Modular and scalable biocatalytic tools for practical safety, health and environmental improvements in the production of speciality chemicals

Biocatalytic tools for both end-of-the-pipe solutions and direct reaction methodology have been developed for the improvement of practical oxidations. The identification of bottlenecks and limitations in biocatalytic Baeyer-Villiger oxidations, and the comparison of scalable process designs to overcome these limitations, have shown the direction for improvements. The first kilogram-scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity has been realized by the combination of a resin-based in-situ SFPR strategy together with micro-bubble aeration. Regioselective asymmetric dihydroxylation of aromatic nitriles has been achieved by recombinant chlorobenzenedioxygenase. The introduction of novel biocatalytic tools for key catalytic asymmetric transformations will change chemical manufacturing in the 21st century.     

Modular and scalable biocatalytic tools for practical safety,health and environmental improvements in the production of speciality chemicals

Biocatalytic tools for both end-of-the-pipe solutions and direct reaction methodology have been developed for the improvement of practical oxidations. The identification of bottlenecks and limitations in biocatalytic Baeyer-Villiger oxidations, and the comparison of scalable process designs to overcome these limitations, have shown the direction for improvements. The first kilogram-scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity has been realized by the combination of a resin-based in-situ SFPR strategy together with micro-bubble aeration. Regioselective asymmetric dihydroxylation of aromatic nitriles has been achieved by recombinant chlorobenzenedioxygenase. The introduction of novel biocatalytic tools for key catalytic asymmetric transformations will change chemical manufacturing in the 21st century.     

Perspectives and industrial potential of PGA selectivity and promiscuity

Penicillin G acylases (PGAs) are robust industrial catalysts used for biotransformation of β-lactams into key intermediates for chemical production of semi-synthetic β-lactam antibiotics by hydrolysis of natural penicillins. They are used also in reverse, kinetically controlled synthetic reactions for large-scale productions of these antibiotics from corresponding beta-lactam nuclei and activated acyl donors. Further biocatalytic applications of PGAs have recently been described: catalysis of peptide syntheses and the resolutions of racemic mixtures for the production of enantiopure active pharmaceutical ingredients that are based on enantioselective acylation or chiral hydrolysis. Moreover, PGAs rank among promiscuous enzymes because they also catalyze reactions such as trans-esterification, Markovnikov addition or Henry reaction. This particular biocatalytic versatility represents a driving force for the discovery of novel members of this enzyme family and further research into the catalytic potential of PGAs. This review deals with biocatalytic applications exploiting enantioselectivity and promiscuity of prokaryotic PGAs that have been recently reported. Biocatalytic applications are discussed and presented with reaction substrates converted into active compounds useful for the pharmaceutical industry.     

Assessment of intraparticle biocatalytic distributions as a tool in rational formulation

Research has shown that the intraparticle biocatalytic distribution has extensive effects on the properties of various (industrial) biocatalytic particles and their performance in (bio-) chemical reactions. In recent years, advances in molecular chemistry have led to the development of many different specific (immuno-) labeling and light-microscopic detection techniques. Furthermore, high-quality image-digitizing devices and enhanced computing power have made image analysis readily accessible. These technologies may lead to the assessment and improvement of the internal biocatalyst profile as an integral part of biocatalytic particle optimization.     

Enzyme catalytic promiscuity: The papain-catalyzed Knoevenagel reaction

Papain as a sustainable and inexpensive biocatalyst was used for the first time to catalyze the Knoevenagel reactions in DMSO/water. A wide range of aromatic, hetero-aromatic and α,β-unsaturated aldehydes could react with less active methylene compounds acetylacetone and ethyl acetoacetate. The products were obtained in moderate to excellent yields with Z/E selectivities of up to 100:0. This case of biocatalytic promiscuity not only widens the application of papain to new chemical transformations, but also could be developed into a potentially valuable method for organic synthesis.     

Engineering novel biocatalytic routes for production of semisynthetic opiate drugs

The morphine alkaloids and their semisynthetic derivatives provide a diverse range of important pharmaceutical drugs. Current production of semisynthetic opiate drugs is by chemical means from naturally occurring morphine, codeine and thebaine. Although various microbial transformations of morphine alkaloids have been identified since the 1960s, more recently there has been considerable effort devoted to engineering biocatalytic routes for producing these important compounds. Such biocatalytic routes are attractive, as they would provide an alternative to the chemical production processes which suffer from limited supply of precursors, often low yields and toxic wastes. The biotransformation of morphine and codeine to the potent analgesic hydromorphone and the mild analgesic/antitussive hydrocodone, respectively, by recombinant Escherichia coli has been demonstrated and the problems encountered when engineering such a system will be discussed.     

