Pickering Emulsions Biocatalysis: Recent Developments and Emerging Trends.

Biocatalysis within biphasic systems is gaining significant attention in the field of synthetic chemistry, primarily for its ability to solve the problem of incompatible solubilities between biocatalysts and organic compounds. By forming an emulsion from these two-phase systems, a larger surface area is created, which greatly improves the mass transfer of substrates to the biocatalysts. Among the various types of emulsions, Pickering emulsions stand out due to their excellent stability, compatibility with biological substances, and the ease with which they can be formed and separated. This makes them ideal for reusing both the emulsifiers and the biocatalysts. This review explores the latest developments in biocatalysis using Pickering emulsions. It covers the structural features, methods of creation, innovations in flow biocatalysis, and the role of interfaces in these processes. Additionally, the challenges and future directions are discussed in combining chemical and biological catalysts within Pickering emulsion frameworks to advance synthetic methodologies.

Integrating Enzymes with Supramolecular Polymers for Recyclable Photobiocatalytic Catalysis.

Chemical modifications of enzymes excel in the realm of enzyme engineering due to its directness, robustness, and efficiency; however, challenges persist in devising versatile and effective strategies. In this study, we introduce a supramolecular modification methodology that amalgamates a supramolecular polymer with Candida antarctica lipase B (CalB) to create supramolecular enzymes (SupEnzyme). This approach features the straightforward preparation of a supramolecular amphiphilic polymer (ß-CD@SMA), which was subsequently conjugated to the enzyme, resulting in a SupEnzyme capable of self-assembly into supramolecular nanoparticles. The resulting SupEnzyme nanoparticles can form micron-scale supramolecular aggregates through supramolecular and electrostatic interactions with guest entities, thus enhancing catalyst recycling. Remarkably, these aggregates maintain 80 % activity after seven cycles, outperforming Novozym 435. Additionally, they can effectively initiate photobiocatalytic cascade reactions using guest photocatalysts. As a consequence, our SupEnzyme methodology exhibits noteworthy adaptability in enzyme modification, presenting a versatile platform for various polymer, enzyme, and biocompatible catalyst pairings, with potential applications in the fields of chemistry and biology.

Enzyme-Polymer-Conjugate-Based Pickering Emulsions for Cell-Free Expression and Cascade Biotransformation.

In this study, we addressed the limitations of conventional enzyme-polymer-conjugate-based Pickering emulsions for interfacial biocatalysis, which traditionally suffer from nonspecific and uncontrollable conjugation positions that can impede catalytic performance. By introducing a non-canonical amino acid (ncAA) at a specific site on target enzymes, we enabled precise polymer-enzyme conjugation. These engineered conjugates then acted as biocatalytically active emulsifiers to stabilize Pickering emulsions, while encapsulating a cell-free protein synthesis (CFPS) system in the aqueous phase for targeted enzyme expression. The resulting cascade reaction system leveraged enzymes expressed in the aqueous phase and on the emulsion interface for optimized chemical biosynthesis. The use of the cell-free system eliminated the need for intact whole cells or purified enzymes, representing a significant advancement in biocatalysis. Remarkably, the integration of Pickering emulsion, precise enzyme-polymer conjugation, and CFPS resulted in a fivefold enhancement in catalytic performance as compared to traditional single-phase reactions. Therefore, our approach harnesses the combined strengths of advanced biochemical engineering techniques, offering an efficient and practical solution for the synthesis of value-added chemicals in various biocatalysis and biotransformation applications.

Metal Alloys-Structured Electrocatalysts: Metal-Metal Interactions, Coordination Microenvironments, and Structural Property-Reactivity Relationships.

