Role of catalyst supports in biocatalysis

Biocatalysis utilizes enzymes and microbial cells as catalysts for a wide range of applications in biotechnology. Immobilization of biocatalysts on various materials has several advantages, including the capacity for reuse, quick reaction termination, easy biocatalyst recovery and operational stability. The present article focuses on the use of material supports for developing immobilized biocatalysts in applications related to energy, environment and chemical synthesis. The work provides a comprehensive overview of a broad class of materials, including organic, inorganic and composites, that have been shown to be prosperous candidates to support the immobilization of enzymes and microbial cells. It also highlights the properties of nanomaterial support such as large surface area and comfort compartment for immobilization. The availability of different types of materials as catalyst support provides an opportunity to understand and develop efficient biocatalytic systems. The choice of selecting a catalyst support will mostly depend on the interaction of the material with the enzyme or microbial cell. Finally, potential challenges, future approaches in developing immobilized biocatalytic systems for various applications and novel material supports are suggested. © 2022 Society of Chemical Industry (SCI).     

The Generation and Exploitation of Protein Mutability Landscapes for Enzyme Engineering

The increasing number of enzyme applications in chemical synthesis calls for new engineering methods to develop the biocatalysts of the future. An interesting concept in enzyme engineering is the generation of large‐scale mutational data in order to chart protein mutability landscapes. These landscapes allow the important discrimination between beneficial mutations and those that are neutral or detrimental, thus providing detailed insight into sequence–function relationships. As such, mutability landscapes are a powerful tool with which to identify functional hotspots at any place in the amino acid sequence of an enzyme. These hotspots can be used as targets for combinatorial mutagenesis to yield superior enzymes with improved catalytic properties, stability, or even new enzymatic activities. The generation of mutability landscapes for multiple properties of one enzyme provides the exciting opportunity to select mutations that are beneficial either for one or for several of these properties. This review presents an overview of the recent advances in the construction of mutability landscapes and discusses their importance for enzyme engineering.     

Curse and Blessing of Non-Proteinogenic Parts in Computational Enzyme Engineering

Enzyme engineering aims to improve or install a new function in biocatalysts for applications ranging from chemical synthesis to biomedicine. For decades, computational techniques have been developed to predict the effect of protein changes and design new enzymes. However, these techniques may have been optimized to deal with proteins composed of the standard amino acid alphabet, while the function of many enzymes relies on non-proteogenic parts like cofactors, nucleic acids, and post-translational modifications. Enzyme systems containing such molecules might be handled or modeled improperly by computational tools, and thus be unsuitable, or require additional tweaking, parameterization, or preparation. In this review, we give an overview of common and recent tools and workflows available to computational enzyme engineers. We highlight the various pitfalls that come with including non-proteogenic compounds in computations and outline potential ways to address common issues. Finally, we showcase successful examples from the literature that computationally engineered such enzymes.     

酶工程研究及应用

酶工程是现代生物技术的重要组成部分，它作为一项高新技术将为各工业的发展起重要推动作用。介绍了酶固定化、基因工程菌（细胞）的固定化、植物细胞培养产酶、酶的化学修饰、核酸酶、抗体酶、酶标药物的理论和技术研究的最新进展以及酶工程在各工业中的应用，对酶工程的发展前景进行了探讨。     

Oleochemicals by Biochemical Reactions?

To obtain oleochemicals via biotransformation or fermentation requires not only appropriate biocatalysts but also good biochemical engineering. Up till now isolated enzymes are mostly used for hydrolytic reactions while entire microorganisms are also employed-for oxidative reactions (especially oxidation of the fatty acid chain). Prejudices that enzymes are expensive, instable catalysts which only work in water and give low space-time-yield are no longer justified. Additional applications of biocatalysts can be expected, where their ability to react with high regio-or enantioselectivity will be exploited. In the literature the standard of biochemical engineering for oleochemicals is mostly rather poor. Kinetic data are incomplete or obtained from conditions not relevant for industrial practice. Product specific biocatalysts consumption especially as a function of conversion is hardly ever given. Mass balances are seldom closed. Bioreactor investigations are mostly performed on too small a scale. Some suggestions are made with respect to classical chemical engineering. Additional reactor concepts are introduced especially for the continuous use of biocatalysts.     

