离子液体中的生物催化反应

本文综述了近年来离子液体中生物催化反应的研究进展。离子液体作为新的绿色溶剂,用于生物催化反应具有以下特点:在离子液体中酶有良好的稳定性、选择性和反应活性。离子液体可溶解极性大的反应物, 产物易分离,酶和离子液体可重复使用。对离子液体中的生物催化进行了展望。     

Reaching New Biocatalytic Reactivity Using Continuous Flow Reactors

The use of flow reactors in biocatalysis has increased significantly in recent years. Chemists have begun to design flow systems that even allow new biocatalytic reactions to take place. This concept article will focus on the design of flow systems that have allowed enzymes to go beyond their limits in batch. The case is made for moving towards fully continuous systems. With flow chemistry increasingly seen as an enabling technology for automated synthesis, and with advancements in AI-assisted enzyme design, there is a real possibility to fully automate the development and implementation of a continuous biocatalytic processes. This will lead to significantly improved enzyme processes for synthesis.     

Highly Mesoporous Metal‐Organic Frameworks as Synergistic Multimodal Catalytic Platforms for Divergent Cascade Reactions

Rational engineering and assimilation of diverse chemo‐ and biocatalytic functionalities in a single nanostructure is highly desired for efficient multistep chemical reactions but has so far remained elusive. Here, we design and synthesize multimodal catalytic nanoreactors (MCNRs) based on a mesoporous metal‐organic framework (MOF). The MCNRs consist of customizable metal nanocrystals and stably anchored enzymes in the mesopores, as well as coordinatively unsaturated cationic metal MOF nodes, all within a single nanoreactor space. The highly intimate and diverse catalytic mesoporous microenvironments and facile accessibility to the active site in the MCNR enables the cooperative and synergistic participation from different chemo‐ and biocatalytic components. This was shown by one‐pot multistep cascade reactions involving a heterogeneous catalytic nitroaldol reaction followed by a [Pd/lipase]‐catalyzed chemoenzymatic dynamic kinetic resolution to yield optically pure (>99 % ee) nitroalcohol derivatives in quantitative yields.     

Highly Mesoporous Metal-Organic Frameworks as Synergistic Multimodal Catalytic Platforms for Divergent Cascade Reactions

Rational engineering and assimilation of diverse chemo- and biocatalytic functionalities in a single nanostructure is highly desired for efficient multistep chemical reactions but has so far remained elusive. Here, we design and synthesize multimodal catalytic nanoreactors (MCNRs) based on a mesoporous metal-organic framework (MOF). The MCNRs consist of customizable metal nanocrystals and stably anchored enzymes in the mesopores, as well as coordinatively unsaturated cationic metal MOF nodes, all within a single nanoreactor space. The highly intimate and diverse catalytic mesoporous microenvironments and facile accessibility to the active site in the MCNR enables the cooperative and synergistic participation from different chemo- and biocatalytic components. This was shown by one-pot multistep cascade reactions involving a heterogeneous catalytic nitroaldol reaction followed by a [Pd/lipase]-catalyzed chemoenzymatic dynamic kinetic resolution to yield optically pure (>99 % ee) nitroalcohol derivatives in quantitative yields.     

Tripeptide Self-Assembly into Bioactive Hydrogels: Effects of Terminus Modification on Biocatalysis

Bioactive hydrogels based on the self-assembly of tripeptides have attracted great interest in recent years. In particular, the search is active for sequences that are able to mimic enzymes when they are self-organized in a nanostructured hydrogel, so as to provide a smart catalytic (bio)material whose activity can be switched on/off with assembly/disassembly. Within the diverse enzymes that have been targeted for mimicry, hydrolases find wide application in biomaterials, ranging from their use to convert prodrugs into active compounds to their ability to work in reverse and catalyze a plethora of reactions. We recently reported the minimalistic l-His–d-Phe–d-Phe for its ability to self-organize into thermoreversible and biocatalytic hydrogels for esterase mimicry. In this work, we analyze the effects of terminus modifications that mimic the inclusion of the tripeptide in a longer sequence. Therefore, three analogues, i.e., N-acetylated, C-amidated, or both, were synthesized, purified, characterized by several techniques, and probed for self-assembly, hydrogelation, and esterase-like biocatalysis. This work provides useful insights into how chemical modifications at the termini affect self-assembly into biocatalytic hydrogels, and these data may become useful for the future design of supramolecular catalysts for enhanced performance.     