Metal alloys-structured electrocatalysts (MAECs) have made essential contributions to accelerating the practical applications of electrocatalytic devices in renewable energy systems. However, due to the complex atomic structures, varied electronic states, and abundant supports, precisely decoding the metal-metal interactions and structure-activity relationships of MAECs still confronts great challenges, which is critical to direct the future engineering and optimization of MAECs. Here, this timely review comprehensively summarizes the latest advances in creating the MAECs, including the metal-metal interactions, coordination microenvironments, and structure-activity relationships. First, the fundamental classification, design, characterization, and structural reconstruction of MAECs are outlined. Then, the electrocatalytic merits and modulation strategies of recent breakthroughs for noble and non-noble metal-structured MAECs are thoroughly discussed, such as solid solution alloys, intermetallic alloys, and single-atom alloys. Particularly, unique insights into the bond interactions, theoretical understanding, and operando techniques for mechanism disclosure are given. Thereafter, the current states of diverse MAECs with a unique focus on structural property-reactivity relationships, reaction pathways, and performance comparisons are discussed. Finally, the future challenges and perspectives for MAECs are systematically discussed. It is believed that this comprehensive review can offer a substantial impact on stimulating the widespread utilization of metal alloys-structured materials in electrocatalysis.

Electronic Structure-Dependent Water-Dissociation Pathways of Ruthenium-Based Catalysts in Alkaline H2 -Evolution.

Ruthenium (Ru)-based catalysts have displayed compelling hydrogen evolution activities, which hold the promising potential to substitute platinum in alkaline H2 -evolution. In the challenging alkaline electrolytes, the water-dissociation process involves multistep reactions, while the profound origin and intrinsic factors of diverse Ru species on water-dissociation pathways and reaction principles remain ambiguous. Here the fundamental origin of water-dissociation pathways of Ru-based catalysts in alkaline media to be from their unique electronic structures in complex coordination environments are disclosed. These theoretical results validate that the modulated electronic structures with delocalization-localization coexistence at their boundaries between the Ru nanocluster and single-atom site have a profound influence on water-dissociation pathways, which push H2 O* migration and binding orientation during the splitting process, thus enhancing the dissociation kinetics. By creating Ru catalysts with well-defined nanocluster, single-atom site, and also complex site, the electrocatalytic data shows that both the nanocluster and single-atom play essential roles in water-dissociation, while the complex site possesses synergistically enhanced roles in alkaline electrolytes. This study discloses a new electronic structure-dependent water-dissociation pathway and reaction principle in Ru-based catalysts, thus offering new inspiration to design efficient and durable catalysts for the practical production of H2 in alkaline electrolytes.

Cell-free metabolic engineering enables selective biotransformation of fatty acids to value-added chemicals.

Fatty acid-derived products such as alkanes, fatty aldehydes, and fatty alcohols have many applications in the chemical industry. These products are predominately produced from fossil resources, but their production processes are often not environmentally friendly. While microbes like Escherichia coli have been engineered to convert fatty acids to corresponding products, the design and optimization of metabolic pathways in cells for high productivity is challenging due to low mass transfer, heavy metabolic burden, and intermediate/product toxicity. Here, we describe an E. coli-based cell-free protein synthesis (CFPS) platform for in vitro conversion of long-chain fatty acids to value-added chemicals with product selectivity, which can also avoid the above issues when using microbial production systems. We achieve the selective biotransformation by cell-free expression of different enzymes and the use of different conditions (e.g., light and heating) to drive the biocatalysis toward different final products. Specifically, in response to blue light, cell-free expressed fatty acid photodecarboxylase (CvFAP, a photoenzyme) was able to convert fatty acids to alkanes with approximately 90% conversion. When the expressed enzyme was switched to carboxylic acid reductase (CAR), fatty acids were reduced to corresponding fatty aldehydes, which, however, could be further reduced to fatty alcohols by endogenous reductases in the cell-free system. By using a thermostable CAR and a heating treatment, the endogenous reductases were deactivated and fatty aldehydes could be selectively accumulated (>97% in the product mixture) without over-reduction to alcohols. Overall, our cell-free platform provides a new strategy to convert fatty acids to valuable chemicals with notable properties of operation flexibility, reaction controllability, and product selectivity.

Tailoring Protein-Polymer Conjugates as Efficient Artificial Enzymes for Aqueous Asymmetric Aldol Reactions.

Artificial enzymes are becoming a powerful toolbox for selective organic syntheses. Herein, we first propose an advanced artificial enzyme by polymeric modularity as an efficient aldolase mimic for aqueous asymmetric aldol reactions. Based on an in-depth understanding of the aldolase reaction mechanism and our previous work, we demonstrate the modular design of protein-polymer conjugates by co-incorporating l-proline and styrene onto a noncatalytic protein scaffold with a high degree of controllability. The tailored conjugates exhibited remarkable catalytic performance toward the aqueous asymmetric aldol reaction of p-nitrobenzaldehyde and cyclohexanone, achieving 94% conversion and excellent selectivity (95/5 diastereoselectivity, 98% enantiomeric excess). In addition, this artificial enzyme showed high tolerance against extreme conditions (e.g., wide pH range, high temperature) and could be reused for more than four times without significant loss of reactivity. Experiments have shown that the artificial enzyme displayed broad specificity for various aldehydes.

