Designing for sustainability with biocatalytic and chemoenzymatic cascade processes

Cascade reactions have been widely recognized to cut costs, decrease solvent usage, and reduce cycle times in chemical processes. Recently, biocatalytic cascades have altered how we design synthetic routes to complex molecules to achieve sustainable commercial processes for pharmaceutical, agricultural, and fine chemical industries. With advancements in protein engineering and an increase in the number of enzyme classes available to chemists, industrial and academic groups alike have endeavored to expand the scope of biocatalysis from single reactions to multi-enzyme cascades to rapidly build complex molecular structures. Recent reports have drawn inspiration from biosynthetic pathways and have applied engineered enzymes to in vitro enzymatic cascades. Furthermore, combining transition-metal catalysis and enzymes in one-pot chemoenzymatic cascades likewise serves to broaden the scope of biocatalysis, enabling traditional chemical reactions to be performed under mild aqueous conditions. In this article, we review recent biocatalytic and chemoenzymatic cascades from 2019 to 2021.     

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.     

Cell–enzyme tandem systems for sustainable chemistry

The combination of isolated enzymes and whole cells for chemical biomanufacturing has recently arose as an alternative with multiple industrial advantages. Both isolated enzymes and whole-cell biocatalysis have benefits of their own that can be synergistically used in more efficient and sustainable bioprocesses. Those benefits range from decreasing the production times to generating products that are otherwise unobtainable. In this review we have studied the reports of cell–enzyme tandem systems applied as biocatalysts focusing on the different architectures used for their coupling. Combination of extracellular enzymes and microorganisms, enzyme display on whole cell walls and integration of enzymes and microorganisms into different materials are presented as the available alternatives for tandem enzyme–cell systems’ biotransformations.     

A generic platform for the immobilisation of engineered biocatalysts

The application of biocatalysis in the pharmaceutical industry is rapidly growing as a result of increased access to enzymes that meet the demands of industrial processes. This expansion of activity has led to a corresponding increase in the demand for immobilised enzymes. EziG? (EnginZyme AB, Sweden) is marketed as a general matrix for enzyme immobilisation on controlled porosity glass. In this work we identified criteria for a “general” enzyme immobilisation technology in the context of the requirements of the pharmaceutical industry. We subsequently evaluated EziG? for generality in a series of case studies for the application of immobilised biocatalysts. In this study we have focussed on the challenges facing both academic and industrial applications such as enzyme stability, multistep reactions and reactions in continuous flow.     

Engineering of Therapeutic and Detoxifying Enzymes

Therapeutic enzymes present excellent opportunities for the treatment of human disease, modulation of metabolic pathways and system detoxification. However, current use of enzyme therapy in the clinic is limited as naturally occurring enzymes are seldom optimal for such applications and require substantial improvement by protein engineering. Engineering strategies such as design and directed evolution that have been successfully implemented for industrial biocatalysis can significantly advance the field of therapeutic enzymes, leading to biocatalysts with new-to-nature therapeutic activities, high selectivity, and suitability for medical applications. This minireview highlights case studies of how state-of-the-art and emerging methods in protein engineering are explored for the generation of therapeutic enzymes and discusses gaps and future opportunities in the field of enzyme therapy.     

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.     

Evolution of polyhydroxyalkanoate (PHA) production system by "enzyme evolution": successful case studies of directed evolution

Biotechnological studies towards the biosynthesis of polyhydroxyalkanoates (PHAs) biopolyesters have extensively progressed through the development of various metabolic engineering strategies. Historically, efficient PHA production has been achieved using the fermentation technology of naturally occurring PHA-producing bacteria based on external substrate manipulation (1st generation), and subsequent reinforcement with recombinant gene technology (2nd generation). More recently, "enzyme evolution" is becoming the 3rd generation approach for PHA production. A break-through in the chemical synthesis of macromolecules with desirable properties was achieved by the development of prominent chemical catalysts via "catalyst evolution", as represented by a series of Ziegler-Natta catalysts. Thus, one can easily accept the concept that the molecular evolution of the biocatalysts (enzymes) relevant to PHA synthesis will provide us with a chance to create novel PHA materials with high performance. The first trial of an in vitro enzyme evolution in PHA biosynthesis was reported by our group in 2001. The following literature data, as well as our own experimental results devoted to this new approach, have been accumulated over a short time. This review article focuses specifically on the concept and current case studies of the application of "enzyme evolution" to PHA biosynthesis.     