Design and evolution of enzymes for non-natural chemistry

Enzymes are used in biocatalytic processes for the efficient and sustainable production of pharmaceuticals, fragrances, fine chemicals, and other products. Most bioprocesses exploit chemistry found in nature, but we are now entering a realm of biocatalysis that goes well beyond. Enzymes have been engineered to catalyze reactions previously only accessible with synthetic catalysts. Because they can be tuned by directed evolution, many of these new biocatalysts have been shown to perform abiological reactions with high activity and selectivity. We discuss recent examples, showcase catalyst improvements achieved using directed evolution, and comment on some current and future implications of non-natural enzyme evolution for sustainable chemical synthesis.     

Cascade Biocatalysis for Sustainable Asymmetric Synthesis: From Biobased l‐Phenylalanine to High‐Value Chiral Chemicals

Sustainable synthesis of useful and valuable chiral fine chemicals from renewable feedstocks is highly desirable but remains challenging. Reported herein is a designed and engineered set of unique non‐natural biocatalytic cascades to achieve the asymmetric synthesis of chiral epoxide, diols, hydroxy acid, and amino acid in high yield and with excellent ee values from the easily available biobased l ‐phenylalanine. Each of the cascades was efficiently performed in one pot by using the cells of a single recombinant strain over‐expressing 4–10 different enzymes. The cascade biocatalysis approach is promising for upgrading biobased bulk chemicals to high‐value chiral chemicals. In addition, combining the non‐natural enzyme cascades with the natural metabolic pathway of the host strain enabled the fermentative production of the chiral fine chemicals from glucose.     

Spatial Localization of Two Enzymes at Pickering Emulsion Droplet Interfaces for Cascade Reactions

Developing biocatalytic cascades in abiological conditions is of utmost significance, but such processes often suffer from low reaction efficiency because of incompatible reaction environments and suppressed intermediate transportation. Herein we report a new type of biocatalytic cascade by localizing two different enzymes separately in the outer and inner interfacial layers of Pickering emulsion droplets. This versatile approach enables the localization of two enzymes in their preferred reaction microenvironments and simultaneously in nanoscale proximity of each other. The thus-designed interfacial biocatalytic cascades show outstanding catalytic efficiency in alkene epoxidation and thioether oxidation with in situ generation of hydrogen peroxide under mild conditions, 6.9–13.6 times higher than the catalytic efficiency of the free enzymes in solution and their multi-enzymatic counterparts. The remarkable interfacial effect of Pickering droplets was found to be responsible for the significantly enhanced cascading efficiency.     

Biomolecular Release from Alginate‐modified Electrode Triggered by Chemical Inputs Processed through a Biocatalytic Cascade – Integration of Biomolecular Computing and Actuation

Biocatalytic cascades involving more than one or two enzyme‐catalyzed step are inefficient inside alginate hydrogel prepared on an electrode surface. The problem originates from slow diffusion of intermediate products through the hydrogel from one enzyme to another. However, enzyme activity can be improved by surface immobilization. We demonstrate that a complex cascade of four consecutive biocatalytic reactions can be designed, with the enzymes immobilized in an LBL‐assembled polymeric layer at the alginate‐modified electrode surface. The product, hydrogen peroxide, then induces dissolution of iron‐cross‐linked alginate, which results in release process of entrapped biomolecular species, here fluorescently marked oligonucleotides, denoted F‐DNA. The enzymatic cascade can be viewed as a biocomputing network of concatenated AND gates, activated by combinations of four chemical input signals, which trigger the release of F‐DNA. The reactions, and diffusion/release processes were investigated by means of theoretical modeling. A bottleneck reaction step associated with one of the enzymes was observed. The developed system provides a model for biochemical actuation triggered by a biocomputing network of reactions.     

Engineering Enzymes for Environmental Sustainability

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO₂) and have been embedded into new efficient metabolic CO₂ fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.     

Urethanases for the Enzymatic Hydrolysis of Low Molecular Weight Carbamates and the Recycling of Polyurethanes

Enzymatic degradation and recycling can reduce the environmental impact of plastics. Despite decades of research, no enzymes for the efficient hydrolysis of polyurethanes have been reported. Whereas the hydrolysis of the ester bonds in polyester-polyurethanes by cutinases is known, the urethane bonds in polyether-polyurethanes have remained inaccessible to biocatalytic hydrolysis. Here we report the discovery of urethanases from a metagenome library constructed from soil that had been exposed to polyurethane waste for many years. We then demonstrate the use of a urethanase in a chemoenzymatic process for polyurethane foam recycling. The urethanase hydrolyses low molecular weight dicarbamates resulting from chemical glycolysis of polyether-polyurethane foam, making this strategy broadly applicable to diverse polyether-polyurethane wastes.     

