Biocatalysis for pharmaceutical intermediates: the future is now

Biocatalysis is continuing to gain momentum and is now becoming a key component in the toolbox of the process chemist, with a place alongside chemocatalysis and chromatographic separations. The pharmaceutical industry demands a speed of development that must be on a parallel with conventional chemistry and high optical purity for complex compounds with multiple chiral centres. This review describes how these demands are being addressed to make biocatalysis successful, particularly by the use of micro-scale technology for high-speed catalyst screening and process development alongside discipline integration of biology and engineering with chemistry. Developments in recombinant technology will further expand the repertoire of biocatalysis in the coming years to new chemistries and enable catalyst design to fit the process. Further development of biocatalysis for green chemistry and high productivity processes can also be expected.     

生物催化与生物转化研究进展

由于生物催化过程具有高效、高选择性、条件温和、环境友好等优点,因此成为可持续发展过程中替代和拓展传统有机化学合成的重要方法。近两年的进展集中于新生物催化剂的发现和改造,以及将生物催化和生物转化应用于工业过程的探索,包括开发新的反应体系,新的固定化方法等。可以预见,在医药中间体等高附加值化工产品的生产过程中,生物催化和生物转化的应用将呈现加速增长趋势。     

Trends and innovations in industrial biocatalysis for the production of fine chemicals

Biocatalysis has become an established technology for the industrial manufacture of fine chemicals. In recent years, a multitude of chemical companies have embraced biocatalysis for the manufacture of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure alpha- and beta-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target molecules. These recent innovations in the manufacture of industrial fine chemicals using biocatalysis are discussed from an industrial perspective.     

Biocatalysis in ionic liquids - advantages beyond green technology

In recent years researchers have started to explore a particular class of organic solvents called room temperature ionic liquids - or simply ionic liquids - to identify their unique advantages for biocatalysis. Because they lack vapour pressure, ionic liquids hold potential as green solvents. Furthermore, unlike organic solvents of comparable polarity, they often do not inactivate enzymes, which simplifies reactions involving polar substrates such as sugars. Biocatalytic reactions in ionic liquids have also shown higher selectivity, faster rates and greater enzyme stability; however, these solvents present other challenges, among them difficulties in purifying ionic liquids and controlling water activity and pH, higher viscosity and problems with product isolation.     

Ionic liquids in biotechnology: applications and perspectives for biotransformations

Ionic liquids are considered as an alternative to organic solvents for catalysis. The literature in this field is reviewed with focus on advantageous use of ionic liquids in biocatalysis and biotransformations. The overview reveals that the exploration and mapping of ionic liquids with respect to biocatalysis is still sketchy. It is apparent that advantages can be gained in view of activity, stability and selectivity. Furthermore, integration of reaction and separation has a high potential in the field. The review presents quantitative data on the productivities, space–time yields, as well as stability as far as they can be extracted from the literature. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Substrate supply for effective biocatalysis

Using biocatalysis for some chemical synthesis steps has unique advantages such as achieving higher product selectivity under ambient process conditions. However, a common limitation with such systems is the inhibition or toxicity posed by the starting substrate as well as limited aqueous solubility in many cases. In this review, we discuss the supply of substrate to bioconversions. The delivery of substrate via an auxiliary, which may be water-miscible, or a second phase such as a water-immiscible organic solvent, adsorbing resin, or a gas, is examined through recent examples in the field. Finally, guidelines for experimental planning and process considerations are suggested to facilitate the choice of substrate delivery method and accelerate process development.     

Modulating enzyme activity using ionic liquids or surfactants

One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme–water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems.     

