Biocatalysis--key to sustainable industrial chemistry

The ongoing trends to process improvements, cost reductions and increasing quality, safety, health and environment requirements of industrial chemical transformations have strengthened the translation of global biocatalysis research work into industrial applications. One focus has been on biocatalytic single-step reactions with one or two substrates, the identification of bottlenecks and molecular as well as engineering approaches to overcome these bottlenecks. Robust industrial procedures have been established along classes of biocatalytic single-step reactions. Multi-step reactions and multi-component reactions (MCRs) enable a bottom-up approach with biocatalytic reactions working together in one compartment and recations hindering each other within different compartments or steps. The understanding of the catalytic functions of known and new enzymes is key for the development of new sustainable chemical transformations.     

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.     

Production of low-lactose milk by means of nonisothermal bioreactors

The effect of the immobilization time on the activity of immobilized beta-galactosidase from K. lactis was investigated. Six biocatalytic membranes, different only for the time of the enzyme immobilization, were obtained by using nylon membranes grafted with glycidyl methacrylate (GMA) and activated by hexamethylenediamine (HMDA) and glutaraldehyde (Glu), used as spacer and coupling agent, respectively. Comparison between the isothermal and nonisothermal yield of these biocatalytic membranes was carried out in the process of lactose hydrolysis in milk. All of the results, reported as a function of the immobilization time, have evidenced the influence of our variable parameter on the activity of the catalytic membranes. The membrane giving highest yield under isothermal and nonisothermal conditions was that obtained with 2 h of immobilization time. The industrial application of these membranes has been discussed in terms of percentage reduction of the production times.     

Enhanced resonance light scattering based on biocatalytic growth of gold nanoparticles for biosensors design

The biocatalytic growth of gold nanoparticles (Au-NPs) has been employed in the design of new optical biosensors based on the enhanced resonance light scattering (RLS) signals. Both absorption spectroscopy and transmission electron microscopy (TEM) analysis revealed Au-NP seeds could be effectively enlarged upon the reaction with H(2)O(2), an important metabolite that could be generated by many biocatalytic reactions. Upon the stepwise enlargement of Au-NPs, the light scattering intensity could be greatly enhanced, which then allowed the quantitative detection of the analyte, H(2)O(2). Further combination of the biocatalytic reaction that can yield H(2)O(2) by using the enzyme, glucose oxidase, with the enlargement of Au-NPs enabled the design of a sensitive glucose biosensor using the RLS technique. In the present study, we could achieve the detection of glucose in a linear range of 1.0 x 10(-6) M to 1.1 x 10(-4) M, with detection limit of 6.8 x 10(-7) M.     

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.     

Biocatalytic membranes for ultrafiltration treatment of wastewater containing dyes

A possibility to prepare the biofunctional membranes showing the biocatalytic properties and use those in post-treatment of wastewater containing synthetic dyes have been established. Selected Pseudomonas mendocina and Bacillus subtilis cultures were used as biocatalysts for dye destruction. It has been established that cells in spore form are able to survive in N-methylpyrrolidone that allow to use method of polymer solution casting for membrane preparation. The optimal conditions for entrapping of whole cells of microorganisms into the polymer matrix have been determined. Membrane biocatalytic activity has been studied depending on method of casting solution preparation, biocatalyst loading and operating parameters. Dye destruction occurs both in membrane pores and on membrane surface. Membrane obtained provide discolouring of treated solutions (permeate). The dye concentration in retentate depends on the trans-membrane fluxes. The concentration in retentate need not be observed at relatively low fluxes (up to 20 l/m² h).     

Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.     

Engineering biocatalytic systems in organic media with low water content

The use of organic media in biocatalysis stems from the fact that in many cases biocatalytic processes can hardly be conducted (if at all) in aqueous solutions because of extremely low solubilities of substrates and/or unfavorable shift of the reaction equilibrium in water. The growing interest in this biotechnological area that has sprung up over the past few years has resulted in various approaches to enzyme stabilization against organic solvents. Thus, the main goal of the present review is to formulate a comprehensive classification of numerous successful nonaqueous biocatalytic systems based on a few fundamental principles. Typical examples are considered, along with the advantages and drawbacks inherent in each of the approaches discussed.     

Investigation of concentration profiles inside operating biocatalytic sensors with scanning electrochemical microscopy (SECM)

Scanning electrochemical microscopy (SECM) with amperometric or potentiometric measuring tips was used to investigate biocatalytic reactions inside the enzyme layer of a biosensor during its operation. The well known glucose oxidase catalyzed oxidation of glucose has been selected for the studies. Local, instantaneous concentration of dissolved oxygen and hydrogen peroxide was studied observing the amperometric current while miniaturized potentiometric tip served for local pH measurements. Liquid enzyme layer immobilized with Cellophane membrane or cross linked polyacrilamide gel membrane containing entrapped enzyme served for biocatalytic media in the SECM imaging. Local maximum of H(2)O(2) and minimum of O(2) profiles were found at approximately 200 microm far from the substrate/enzyme layer boundary. From the experimental findings guidelines to design well functioning biocatalytic sensors could be concluded. The concentration profiles obtained with SECM techniques were compared with the results of simple model calculations carried out with the method of finite changes. Most of earlier made SECM studies dealing with enzyme reactions imaged the electrolyte being in contact with the immobilized enzyme. The data in our investigation, however, were collected inside the working catalytic layer.     