On‐Demand Production of Flow‐Reactor Cartridges by 3D Printing of Thermostable Enzymes

The compartmentalization of chemical reactions is an essential principle of life that provides a major source of innovation for the development of novel approaches in biocatalysis. To implement spatially controlled biotransformations, rapid manufacturing methods are needed for the production of biocatalysts that can be applied in flow systems. Whereas three‐dimensional (3D) printing techniques offer high‐throughput manufacturing capability, they are usually not compatible with the delicate nature of enzymes, which call for physiological processing parameters. We herein demonstrate the utility of thermostable enzymes in the generation of biocatalytic agarose‐based inks for a simple temperature‐controlled 3D printing process. As examples we utilized an esterase and an alcohol dehydrogenase from thermophilic organisms as well as a decarboxylase that was thermostabilized by directed protein evolution. We used the resulting 3D‐printed parts for a continuous, two‐step sequential biotransformation in a fluidic setup.     

生物催化在高分子合成中的应用

生物催化剂已被越来越多地用于高分子学中,产生了许多新的反应、工艺和商业用途.酶和全细胞工艺都引起了众多关注,生物催化剂的立体选择性是它们的主要优势之一,新的或改良的方法层出不穷.生物催化的进程主要集中在几个方面上:聚合反应、聚合物修饰反应、聚合物降解反应以及单体和低聚物的合成.在这篇文章中,我们总结了生物催化在高分子合成中的最新应用.     

In Vivo Biocatalytic Cascades Featuring an Artificial-Enzyme-Catalysed New-to-Nature Reaction**

Artificial enzymes utilizing the genetically encoded non-proteinogenic amino acid p-aminophenylalanine (pAF) as a catalytic residue are able to react with carbonyl compounds through an iminium ion mechanism to promote reactions that have no equivalent in nature. Herein, we report an in vivo biocatalytic cascade that is augmented with such an artificial enzyme-catalysed new-to-nature reaction. The artificial enzyme in this study is a pAF-containing evolved variant of the lactococcal multidrug-resistance regulator, designated LmrR_V15pAF_RMH, which efficiently converts benzaldehyde derivatives produced in vivo into the corresponding hydrazone products inside E. coli cells. These in vivo biocatalytic cascades comprising an artificial-enzyme-catalysed reaction are an important step towards achieving a hybrid metabolism.     

Ene-Reductase: A Multifaceted Biocatalyst in Organic Synthesis

Biocatalysis integrate microbiologists, enzymologists, and organic chemists to access the repertoire of pharmaceutical and agrochemicals with high chemoselectivity, regioselectivity, and enantioselectivity. The saturation of carbon-carbon double bonds by biocatalysts challenges the conventional chemical methodology as it bypasses the use of precious metals (in combination with chiral ligands and molecular hydrogen) or organocatalysts. In this line, Ene-reductases (ERs) from the Old Yellow Enzymes (OYEs) family are found to be a prominent asymmetric biocatalyst that is increasingly used in academia and industries towards unparalleled stereoselective trans-hydrogenations of activated C=C bonds. ERs gained prominence as they were used as individual catalysts, multi-enzyme cascades, and in conjugation with chemical reagents (chemoenzymatic approach). Besides, ERs’ participation in the photoelectrochemical and radical-mediated process helps to unlock many scopes outside traditional biocatalysis. These up-and-coming methodologies entice the enzymologists and chemists to explore, expand and harness the chemistries displayed by ERs for industrial settings. Herein, we reviewed the last five year's exploration of organic transformations using ERs.     