Designer metalloenzymes for synthetic biology: Enzyme hybrids for catalysis

Combining organometallics and biology has generated broad interest from scientists working on applications from in situ drug release to biocatalysis. Engineered enzymes and biohybrid catalysts (also referred to as artificial enzymes) have introduced a wide range of abiotic chemistry into biocatalysis. Predominantly, this work has concentrated on using these catalysts for single step in vitro reactions. However, the promise of using these hybrid catalysts in vivo and combining them with synthetic biology and metabolic engineering is vast. This report will briefly review recent advances in artificial metalloenzyme design, followed by summarising recent studies that have looked at the use of these hybrid catalysts in vivo and in enzymatic cascades, therefore exploring their potential for synthetic biology.     

Directed evolution for enzyme development in biocatalysis

As an important sector of the chemical industry, biocatalysis requires the continuous development of enzymes with tailor-made activity, selectivity, stability, or tolerance to unnatural environments. This is now routinely achieved by directed evolution based on iterative cycles of genetic diversification and activity screening. Here, we highlight its recent developments. First, the design of “smarter” libraries by focused mutagenesis may be a crucial start-up for a fast and successful outcome. Then library assembly and expression are also key steps that benefits from modern molecular biology progresses. Finally, various strategies may be considered for library screening depending on the final objective: while low-throughput direct assays have been very successful in generating enzymes for important biocatalytic processes, even in bringing completely new chemistries to the enzyme world, ultrahigh-throughput screening methods are emerging as powerful approaches for engineering the next generation of industrial enzymes.     

Biocatalytic transformations in ionic liquids

Room temperature ionic liquids are non-volatile, thermally stable and highly polar; they are also moderately hydrophilic solvents. Here, we discuss their use as reaction media for biocatalysis. Enzymes of widely diverging types are catalytically active in ionic liquids or aqueous biphasic ionic liquid systems. Lipases, in particular, maintain their activity in anhydrous ionic liquid media; the (enantio)selectivity and operational stability are often better than in traditional media. The unconventional solvent properties of ionic liquids have been exploited in biocatalyst recycling and product recovery schemes that are not feasible with traditional solvent systems.     

Biodiversity for biocatalysis: A review of the α/β-hydrolase fold superfamily of esterases-lipases discovered in metagenomes

Natural biodiversity undoubtedly inspires biocatalysis research and innovation. Biotransformations of interest also inspire the search for appropriate biocatalysts in nature. Indeed, natural genetic resources have been found to support the hydrolysis and synthesis of not only common but also unusual synthetic scaffolds. The emerging tool of metagenomics has the advantage of allowing straightforward identification of activity directly applicable as biocatalysis. However, new enzymes must not only have outstanding properties in terms of performance but also other properties superior to those of well-established commercial preparations in order to successfully replace the latter. Esterases (EST) and lipases (LIP) from the α/β-hydrolase fold superfamily are among the enzymes primarily used in biocatalysis. Accordingly, they have been extensively examined with metagenomics. Here we provided an updated (October 2015) overview of sequence and functional data sets of 288 EST–LIP enzymes with validated functions that have been isolated in metagenomes and (mostly partially) characterized. Through sequence, biochemical, and reactivity analyses, we attempted to understand the phenomenon of variability and versatility within this group of enzymes and to implement this knowledge to identify sequences encoding EST–LIP which may be useful for biocatalysis. We found that the diversity of described EST–LIP polypeptides was not dominated by a particular type of protein or highly similar clusters of proteins but rather by diverse nonredundant sequences. Purified EST–LIP exhibited a wide temperature activity range of 10–85?°C, although a preferred bias for a mesophilic temperature range (35–40?°C) was observed. At least 60% of the total characterized metagenomics-derived EST–LIP showed outstanding properties in terms of stability (solvent tolerance) and reactivity (selectivity and substrate profile), which are the features of interest in biocatalysis. We hope that, in the future, the search for and utilization of sequences similar to those already encoded and characterized EST–LIP enzymes from metagenomes may be of interest for promoting unresolved biotransformations in the chemical industry. Some examples are discussed in this review.     