Temperature-responsive Pickering high internal phase emulsions for recyclable efficient interfacial biocatalysis

The field of biocatalysis is expanding owing to the increasing demand for efficient low-cost green chemical processes. However, a feasible strategy for achieving product separation, enzyme recovery, and high catalytic efficiency in biocatalysis remains elusive. Herein, we present thermoresponsive Pickering high internal phase emulsions (HIPEs) as controllable scaffolds for efficient biocatalysis; these HIPEs demonstrate a transition between emulsification and demulsification depending on temperature. Ultra-high-surface-area Pickering HIPEs were stabilized by Candida antarctica lipase B immobilized on starch particles modified with butyl glycidyl ether and glycidyl trimethyl ammonium chloride, thus simplifying the separation and reuse processes and significantly improving the catalytic efficiency. In addition, the switching temperature can be precisely tuned by adjusting the degree of substitution of the modified starches to meet the temperature demands of various enzymes. We believe that this system provides a green platform for various interfacial biocatalytic processes of industrial interest.

The thermoresponsive Pickering high internal phase emulsions stabilized by starch particles as controllable scaffolds for efficient biocatalysis, which simplified the separation and reuse processes and significantly improved the catalytic efficiency.     

Engineering of biocatalysts - from evolution to creation

Enzymes are increasingly being used in an industrial setting as a cheap and environmentally-friendly alternative to chemical catalysts. In order to produce the ideal biocatalyst, natural enzymes often require optimization to increase their catalytic efficiencies and specificities under a particular range of reaction conditions. A number of enzyme engineering strategies are currently employed to modify biocatalysts, improving their suitability for large-scale industrial applications. These include various directed evolution techniques, semi-rational design techniques, and more recently, the de novo design of novel enzymes. Advances in mutant library design, high-throughput selection processes, and the introduction of powerful computer algorithms have all contributed to the current exponential growth of the field of enzyme engineering. This review article aims to present some of the currently employed strategies for enzyme engineering and attempts to highlight the most recent advances in methodology.     

Recent update on use of ionic liquids for enzyme immobilization,activation, and catalysis: A partnership for sustainability

Recent literature survey suggested that, ionic liquid not only possesses potential as a green solvent, but also plays a significant role in enzyme immobilization, activation, stabilization, and catalysis. Furthermore, biocatalysis in ionic liquids (IL) may be a key sustainable solution for the next generation chemical processes, which requisite extensive research efforts to expand the knowledge horizons in this field. In view of this, the present review highlights the recent update of potential applications of IL in biocatalysis for (i) biomass pretreatment/hydrolysis, (ii) enzyme immobilization-activation, (iii) organic transformation, (iv) bioremediation, and (v) biosensing. Moreover, this review also addresses the challenging issues and future outlook of this research area for the industrial development in near future.     

高通量筛选策略在细胞色素P450单加氧酶定向进化中的应用

细胞色素P450单加氧酶具有催化活性混杂性的特点,可以催化多种氧化反应,因而在生物催化领域受到了极大的关注。然而P450单加氧酶往往存在催化活性低、稳定性差、区域和立体选择性不理想等问题,从而限制了其在生物催化领域的广泛运用。蛋白质定向进化的发展与运用为改善P450单加氧酶的催化性能提供了有效的途径,而一种高效的高通量筛选策略是保证酶蛋白定向进化成功实施的关键。本文综述了P450单加氧酶定向进化过程中高通量筛选策略的最新进展。     

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.     

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.     

High‐throughput droplet‐based microfluidics for directed evolution of enzymes

Natural enzymes have evolved over millions of years to allow for their effective operation within specific environments. However, it is significant to note that despite their wide structural and chemical diversity, relatively few natural enzymes have been successfully applied to industrial processes. To address this limitation, directed evolution (DE) (a method that mimics the process of natural selection to evolve proteins toward a user‐defined goal) coupled with droplet‐based microfluidics allows the detailed analysis of millions of enzyme variants on ultra‐short timescales, and thus the design of novel enzymes with bespoke properties. In this review, we aim at presenting the development of DE over the last years and highlighting the most important advancements in droplet‐based microfluidics, made in this context towards the high‐throughput demands of enzyme optimization. Specifically, an overview of the range of microfluidic unit operations available for the construction of DE platforms is provided, focusing on their suitability and benefits for cell‐based assays, as in the case of directed evolution experimentations.     