Tools for Selective Enzyme Reaction Steps in the Synthesis of Laboratory Chemicals

The need for more selective reactions steps and the compatibility between process steps which follow on from each other has been a major driving force for organic synthesis. The synthesis of chiral compounds, metabolites, new chemical entities and natural products by a combination of chemical and enzyme reaction steps has become well established, due the existence of stable enzymes as selective catalysts which are inherently chiral by nature. Auxiliary tools such as suitable transfer reagents for reaching complete conversion, easy and robust reaction control as well as tools for straightforward workup and purification of the final product have been developed. Selective enzyme reaction steps in the area of hydrolyses, oxidation steps including hydroxylation and the Baeyer‐Villiger oxidation, carbon‐carbon bond formation and glycosylation reactions have compared favorably with existing methods of classical organic synthesis. The tools developed during optimization and scale‐up of these enzyme reaction steps have the potential to shorten development time. The introduction of selective enzyme reactions into an entire synthetic process has resulted in harmonization of improvements in economic efficiency with resultant solutions to health, safety and environment problems. This will become even more important in industrial synthetic chemistry in the future, for convenient solutions to certain intractable synthetic problems and for expanding the repertoire of chemistry by modular biocatalysts. Efficient isolation procedures for the final product are essential to take full advantage of the biocatalytic conversion to obtain high product yields.     

人参皂苷单体定向转化的生物催化及应用进展

人参是我国传统中药,药效显著、应用广泛。通过定向修饰与转化人参皂苷糖基可产生高抗癌活性稀有人参皂苷。传统化学法由于制备工艺极其复杂、成本过高,不能应用于临床,微生物及其酶系转化成为解决该瓶颈问题的最可行手段。有关全细胞催化、糖苷酶重组表达、固定化及其催化分子识别机制和溶剂工程的生物转化已有大量综述报道,但尚无在人参皂苷转化应用中的系统研究。文中通过对人参皂苷单体生物转化理论和应用研究最新进展的回顾,结合目前广泛采用的生物催化方法的讨论,系统梳理归纳了能够改善产物专一性、提高催化效率,且具有工业应用前景的人参皂苷单体定向转化方法。基于酶分子设计以及离子液体溶剂工程,对人参皂苷单体抗癌药物和食品、保健品市场的开发、规模化制备进行了展望。     

Principles of antibody catalysis

Antibodies have now been shown to catalyze a variety of chemical transformations, including hydrolytic, concerted, and bimolecular reactions. The inherent chirality of the antibody binding pocket has been exploited to exert precise stereochemical control over their catalyzed reactions. The mechanisms by which antibodies catalyze reactions are not expected to differ in any general way from those of natural enzymes. Antibodies use their binding energy to stabilize species of higher free energy which appear along the reaction coordinate or effect general acid/base catalysis. The advent of catalytic antibodies promises new catalysts that extend the range of catalysis by proteins to chemical transformations that were not required during the evolution of enzymes.     

High-throughput screening of biocatalytic activity: applications in drug discovery

Enzymes catalyze a diverse set of reactions that propel life's processes and hence serve as valuable therapeutic targets. High-throughput screening methods have become essential for sifting through large chemical libraries in search of drug candidates, and several sensitive and reliable analytical techniques have been specifically adapted to high-throughput measurements of biocatalytic activity. High-throughput biocatalytic assay platforms thus enable rapid screening against enzymatic targets, and have vast potential to impact various stages of the drug discovery process, including lead identification and optimization, and ADME/Tox assessment. These advances are paving the way for the adoption of high-throughput biocatalytic assays as an indispensable tool for the pharmaceutical industry.     