Biocatalytic Synthesis Using Self-Assembled Polymeric Nano- and Microreactors.

Biocatalysis is increasingly being explored for the sustainable development of green industry. Though enzymes show great industrial potential with their high efficiency, specificity, and selectivity, they suffer from poor usability and stability under abiological conditions. To solve these problems, researchers have fabricated nano- and micro-sized biocatalytic reactors based on the self-assembly of various polymers, leading to highly stable, functional, and reusable biocatalytic systems. This Review highlights recent progress in self-assembled polymeric nano- and microreactors for biocatalytic synthesis, including polymersomes, reverse micelles, polymer emulsions, Pickering emulsions, and static emulsions. We categorize these reactors into monophasic and biphasic systems and discuss their structural characteristics and latest successes with representative examples. We also consider the challenges and potential solutions associated with the future development of this field.

Copper-Containing Artificial Polyenzymes as a Clickase for Bioorthogonal Chemistry.

Artificial polyenzymes (ArPoly) are tailored combinations of universal protein scaffolds and polymers newly proposed as promising alternatives to natural enzymes to expand the biocatalyst toolbox. The concept of ArPoly has been continuously extended to metal-containing ArPoly to overcome the drawbacks faced by conventional artificial metalloenzymes. Herein, we present a sustainable route to synthesize a novel water-soluble metalloenzyme for copper-catalyzed azide-alkyne cycloadditions in water with remarkable selectivity. In this case, synthetic l-proline monomers were polymerized onto bovine serum albumen in an aqueous medium via copper-mediated "grafting-from" atom-transfer radical polymerization, resulting in protein-polymer-copper conjugates named ArPolyclickase. The copper in ArPolyclickase plays pivotal bifunctional roles, not only as the catalyst for polymerization but also as the coordinated active site for alkyne-azide click catalysis. ArPolyclickase showcases high efficiency, substrate generality, regioselectivity, and ease of product separation for "click chemistry" in water. Notably, ArPolyclickase displays good biocompatibility without imposing copper toxicity on living cells, which offers the prospect for the upcoming bioorthogonal chemistry.

Cationic Covalent Organic Polymer Thin Film for Label-free Electrochemical Bacterial Cell Detection.

Numerous species of bacteria pose a serious threat to human health and cause several million deaths annually. It is therefore essential to have quick, efficient, and easily operable methods of bacterial cell detection. Herein, we synthesize a novel cationic covalent organic polymer (COP) named CATN through the Menshutkin reaction and evaluate its potential as an impedance sensor for Escherichia coli cells. On account of its positive surface charge (ζ-potential = +21.0 mV) and pyridinium moieties, CATN is expected to interact favorably with bacteria that possess a negatively charged cell surface through electrostatic interactions. The interdigitated electrode arrays were coated with CATN using a simple yet non-traditional method of electrophoresis and then used in two-electrode electrochemical impedance spectroscopy (EIS) measurements. The impedance response showed a linear relationship with the increasing concentration of E. coli. The system was sensitive to bacterial concentrations as low as ∼30 CFU mL-1, which is far below the concentration considered to cause illnesses. The calculated limit of detection was as low as 2 CFU mL-1. This work is a rare example of a COP used in this type of bacteria sensing and is anticipated to stimulate further interest in the synthesis of organic polymers for EIS-based sensors.

Artificially sporulated Escherichia coli cells as a robust cell factory for interfacial biocatalysis.