Biochemical basis of free and immobilised enzyme applications in industry,analysis, synthesis and therapy

Biotechnological use of enzymes has been started with cheap enzymes in soluble form, which were chosen from those that do not require the expense of added cofactors. These simple uses have been mainly in food industry applications, often for hydrolytic purposes in order to improve a process or product. Purified enzymes are finding application in analysis, including immunoassay, stereospecific synthesis and in therapy. In these cases, however, much more enzymological knowledge is needed to understand and control the use of the soluble or immobilised enzyme form. It is the appreciation of enzyme structure-mechanism relationships that is essential to progress. This, and the control of enzyme stability, as determined by changes in protein conformation and aggregation, is of the greatest importance for the ultimate goal of designing new and better enzymes and enzyme analogues.     

酶工程在医药工业中的应用

酶工程是现代生物技术的重要组成部分,它作为一项高新技术将为各工业的发展起重要推动作用。介绍了酶固定化、基因工程菌(细胞)的固定化、植物细胞培养产酶、酶的化学修饰、核酸酶、抗体酶、酶标药物的理论和技术研究的最新进展以及酶工程在医药工业中的应用,对酶工程的发展前景进行了探讨。     

Insight into the Structure and Activity of Surface-Engineered Lipase Biofluids

Despite a successful application of solvent-free liquid protein (biofluids) concept to a number of commercial enzymes, the technical advantages of enzyme biofluids as hyperthermal stable biocatalysts cannot be fully utilized as up to 90–99% of native activities are lost when enzymes were made into biofluids. With a two-step strategy (site-directed mutagenesis and synthesis of variant biofluids) on Bacillus subtilis lipase A (BsLA), we elucidated a strong dependency of structure and activity on the number and distribution of polymer surfactant binding sites on BsLA surface. Here, it is demonstrated that improved BsLA variants can be engineered via site-mutagenesis by a rational design, either with enhanced activity in aqueous solution in native form, or with improved physical property and increased activity in solvent-free system in the form of a protein liquid. This work answered some fundamental questions about the surface characteristics for construction of biofluids, useful for identifying new strategies for developing advantageous biocatalysts.     

Perspectives on processing methods,equipment and procedures

World population growth and increasing per capita consumption will place significant demands on the food oils industry in the coming years to maimize efficiency of raw material use and to optimize processing operations. Other demands on the processor of food oils center on resolving issues that are already impacting on the industry, i.e., energy conservation, pollution abatement, and diminishing reserve of petroleum-based resources. Research now in progress in the laboratory may form the basis for the industry response to these challenges. Innovative methods of raw material preparation will be needed to obtain a higher quality oil. Alternative solvent processing could use alcohol or aqueous extraction or supercritical fluids. Each of the processing techniques used to produce a finished edible oil from crude oils, from degumming through alkali refining and bleaching to deodorization, is subject to change, and the form of these changes can be perceived from the directions of current research. Formulation of solid fats from liquid oils may see a shift from metal-catalyzed reactions to the use of immobilized enzymes. Implementation of many of the process changes will depend on equipment development and application of advanced engineering concepts to assure their assimilation into the food oil industry. By projecting the successful integration of the chemical, process design and engineering sciences, a realistic picture of the year 2000 can be formulated.     

Engineering of Immobilized Enzyme Systems

The advent of enzyme immobilization, allowing re-use of enzymes, and essentially eliminating product contamination, has greatly increased the potential of enzymatic processes for industrial use. Although this is a relatively new field of study, the literature is rapidly expanding. The compendium of references prepared by the New England Research Applications Center for Corning Glass Works now lists over 800 papers concerning immobilized enzymes. For this reason the scope of this paper is limited to immobilized enzyme engineering, including reactor design and performance. Although medical and analytical applications of immobilized enzymes are important, they will not be discussed as such here. Another large body of information concerning enzyme immobilization techniques will be treated only briefly as an introduction. Other general articles and reviews [1-5] may be useful in providing additional background information.     