Tools and ingredients for the biocatalytic synthesis of metabolites

Metabolic networks have been an interesting starting point not only for the design of synthetic routes in a similar sequence of reactions, e.g., in biomimetic syntheses, but also for assembling a number of biocatalytic steps by preparing the required enzymes and auxiliary reagents. Retrosynthetic analysis involving multiple biocatalytic reactions steps therefore needs to consider the practically realized biocatalytic single steps. The opportunities for route selection are enlarged if novel synthetic reactions connecting easily available starting materials and products are found, and/or both biocatalytic and classical reactions of organic chemistry are utilized. Tools and ingredients for biocatalytic synthesis are of special interest for reactions difficult to achieve by classical organic synthesis. Densely and differentially functionalized small molecules do not allow much space for protecting or activating groups. Biocatalytic reactions have therefore performed well for a number of useful metabolites in enantiopure form to achieve full functionality. Although many well-known metabolites from classical biochemistry have only been prepared in racemic form, it is of fundamental interest to have these available in enantiomerically pure form. Biocatalytic reactions with nature's privileged chiral catalysts appear to be a promising synthetic strategy towards these metabolites, especially when sensitive or stable-isotope-labeled metabolites are to be prepared. The main applications for these metabolites are as references materials in metabolomics, as enzyme substrates for the characterization of metabolic enzyme activities and as potential pharmaceuticals in biomedical research. The use of stable-isotope-labeled metabolites can thereby simplify in vivo applications and metabolic flux analyses.     

Biocatalysis as an alternative for the production of chiral epoxides: A comparative review

Enantiopure epoxides are remarkably versatile intermediates for the synthesis of numerous biologically active targets, to which considerable efforts have been devoted either chemically or biologically during the past few decades. This review will emphasize the application of biocatalysis as an efficient alternative that complements conventional chemical reactions, with a special focus on the epoxidation reactions catalyzed with monooxygenases and chloroperoxidases and the hydrolytic kinetic resolution catalyzed with epoxide hydrolases. Their scopes and limitations will be elaborately discussed as compared with their chemical counterparts. These biocatalytic approaches have not only provided environmentally friendly alternatives, but also displayed advantages for certain types of enantiopure epoxides, and could serve as potential tools for synthetic chemists.     

Applications of oxidoreductases.

Oxidoreductases comprise the large class of enzymes that catalyze biological oxidation/reduction reactions. Because many chemical and biochemical transformations involve oxidation/reduction processes, developing practical biocatalytic applications of oxidoreductases has long been an important goal in biotechnology. During the past year, significant progress has been made in the development of oxidoreductase-based diagnostic tests and improved biosensors, in the design of innovative systems for regeneration of essential coenzymes, in the construction bioreactors for biodegradation of pollutants and for biomass processing, and in the development of oxidoreductase-based approaches for synthesis of polymers and oxyfunctionalized organic substrates.     

Chemical biotechnology for the specific oxyfunctionalization of hydrocarbons on a technical scale

Oxygenases catalyze, among other interesting reactions, highly selective hydrocarbon oxyfunctionalizations, which are important in industrial organic synthesis but difficult to achieve by chemical means. Many enzymatic oxygenations have been described, but few of these have been scaled up to industrial scales, due to the complexity of oxygenase based biocatalysts and demanding process implementation. We have combined recombinant whole-cell catalysis in a two-liquid phase system with fed-batch cultivation in an optimized medium and developed an industrially feasible process for the kinetically controlled and complex multistep oxidation of pseudocumene to 3,4-dimethylbenzaldehyde using the xylene monooxygenase of Pseudomonas putida mt-2 in Escherichia coli. Successful scale up to 30 L working volume using downscaled industrial equipment allowed a productivity of 31 g L(-1) d(-1) and a product concentration of 37 g L(-1). These performance characteristics meet present industry requirements. Product purification resulted in the recovery of 469 g of 3,4-dimethyl- benzaldehyde at a purity of 97% and an overall yield of 65%. This process illustrates the general feasibility of industrial biocatalytic oxyfunctionalization.     

Review: Biocatalytic transformations of ferulic acid: An abundant aromatic natural product

In this review we examine the fascinating array of microbial and enzymatic transformations of ferulic acid. Ferulic acid is an extremely abundant, preformed phenolic aromatic chemical found widely in nature. Ferulic acid is viewed as a commodity scale, renewable chemical feedstock for biocatalytic conversion to other useful aromatic chemicals. Most attention is focused on bioconversions of ferulic acid itself. Topics covered include cinnamoyl side-chain cleavage; nonoxidative decarboxylation; mechanistic details of styrene formation; purification and characterization of ferulic acid decarboxylase; conversion of ferulic acid to vanillin;O-demethylation; and reduction reactions. Biotransformations of vinylgualacol are discussed, and selected biotransformations of vanillic acid including oxidative and nonoxidative decarboxylation are surveyed. Finally, enzymatic oxidative dimerization and polymerization reactions are reviewed.     