酶复合催化剂原位合成新方法及生物催化应用

工业生物催化面临两大重要挑战,一是可工业应用的酶催化反应类型仍然比较有限,远少于化学催化剂,因此需要拓展酶催化的反应类型;二是酶在苛刻的工业催化反应条件下尤其是在高温、有机溶剂、不适宜的pH等环境下稳定性较差,因此需要提高工业酶催化剂的稳定性.研究者已经开发了很多方法,以解决这两方面难题,例如酶的定向进化、定点突变、酶的计算机从头设计和构建人工金属酶等.本文系统介绍了本课题组开发的酶复合催化剂原位合成方法及其生物催化应用,期望为解决工业生物催化的上述挑战提供新思路.原位合成是构建酶-无机晶体复合催化剂的一种简便、高效、普适的方法.酶-无机晶体复合物中,限域包埋使酶具有高于常规固定化酶的催化活性和稳定性.该方法可以简便拓展至其它多种类型的无机晶体材料,显著提高酶的稳定性.无机晶体的限域包埋对酶分子结构和性能有着重要影响,通过理性设计复合催化剂的结构,可实现对酶的活性、稳定性以及多酶反应级联效率的有效调控.本课题组采用分子模拟和实验相结合的方法阐释了多酶-无机晶体复合催化剂所驱动的级联反应效率提高的关键因素.通过调控原位合成中金属离子和有机配体的浓度,实现了酶分子在缺陷型甚至无定形载体中的包埋.在此基础上,深入探讨了缺陷对酶分子结构和催化活性的调控机制,为酶复合催化剂的理性设计提供了依据.同样基于原位合成方法,本课题组构建了酶-金属团簇复合催化剂,实现了温和条件下酶催化和金属催化的高效耦合和协同.以脂肪酶-钯团簇复合催化剂为例,阐明了酶-金属团簇复合催化剂中二者相互作用对酶分子结构和活性以及金属催化活性的影响机制,为酶催化和金属催化相融合的研究提供了重要基础.我们对这一领域存在的挑战和未来重要的研究方向也进行了讨论,希望本文可以从催化剂工程角度为高效酶催化剂的设计以及生物催化应用领域的拓展提供新思路,推动该领域发展.     

A dynamic combinatorial screen for novel imine reductase activity

New imine reductase activity has been discovered in the anaerobic bacterium Acetobacterium woodii by screening a dynamic combinatorial library of virtual imine substrates, using a biphasic water-tetradecane solvent system. Benzylidine aniline and butylidine aniline were reduced to the corresponding amines by caffeate-induced cells, whereas uninduced cells reduced butylidine aniline only. The reductions were detected despite side reactions that consumed some of the starting materials. The new screen can now be extended to discover synthetically useful imine reductases and enzymes that catalyse reactions for which biocatalytic equivalents of the chemical reactions have not yet been discovered.     

Proteins in mesoporous silicates

Mesoporous silicates (MPS) have an ordered pore structure with dimensions comparable to many biological molecules. They have been extensively explored as supports for proteins and enzymes in biocatalytic applications. Since their initial discovery, novel syntheses methods have led to precise control over pore size and structure, particle size, chemical composition, and stability, thus allowing the adsorption of a wide variety of biological macromolecules, such as heme proteins, lipases, antibody fragments, and proteases, into their structures. This Review discusses the application of ordered, large-pore, functionalized mesoporous silicates to immobilize proteins for biocatalysis.     

One-Pot Synthesis of Chiral N-Arylamines by Combining Biocatalytic Aminations with Buchwald–Hartwig N-Arylation

The combination of biocatalysis and chemo-catalysis increasingly offers chemists access to more diverse chemical architectures. Here, we describe the combination of a toolbox of chiral-amine-producing biocatalysts with a Buchwald–Hartwig cross-coupling reaction, affording a variety of α-chiral aniline derivatives. The use of a surfactant allowed reactions to be performed sequentially in the same flask, preventing the palladium catalyst from being inhibited by the high concentrations of ammonia, salts, or buffers present in the aqueous media in most cases. The methodology was further extended by combining with a dual-enzyme biocatalytic hydrogen-borrowing cascade in one pot to allow for the conversion of a racemic alcohol to a chiral aniline.     

Recent Trends in Enzyme Immobilization—Concepts for Expanding the Biocatalysis Toolbox

Enzymes have been exploited by humans for thousands of years in brewing and baking, but it is only recently that biocatalysis has become a mainstream technology for synthesis. Today, enzymes are used extensively in the manufacturing of pharmaceuticals, food, fine chemicals, flavors, fragrances and other products. Enzyme immobilization technology has also developed in parallel as a means of increasing enzyme performance and reducing process costs. The aim of this review is to present and discuss some of the more recent promising technical developments in enzyme immobilization, including the supports used, methods of fabrication, and their application in synthesis. The review highlights new support technologies such as the use of well-established polysaccharides in novel ways, the use of magnetic particles, DNA, renewable materials and hybrid organic–inorganic supports. The review also addresses how immobilization is being integrated into developing biocatalytic technology, for example in flow biocatalysis, the use of 3D printing and multi-enzymatic cascade reactions.