The natural bacterial spores have inspired the development of artificial spores, through coating cells with protective materials, for durable whole-cell catalysis. Despite attractiveness, artificial spores developed to date are generally limited to a few microorganisms with their natural endogenous enzymes, and they have never been explored as a generic platform for widespread synthesis. Here, we report a general approach to designing artificial spores based on Escherichia coli cells with recombinant enzymes. The artificial spores are simply prepared by coating cells with polydopamine, which can withstand UV radiation, heating and organic solvents. Additionally, the protective coating enables living cells to stabilize aqueous-organic emulsions for efficient interfacial biocatalysis ranging from single reactions to multienzyme cascades. Furthermore, the interfacial system can be easily expanded to chemoenzymatic synthesis by combining artificial spores with metal catalysts. Therefore, this artificial-spore-based platform technology is envisioned to lay the foundation for next-generation cell factory engineering.

Designing Modular Cell-free Systems for Tunable Biotransformation of l-phenylalanine to Aromatic Compounds.

Cell-free systems have been used to synthesize chemicals by reconstitution of in vitro expressed enzymes. However, coexpression of multiple enzymes to reconstitute long enzymatic pathways is often problematic due to resource limitation/competition (e.g., energy) in the one-pot cell-free reactions. To address this limitation, here we aim to design a modular, cell-free platform to construct long biosynthetic pathways for tunable synthesis of value-added aromatic compounds, using (S)-1-phenyl-1,2-ethanediol ((S)-PED) and 2-phenylethanol (2-PE) as models. Initially, all enzymes involved in the biosynthetic pathways were individually expressed by an E. coli-based cell-free protein synthesis (CFPS) system and their catalytic activities were confirmed. Then, three sets of enzymes were coexpressed in three cell-free modules and each with the ability to complete a partial pathway. Finally, the full biosynthetic pathways were reconstituted by mixing two related modules to synthesize (S)-PED and 2-PE, respectively. After optimization, the final conversion rates for (S)-PED and 2-PE reached 100 and 82.5%, respectively, based on the starting substrate of l-phenylalanine. We anticipate that the modular cell-free approach will make a possible efficient and high-yielding biosynthesis of value-added chemicals.

Responsive Emulsions for Sequential Multienzyme Cascades.

Multienzyme cascade biocatalysis is an efficient synthetic process, avoiding the isolation/purification of intermediates and shifting the reaction equilibrium to the product side.. However, multienzyme systems are often limited by their incompatibility and cross-reactivity. Herein, we report a multi-responsive emulsion to proceed multienzyme reactions sequentially for high reactivity. The emulsion is achieved using a CO2 , pH, and thermo-responsive block copolymer as a stabilizer, allowing the on-demand control of emulsion morphology and phase composition. Applying this system to a three-step cascade reaction enables the individual optimal condition for each enzyme, and a high overall conversion (ca. 97 % of the calculated limit) is thereby obtained. Moreover, the multi-responsiveness of the emulsion allows the facile and separate yielding/recycling of products, polymers and active enzymes. Besides, the system could be scaled up with a good yield.

Tailoring Particle-Enzyme Nanoconjugates for Biocatalysis at the Organic-Organic Interface.

Nonaqueous Pickering emulsions (PEs) are a powerful platform for catalysis design, offering both a large interface contact and a preferable environment for water-sensitive synthesis. However, up to now, little progress has been made to incorporate insoluble enzymes into the nonaqueous system for biotransformation. Herein, we present biocatalytically active nonaqueous PEs, stabilized by particle-enzyme nanoconjugates, for the fast transesterification and esterification, and eventually for biodiesel synthesis. Our nanoconjugates are the hybrid biocatalysts tailor-made by loading hydrophilic Candida antarctica lipase B onto hydrophobic silica nanoparticles, resulting in not only catalytically active but highly amphiphilic particles for stabilization of a methanol-decane emulsion. The enzyme activity in these PEs is significantly enhanced, ca. 375-fold higher than in the nonaqueous biphasic control. Moreover, the PEs can be multiply reused without significant loss of enzyme performance. With this proof-of-concept, this system can be expanded for many advanced syntheses using different enzymes in the future.

Cell-free protein synthesis enables one-pot cascade biotransformation in an aqueous-organic biphasic system.