Microbial P450 enzymes in biotechnology

Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes since they allow the introduction of oxygen into non-activated carbon atoms in a sterically and optically selective manner that is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. Surprisingly, cytochrome P450 monooxygenases have scarcely been studied for use in biooxidations, although they are one of the largest known superfamilies of enzyme proteins. Their gene sequences have been identified in various organisms such as humans, bacteria, algae, fungi, and plants. The reactions catalyzed by P450s are quite diverse and range from biosynthetic pathways (e.g. those of animal hormones and secondary plant metabolites) to the activation or biodegradation of hydrophobic xenobiotic compounds (e.g. those of various drugs in the liver of higher animals). From a practical point of view, the great potential of P450s is limited by their functional complexity, low activity, and limited stability. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways that could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes, and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are both covered in this review.     

Development of biobased products

Research conducted over the past seven years by the biotechnology byproducts consortium (BBC) addresses its mission to investigate the opportunities to add value to agricultural products, byproducts and coproducts and to manage the wastewater arising from agribusinesses in an environmentally favorable way. Since a wide variety of research approaches have been taken, the results are collected in five topic groups: (1) bioremediation that includes anaerobic fermentations of wastes to produce methane and hydrogen, the genetics of methanogenesis and in situ remediation of contaminated aquifer systems, landfill leachates and industrial effluents; (2) land application of fermentation byproducts and their use in animal feeds; (3) biocatalytic studies of transformations of components of corn and soybean oils, peroxidases present in plant products, such as soybean hulls; (4) biochemical reactions for the production of de-icers from industrial water streams, biodiesel production from fats and greases, biodegradable plastics from polymerizable sugar derivatives, single cell foods derived from fungal growth on waste streams, and bacterial polysaccharides from Erwinia species; (5) separation and recovery of components by membrane technologies.     

Promiscuous protease-catalyzed aldol reactions: a facile biocatalytic protocol for carbon-carbon bond formation in aqueous media

Several proteases, especially pepsin, were observed to directly catalyze asymmetric aldol reactions. Pepsin, which displays well-documented proteolytic activity under acidic conditions, exhibited distinct catalytic activity in a crossed aldol reaction between acetone and 4-nitrobenzaldehyde with high yield and moderate enantioselectivity. Fluorescence experiments indicated that under neutral pH conditions, pepsin maintains its native conformation and that the natural structure plays an important role in biocatalytic promiscuity. Moreover, no significant loss of enantioselectivity was found even after four cycles of catalyst recycling, showing the high stability of pepsin under the selected aqueous reaction conditions. This case of biocatalytic promiscuity not only expands the application of proteases to new chemical transformations, but also could be developed into a potentially valuable method for green organic synthesis.     

Environmental metagenomics: An innovative resource for industrial biocatalysis

The current existing enzymes have been identified from cultivable micro-organisms, most frequently from bacteria. These bacterial biocatalytic capabilities have been widely used for biotransformations, resulting in the development of profitable industrial bioprocesses in the fields of feed and food processing, textiles, agro-chemistry, cosmetics, pharmaceuticals and fine chemistry. However, the originality of this bioresource is progressively drying up, while requests from industry for novel biocatalytic activities are increasing in the face of economic and environmental pressure. Metagenomics, through access to the huge reservoir of uncultivated bacteria which represents the majority of the present biodiversity, opens the door to new industrial sources of enzymes. Surmounting hurdles encountered with this technology (e.g. DNA extraction to obtain high quality DNA libraries with proper statistical representativity, setting up of relevant high throughput screenings assays, combining functional and genome-based identifications), gives unique opportunities to access novel biocatalysts that better fit with the required industrial specifications, thus providing new biocatalysis tool boxes.     

Environmental metagenomics: An innovative resource for industrial biocatalysis

The current existing enzymes have been identified from cultivable micro-organisms, most frequently from bacteria. These bacterial biocatalytic capabilities have been widely used for biotransformations, resulting in the development of profitable industrial bioprocesses in the fields of feed and food processing, textiles, agro-chemistry, cosmetics, pharmaceuticals and fine chemistry. However, the originality of this bioresource is progressively drying up, while requests from industry for novel biocatalytic activities are increasing in the face of economic and environmental pressure. Metagenomics, through access to the huge reservoir of uncultivated bacteria which represents the majority of the present biodiversity, opens the door to new industrial sources of enzymes. Surmounting hurdles encountered with this technology (e.g. DNA extraction to obtain high quality DNA libraries with proper statistical representativity, setting up of relevant high throughput screenings assays, combining functional and genome-based identifications), gives unique opportunities to access novel biocatalysts that better fit with the required industrial specifications, thus providing new biocatalysis tool boxes.