Properties and Synthetic Applications of Enzymes in Organic Solvents

Biotransformations already represent an effective and sometimes preferable alternative to chemical synthesis for the production of fine chemicals and optically active compounds. To further widen the versatility of the biological approach, the so-called "nonaqueous enzymology", which now represents an important area of research and biotechnological development, has emerged in the last ten years or so. This new methodology is especially suitable for the modification of precursors of pharmaceutical compounds and fine chemicals, which, in most cases, are insoluble or poorly soluble in water. Even though the idea of carrying out an enzymatic process in organic solvent was initially considered with scepticism, biocatalysis in such media is now investigated and exploited in numerous academic and industrial laboratories. One of the reasons that makes enzymatic catalysis in nonaqueous media so appealing, is the important new properties that enzymes exhibit in organic solvents. For example, they are often more stable and can catalyze reactions that are impossible or difficult in water. Furthermore, enzyme selectivity can also differ from that in water and can change, or even reverse, from one solvent to another. This phenomenon, which can be called "medium engineering", can be exploited as a valid alternative to protein engineering. The first part of this review examines the thermodynamic, kinetic, spectroscopic, and physical approaches that have been adopted to investigate the factors that affect activity, stability, structure, and selectivity of enzymes in organic solvents. These combined studies have brought the understanding of enzyme catalysis in organic solvents to a level almost comparable to that reached for biocatalysis in aqueous media. The second part surveys a number of the synthetic applications of enzymes in organic media, which span from the preparation of milligrams of specifically labeled compounds to the modification of fats on multiton scale and from the preparation of complex key intermediates for the pharmaceutical industry to the synthesis of polymers.     

Biochemical applications of ultrathin films of enzymes, polyions and DNA

This feature article summarizes recent applications of ultrathin films of enzymes and DNA assembled layer-by-layer (LbL). Using examples mainly from our own research, we focus on systems developed for biocatalysis and biosensors for toxicity screening. Enzyme-poly(L-lysine) (PLL) films, especially when stabilized by crosslinking, can be used for biocatalysis at unprecedented high temperatures or in acidic or basic solutions on electrodes or sub-micron sized beads. Such films have bright prospects for chiral synthesis and biofuel cells. Excellent bioactivity and retention of enzyme structure in these films facilitates their use in detailed kinetic studies. Biosensors and arrays employing DNA-enzyme films show great promise in predicting genotoxicity of new drug and chemical product candidates. These devices combine metabolic biocatalysis, reactive metabolite-DNA reactions, and DNA damage detection. Catalytic voltammetry or electrochemiluminescence (ECL) can be used for high throughput arrays utilizing multiple LbL "spots" of DNA, enzyme and metallopolymer. DNA-enzyme films can also be used to produce nucleobase adduct toxicity biomarkers for detection by LC-MS. These approaches provide valuable high throughput tools for drug and chemical product development and toxicity prediction.     

Miniaturizing chemistry and biology in microdroplets

By compartmentalizing reactions in aqueous microdroplets of water-in-oil emulsions, reaction volumes can be reduced by factors of up to 10(9) compared to conventional microtitre-plate based systems. This allows massively parallel processing of as many as 10(10) reactions in a total volume of only 1 ml of emulsion. This review describes the use of emulsions for directed evolution of proteins and RNAs, and for performing polymerase chain reactions (PCRs). To illustrate these applications we describe certain specific experiments, each of which exemplifies a different facet of the technique, in some detail. These examples include directed evolution of Diels-Alderase and RNA ligase ribozymes and several classes of protein enzymes, including DNA polymerases, phosphotriesterases, beta-galactosidases and thiolactonases. We also describe the application of emulsion PCR to screen for rare mutations and for new ultra-high throughput sequencing technologies. Finally, we discuss the recent development of microfluidic tools for making and manipulating microdroplets and their likely impact on the future development of the field.     

Ionic liquids in enzymatic catalysis and biochemical methods of analysis: Capabilities and prospects

The first Russian review systematizes and discusses the most important and promising published data on the use of ionic liquids in biocatalysis and, especially, biochemical methods of analysis. Studies on the use of ionic liquids as solvents for enzymes, new reaction media for enzymatic reactions, and components of the biosensitive layers of sensors are analyzed. The physical and chemical properties of ionic liquids used in biocatalysis are discussed. The advantages of ionic liquids over the usual solvents in homogeneous and heterogeneous reactions with the participation of enzymes from various classes are demonstrated, procedures for the coimmobilization of biocatalysts and ionic liquids with cellulose onto polymer supports and electrodes are described, and prospects for the use of enzyme-ionic liquid compositions in biochemical methods of analysis are considered.     

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.     

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.