Enzyme flow microcalorimetry—a useful tool for screening of immobilized penicillin G acylase

Screening of a representative series of immobilized penicillin G acylase biocatalysts (enzyme, cells) using enzyme flow microcalorimetry is described. Immobilized penicillin G acylase biocatalysts were either prepared in the laboratory by various techniques or obtained from four commercial manufacturers. An industrial strain of Escherichia coli was entrapped in (poly)acrylamide gel or hardened calcium pectate gel. Semi-purified enzyme was immobilized in various ways—either by covalent binding to oxirane-acrylic beads or chlorotriazine bead cellulose or by entrapment in (poly)acrylamide gel. The validity of the enzyme flow microcalorimetry results was corroborated by a pH-stat method, showing enzyme flow microcalorimetry to be a suitable method for rapid screening of immobilized biocatalysts regardless of the immobilization technique, carrier type or the biocatalyst source. © 1998 Society of Chemical Industry     

Potential of Different Enzyme Immobilization Strategies to Improve Enzyme Performance

Enzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual pre‐requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre‐existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports [enzyme crosslinked aggregates (CLEAs) or crystals (CLECs)] are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc).     

酶工程：从人工设计到人工智能

计算机在酶工程中的应用使得酶的序列空间探索度不断被扩大。随着不同分子力场参数的建立,涌现出诸多以计算分子能量为基础的算法,并被用于酶的催化活性、稳定性、底物特异性等的改造与筛选。伴随计算机硬件的提升与算法的优化,从头设计全新功能的人工酶取得成功并得以发展。近年来,人工智能在蛋白质结构预测上不断获得突破,同时也被应用到酶的设计中。介绍了分子力场基础和酶设计与筛选的算法,重点阐述了从头设计的方法和成功案例,以及机器学习设计酶的流程和最新的研究进展,展望了人工智能在酶工程领域的未来发展,为酶的改造与全新功能的生物催化剂的设计助力。     

Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases.     

Die Bedeutung der Biotechnologie für die industrielle Chemie

The significance of biotechnology for industrial chemistry. In the next few decades biotechnology will bring about fundamental innovations in the fields of health, nutrition, and agriculture as well as in important branches of industrial production, such as chemistry, which may well be comparable in importance with the present revolutionary technical progress based on electronics. Justification for these forecasts comes mainly from pioneering scientific advances in the fields of molecular biology, biochemistry, and microbiology. Particular mention should be made here of the fascinating potential of genetic engineering methods presently undergoing rapid development. However, less spectacular techniques, such as cell fusion and the immobilization of enzymes or whole living cells on polymeric substrates to give manageable, long-lived biocatalysts, will also contribute to this wave of innovation. Some of these new developments are now gaining acceptance in industrial production.     

Novel biocatalysts: Recent developments

The limited number of suitably well characterized biocatalysts continues to limit progress in the application of biological routes in the synthesis of compounds for novel pharmaceuticals, materials, or performance chemicals. In this situation, the discovery of novel biocatalysts or novel functionalities or substrates on existing ones is an important task. This work describes a range of novel biocatalysts obtained recently through one of three techniques: environmental sampling or screening, protein engineering on existing enzymes, or extension of the catalytic profile of existing catalysts.     

