica nanoparticles (SiNPs) has been investigated using two complementary and supporting kinetic approaches: enzyme excess and reactant (NAD+) excess. Kinetic models developed for these two approaches characterize several critical reaction steps including reversible enzyme adsorption, complexation, decomplexation, and catalysis of the surface-bound enzyme/NAD+ complex. The analysis reveals a concentrating effect resulting in a very high local concentration of enzyme and cofactor on the particle surface, in which the enzyme is saturated by surface-bound NAD, facilitating a rate enhancement of enzyme/NAD+ complexation and catalysis. This resulted in high enzyme efficiency within the tethered NAD+ system compared to that of the free enzyme/NAD+ system, which increases with decreasing enzyme concentration. The role of enzyme adsorption onto solid substrates with a tethered catalyst (such as NAD+) has potential for creating highly efficient flow biocatalytic systems.

Highly selective whole-cell 25-hydroxyvitamin D3 synthesis using molybdenum-dependent C25-steroid dehydrogenase and cyclodextrin recycling

BackgroundThe global prevalence of vitamin D (VitD) deficiency associated with numerous acute and chronic diseases has led to strategies to improve the VitD status through dietary intake of VitD-fortified foods and VitD supplementation. In this context, the circulating form of VitD3 (cholecalciferol) in the human body, 25-hydroxy-VitD3 (calcifediol, 25OHVitD3), has a much higher efficacy in improving the VitD status, which has motivated researchers to develop methods for its effective and sustainable synthesis. Conventional monooxygenase-/peroxygenase-based biocatalytic platforms for the conversion of VitD3 to value-added 25OHVitD3 are generally limited by a low selectivity and yield, costly reliance on cyclodextrins and electron donor systems, or by the use of toxic co-substrates.ResultsIn this study, we used a whole-cell approach for biocatalytic 25OHVitD3 synthesis, in which a molybdenum-dependent steroid C25 dehydrogenase was produced in the denitrifying bacterium Thauera aromatica under semi-aerobic conditions, where the activity of the enzyme remained stable. This enzyme uses water as a highly selective VitD3 hydroxylating agent and is independent of an electron donor system. High density suspensions of resting cells producing steroid C25 dehydrogenase catalysed the conversion of VitD3 to 25OHVitD3 using either O2 via the endogenous respiratory chain or externally added ferricyanide as low cost electron acceptor. The maximum 25OHVitD3 titer achieved was 1.85 g L–1 within 50 h with a yield of 99%, which is 2.2 times higher than the highest reported value obtained with previous biocatalytic systems. In addition, we developed a simple method for the recycling of the costly VitD3 solubiliser cyclodextrin, which could be reused for 10 reaction cycles without a significant loss of quality or quantity.ConclusionsThe established steroid C25 dehydrogenase-based whole-cell system for the value-adding conversion of VitD3 to 25OHVitD3 offers a number of advantages in comparison to conventional oxygenase-/peroxygenase-based systems including its high selectivity, independence from an electron donor system, and the higher product titer and yield. Together with the established cyclodextrin recycling procedure, the established system provides an attractive platform for large-scale 25OHVitD3 synthesis.

Monoliths enabling biocatalysis in flow chemistry

Biocatalysis in flow improves space time yields and enables straightforward downstream processing. The underlying principles of monolithic flow reactors and their application in mono- and bi-phasic biocatalytic systems are discussed.

Electrochemical and Biocatalytic Signal-Controlled Payload Release from a Metal-Organic Framework.