The Promises and the Challenges of Biotransformations in Microflow

The challenges of transition toward the postpetroleum world shed light on the biocatalysis as the most sustainable way for the valorization of biobased raw materials. However, its industrial exploitation strongly relies on integration with innovative technologies such as microscale processing. Microflow devices remarkably accelerate biocatalyst screening and engineering, as well as evaluation of process parameters, and intensify biocatalytic processes in multiphase systems. The inherent feature of microfluidic devices to operate in a continuous mode brings additional interest for their use in chemoenzymatic cascade systems and in connection with the downstream processing units. Further steps toward automation and analytics integration, as well as computer‐assisted process development, will significantly affect the industrial implementation of biocatalysis and fulfill the promises of the bioeconomy. This review provides an overview of recent examples on implementation of microfluidic devices into various stages of biocatalytic process development comprising ultrahigh‐throughput biocatalyst screening, highly efficient biocatalytic process design including specific immobilization techniques for long‐term biocatalyst use, integration with other (bio)chemical steps, and/or downstream processing.     

Microbial steroid transformations: current state and prospects

Studies of steroid modifications catalyzed by microbial whole cells represent a well-established research area in white biotechnology. Still, advances over the last decade in genetic and metabolic engineering, whole-cell biocatalysis in non-conventional media, and process monitoring raised research in this field to a new level. This review summarizes the data on microbial steroid conversion obtained since 2003. The key reactions of structural steroid functionalization by microorganisms are highlighted including sterol side-chain degradation, hydroxylation at various positions of the steroid core, and redox reactions. We also describe methods for enhancement of bioprocess productivity, selectivity of target reactions, and application of microbial transformations for production of valuable pharmaceutical ingredients and precursors. Challenges and prospects of whole-cell biocatalysis applications in steroid industry are discussed.     

A window into biocatalysis and biotransformations

Eight papers were presented in this year's symposium "Advances in Biocatalysis" at the 232nd ACS National Meeting, accentuating the most recent development in biocatalysis. Researchers from both industry and academia are addressing several fundamental problems in biocatalysis, including the limited number of commercially available enzymes that can be provided in bulk quantities, the limited enzyme stability and activity in nonaqueous environments, and the permeability issue and cell localization problems in whole-cell systems. A trend that can be discerned from these eight talks is the infusion of new tools and technologies in addressing various challenges facing biocatalysis. Nanotechnology, bioinformatics, cellular membrane engineering and metabolic engineering (for engineering whole-cell catalysts), and protein engineering (to improve enzymes and create novel enzymes) are becoming more routinely used in research laboratories and are providing satisfactory solutions to the problems in biocatalysis. Significant progress in various aspects of biocatalysis from discovery to industrial applications was highlighted in this symposium.     

Strategies for discovery and improvement of enzyme function: state of the art and opportunities

Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non‐conventional media, using new substrates for old enzymes, active‐site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities.     

Editorial

The field of biocatalysis and biotransformations has grown considerably in recent years, as witnessed at Biotrans '99 (the 4th international symposium on Biocatalysis and Biotransformations, held in Sicily) which was an outstanding scientific success. BIOCATALYSIS AND BIOTRANSFORMA-TION (BCBT), which is the official journal of the Working Party on Applied Biocatalysis of the European Federation of Biotechnology, intends to be at the forefront of developments in this field and, together with its established coverage, is particularly keen to present work on novel biocatalysis (nucleozymes, catalytic antibodies, synzymes etc.) and biomimetic chemical catalysts which could help in understanding the functioning of biological catalysts.     

Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.     

Combinatorial biocatalysis: taking the lead from nature

Combinatorial biocatalysis is an emerging technology in the field of drug discovery. The biocatalytic approach to combinatorial chemistry uses enzymatic, chemoenzymatic, and microbial transformations to generate libraries from lead compounds. Important recent advances in combinatorial biocatalysis include iterative derivatization of small molecules and complex natural products, regioselectively controlled libraries, novel one-pot library syntheses, process automation, and biocatalyst enhancements.     

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature’s biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.