Oxidation Catalysis by Rationally Designed Artificial Metalloenzymes

The principle of enzyme mimics has been raised to its pinnacle by the design of hybrids made from inorganic complexes embedded into biomolecules. The present review focuses on the design of artificial metalloenzymes for oxidation reactions by oxygen transfer reactions, with a special focus on proteins anchoring inorganic complexes or metal ions via supramolecular interactions. Such reactions are of great interest for the organic synthesis of building blocks. In the first part, following an overview of the different design of artificial enzymes, the review presents contributions to the rational design of efficient hybrid biocatalysts via supramolecular host/guest approaches, based on the nature of the inorganic complex and the nature of the protein, with special attention to the substrate binding. In the second part, the original purpose of artificial metalloenzymes has been twisted to enable the observation of transient intermediates, to decipher metal-based oxidation mechanisms. The host protein crystals have been used as crystalline molecular-scale vessels, within which inorganic catalytic reactions have been followed, thanks to X-ray crystallography. These hybrids should be an alternative to enzymes for sustainable chemistry.     

Harnessing P450 Enzyme for Biotechnology and Synthetic Biology

Cytochrome P450 enzymes (P450s, CYPs) catalyze the oxidative transformation of a wide range of organic substrates. Their functions are crucial to xenobiotic metabolism and steroid transformation in humans and other organisms. The enzymes are promising for synthetic biology applications but limited by several drawbacks including low turnover rates, poor stability, the dependance of expensive cofactors and redox partners, and the narrow substrate scope. To conquer these obstacles, emerging strategies including substrate engineering, usage of decoy and decoy-based small molecules auxiliaries, designing of artificial enzyme cascades and the incorporation of materials have been explored based on the unique properties of P450s. These strategies can be applied to a wide range of P450s and can be combined with protein engineering to improve the enzymatic activities. This minireview will focus on some recent developments of these strategies which have been used to leverage P450 catalysis. Remaining challenges and future opportunities will also be discussed.     

Flow Biocatalysis: A Challenging Alternative for the Synthesis of APIs and Natural Compounds

Biocatalysts represent an efficient, highly selective and greener alternative to metal catalysts in both industry and academia. In the last two decades, the interest in biocatalytic transformations has increased due to an urgent need for more sustainable industrial processes that comply with the principles of green chemistry. Thanks to the recent advances in biotechnologies, protein engineering and the Nobel prize awarded concept of direct enzymatic evolution, the synthetic enzymatic toolbox has expanded significantly. In particular, the implementation of biocatalysts in continuous flow systems has attracted much attention, especially from industry. The advantages of flow chemistry enable biosynthesis to overcome well-known limitations of “classic” enzymatic catalysis, such as time-consuming work-ups and enzyme inhibition, as well as difficult scale-up and process intensifications. Moreover, continuous flow biocatalysis provides access to practical, economical and more sustainable synthetic pathways, an important aspect for the future of pharmaceutical companies if they want to compete in the market while complying with European Medicines Agency (EMA), Food and Drug Administration (FDA) and green chemistry requirements. This review focuses on the most recent advances in the use of flow biocatalysis for the synthesis of active pharmaceutical ingredients (APIs), pharmaceuticals and natural products, and the advantages and limitations are discussed.     

Cell-Free Protein Synthesis for the Screening of Novel Azoreductases and Their Preferred Electron Donor

Azoreductases are potent biocatalysts for the cleavage of azo bonds. Various gene sequences coding for potential azoreductases are available in databases, but many of their gene products are still uncharacterized. To avoid the laborious heterologous expression in a host organism, we developed a screening approach involving cell-free protein synthesis (CFPS) combined with a colorimetric activity assay, which allows the parallel screening of putative azoreductases in a short time. First, we evaluated different CFPS systems and optimized the synthesis conditions of a model azoreductase. With the findings obtained, 10 azoreductases, half of them undescribed so far, were screened for their ability to degrade the azo dye methyl red. All novel enzymes catalyzed the degradation of methyl red and can therefore be referred to as azoreductases. In addition, all enzymes degraded the more complex and bulkier azo dye Brilliant Black and four of them also showed the ability to reduce p-benzoquinone. NADH was the preferred electron donor for the most enzymes, although the synthetic nicotinamide co-substrate analogue 1-benzyl-1,4-dihydronicotinamide (BNAH) was also accepted by all active azoreductases. This screening approach allows accelerated identification of potential biocatalysts for various applications.