Biocatalytic cascade reactions have become increasingly important and useful for chemical synthesis. However, biocatalysts are often incompatible with organic solvents, which prohibits many cascade reactions involving nonpolar substrates. In this study, we used cell-free protein synthesis (CFPS) to express enzymes in an aqueous-organic biphasic system for the construction of an artificial enzymatic pathway. CFPS-expressed enzymes without purification performed efficiently to convert styrene (below 20 mM) to (S)-1-phenyl-1,2-ethanediol (two steps in one pot) with 100% conversion. In addition, our CFPS system showed great tolerance to different organic solvents, and, importantly, the entire biocatalytic system can be consistently scaled up without a reduction of the substrate conversion rate. We, therefore, anticipate that our cell-free approach will make a possible cost-effective, high-yielding synthesis of valuable chemicals.

Facile Synthesis of Enzyme-Embedded Metal-Organic Frameworks for Size-Selective Biocatalysis in Organic Solvent.

In situ immobilization of enzyme into metal-organic frameworks (MOFs) is performed through a one-step and facile method. Candida antarctica lipase B (CalB) is directly embedded in zeolitic imidazolate framework (ZIF)-8 by simply mixing an aqueous solution of 2-methylimidazole and zinc nitrate hexahydrate [Zn(NO3)2⋅6H2O] containing CalB at room temperature. Due to the intrinsic micropores of ZIF-8, the obtained CalB@ZIF composite is successfully applied in size-selective transesterification reaction in organic solvent. CalB@ZIF not only shows much higher catalytic activity but also exhibits higher thermal stability than free CalB. Besides, the robust ZIF-8 shell also offers the hybrid composites excellent reusability.

Association of Serum Lipoprotein-Associated Phospholipase A2 and A379V Gene Polymorphisms with Carotid Plaques.

Objective: Lipoprotein-associated phospholipase A2 (LP-PLA2) is closely related to the development of atherosclerosis. The A379V gene polymorphism, located in exon 11 of the PLA2G7 gene, can affect LP-PLA2 levels and the inflammatory response. However, the association between the A379V polymorphism and formation of carotid plaques is unclear. Materials and Methods: A total of 516 ischemic stroke patients were classified according to carotid intima-media thickness as measured by ultrasound into the plaque group (n = 375, including 258 and 117 cases having vulnerable and stable plaques, respectively) and the nonplaque group (n = 141). The LP-PLA2 gene A379V polymorphism was determined by DNA sequencing, and Lp-PLA2 serum protein levels were determined simultaneously. Results: The serum Lp-PLA2 levels (p < 0.0005), CT+TT genotype frequency (odds ratio [OR]: 1.730, 95% confidence interval [CI]: 1.114-2.686, p = 0.014), and T allele frequency (OR: 1.592, 95% CI: 1.082-2.342, p = 0.018) in the plaque group were significantly higher than those in the nonplaque group. Lp-PLA2 serum levels in the vulnerable plaque subgroup were significantly higher than those in the stable plaque subgroup (p = 0.003). However, there were no significant differences in the frequency of the A379V polymorphism between the vulnerable and stable plaque subgroups. For all subjects, Lp-PLA2 serum levels for patients having a CC genotype were significantly lower than those for patients having a CT (p = 0.003), TT (p = 0.014), or CC+TT genotype (p = 0.001). Logistic regression showed that the Lp-PLA2 level was a risk factor for carotid plaque formation (OR: 1.024, 95% CI: 1.011-1.030, p = 0.001), but the A379V gene polymorphism was not (OR: 1.037, 95% CI: 0.357-3.012, p = 0.947). Conclusion: The A379V gene polymorphism might be associated with serum Lp-PLA2 levels and carotid plaque formation, but not with plaque vulnerability in a Chinese Han population. Serum Lp-PLA2 level was shown to be a risk factor for carotid plaque formation.

Living whole-cell catalysis in compartmentalized emulsion.

Whole-cell biocatalysis plays an important role in biotransformation with unique features such as good tolerance of solvents and easy recycling. However, the relatively low catalytic efficiency limits their use in real production. In this study, a multi-compartmentalized emulsion in organic solvent was constructed to encapsulate living cells for enhanced catalytic performance. Extraordinary large interfacial area of the emulsion improved the bioactivity of Escherichia coli (E. Coli) cells up to 137 times compared to a standard biphasic system. The emulsion was stabilized by a biocompatible polymer and prepared by gentle shaking by hand, which resulted in good cell viability. Moreover, the encapsulated cells could be easily recycled, and the activity remained more than 70% after five cycles. This work provides a promising approach for utilizing whole-cell catalysts for efficient organic catalysis.