A metal-organic framework (MOF), ZIF-8, which is stable at neutral and slightly basic pH values in aqueous solutions and destabilized/dissolved under acidic conditions, is loaded with a pH-insensitive fluorescent dye, rhodamine-B isothiocyanate, as a model payload species. Then, the MOF species are immobilized at an electrode surface. The local (interfacial) pH value is rapidly decreased by means of an electrochemically stimulated ascorbate oxidation at +0.4V (Ag/AgCl/KCl). Oxygen reduction upon switching the applied potential to -0.8V allows to return the local pH to the neutral/basic pH, then stopping rapidly the release process. The developed method allows electrochemical control over stimulated or inhibited payload release processes from the MOF. The pH variation proceeds in a thin film of the solution near the electrode surface. The switchable release process is realized in a buffer solution and undiluted human serum. As the second option, the pH decrease stimulating the release process is achieved upon an enzymatic reaction using esterase and ester substrate. This approach potentially allows the release activation controlled by numerous enzymes assembled in complex biocatalytic cascades. It is expected that related electrochemical or biocatalytic systems can represent novel signal-responding materials with switchable features for delivering (bio)molecules within biomedical applications.

Synthesis of Metabolites and Metabolite-like Compounds Using Biocatalytic Systems.

Methodologies for the synthesis and purification of metabolites, which have been developed following their discovery, analysis, and structural identification, have been involved in numerous life science milestones. The renewed focus on the small molecule domain of biological cells has also created an increasing awareness of the rising gap between the metabolites identified and the metabolites which have been prepared as pure compounds. The design and engineering of resource-efficient and straightforward synthetic methodologies for the production of the diverse and numerous metabolites and metabolite-like compounds have attracted much interest. The variety of metabolic pathways in biological cells provides a wonderful blueprint for designing simplified and resource-efficient synthetic routes to desired metabolites. Therefore, biocatalytic systems have become key enabling tools for the synthesis of an increasing number of metabolites, which can then be utilized as standards, enzyme substrates, inhibitors, or other products, or for the discovery of novel biological functions.

Conjugated Molecules Based Multi-Component Artificial Photosynthesis System for Producing Multi-Objective Products.

The development of artificial photosynthesis systems that mimics natural photosynthesis can help address the issue of energy scarcity by efficiently utilizing solar energy. Here, it presents liposomes-based artificial photosynthetic nanocapsules (PSNC) integrating photocatalytic, chemical catalytic, and biocatalytic systems through one-pot method. The PSNC contains 5,10,15,20-tetra(4-pyridyl) cobalt-porphyrin, tridipyridyl-ruthenium nitrate, oligo-pphenyl-ethylene-rhodium complex, and creatine kinase, efficiently generating oxygen, nicotinamide adenine dinucleotide (NADH), and adenosine triphosphate with remarkable enhancements of 231%, 30%, and 86%, compared with that of molecules mixing in aqueous solution. Additionally, the versatile PSNC enables simulation of light-independent reactions, achieving a controllable output of various target products. The regenerated NADH within PSNC further facilitates alcohol dehydrogenase, yielding methanol with a notable efficiency improvement of 37%. This work introduces a promising platform for sustainable solar energy conversion and the simultaneous synthesis of multiple valuable products in an ingenious and straightforward way.

Biocatalysis in Water or in Non-Conventional Media? Adding the CO2 Production for the Debate.

Biocatalysis can be applied in aqueous media and in different non-aqueous solutions (non-conventional media). Water is a safe solvent, yet many synthesis-wise interesting substrates cannot be dissolved in aqueous solutions, and thus low concentrations are often applied. Conversely, non-conventional media may enable higher substrate loadings but at the cost of using (fossil-based) organic solvents. This paper determines the CO2 production-expressed as kg CO2·kg product-1-of generic biotransformations in water and non-conventional media, assessing both the upstream and the downstream. The key to reaching a diminished environmental footprint is the type of wastewater treatment to be implemented. If the used chemicals enable a conventional (mild) wastewater treatment, the production of CO2 is limited. If other (pre)treatments for the wastewater are needed to eliminate hazardous chemicals and solvents, higher environmental impacts can be expected (based on CO2 production). Water media for biocatalysis are more sustainable during the upstream unit-the biocatalytic step-than non-conventional systems. However, processes with aqueous media often need to incorporate extractive solvents during the downstream processing. Both strategies result in comparable CO2 production if extractive solvents are recycled at least 1-2 times. Under these conditions, a generic industrial biotransformation at 100 g L-1 loading would produce 15-25 kg CO2·kg product-1 regardless of the applied media.