New applications of biocatalysts.

The development of new biocatalytic applications continues to advance in several directions. Over the past year, new enzymes have been discovered and their potential in biocatalyst applications has been researched. In addition, new chemical and genetic modifications have been made in the development of novel fermentation processes.     

Substrate range and enantioselectivity of epoxidation reactions mediated by the ethene-oxidising Mycobacterium strain NBB4

Mycobacterium strain NBB4 is an ethene-oxidising micro-organism isolated from estuarine sediments. In pursuit of new systems for biocatalytic epoxidation, we report the capacity of strain NBB4 to convert a diverse range of alkene substrates to epoxides. A colorimetric assay based on 4-(4-nitrobenzyl)pyridine) has been developed to allow the rapid characterisation and quantification of biocatalytic epoxide synthesis. Using this assay, we have demonstrated that ethene-grown NBB4 cells epoxidise a wide range of alkenes, including terminal (propene, 1-butene, 1-hexene, 1-octene and 1-decene), cyclic (cyclopentene, cyclohexene), aromatic (styrene, indene) and functionalised substrates (allyl alcohol, dihydropyran and isoprene). Apparent specific activities have been determined and range from 2.5 to 12.0 nmol min^?1 per milligram of cell protein. The enantioselectivity of epoxidation by Mycobacterium strain NBB4 has been established using styrene as a test substrate; (R)-styrene oxide is produced in enantiomeric excesses greater than 95%. Thus, the ethene monooxygenase of Mycobacterium NBB4 has a broad substrate range and promising enantioselectivity, confirming its potential as a biocatalyst for alkene epoxidation.     

Biocatalytic conversion of lignocellulose to platform chemicals

Naturally occurring lignocellulose can be used as a renewable resource for the sustainable production of platform chemicals that can in turn be converted to valuable fine chemicals, polymers, and fuels. The biocatalytic conversion of lignocellulose is a very promising approach due to its high selectivity, mild conditions, and low exergy loss. However, such biocatalytic processes are still seldom applied at the industrial scale since the single conversion steps (pretreatment, hydrolysis, and fermentation) may exhibit low conversion rates, low efficiencies, or high costs. The biocatalytic conversion of lignocellulose to platform chemicals is reviewed in this work. Structures and production rates of lignocellulose are described, and platform chemicals that may be produced from lignocellulose are summarized. Biocatalytic conversion of lignocellulose is distinguished from conventional non-selective approaches. All essential conversion steps used in biocatalytic approaches (pretreatment, hydrolysis, and fermentation) are reviewed in detail. Finally, potential interactions between these conversion steps are highlighted and the advantages as well as disadvantages of integrated process configurations are elucidated. In conclusion, a comprehensive understanding of the biocatalytic conversion of lignocellulose is provided in this review.     

Asymmetric synthesis of arylselenoalcohols by means of the reduction of organoseleno acetophenones by whole fungal cells

A series of novel organoseleno acetophenones (3a–f) have been synthesized. The microbial reduction of the seleno ketones (3) has been evaluated using whole cells of Rhizopus oryzae CCT 4964, Aspergillus terreus CCT 3320, A. terreus CCT 4083 and Emericella nidulans CCT 3119. These microorganisms showed Prelog and anti-Prelog stereoselectivity, leading to the arylselenoalcohols in moderate to high enantiomeric excesses. The organoselenium compounds were compatible with the biocatalytic conditions employed.     

Enzymatic synthesis of beta-lactam antibiotics. Analytical review]

The paper presents an analytical review of the literature on enzymatic synthesis of semisynthetic beta-lactam antibiotics. The results of the studies on the thermodynamics and kinetics of beta-lactams synthesis are generalized and the approaches to increasing the efficiency of the biocatalytic processes based on both thermodynamically controlled synthesis (direct) and kinetically controlled synthesis (acyl transfer) are systematized. Characteristic features of the processes for separation of the reaction mass components and recovery of the final products of the biocatalytic synthesis of beta-lactam antibiotics are considered and the pathways to increasing the economic efficiency of biocatalytic processes used in design of the technologies and their introduction to manufacture are discussed.     

Tools and ingredients for the biocatalytic synthesis of carbohydrates and glycoconjugates

The presence of multiple functional groups and stereocentres in carbohydrates and glycoconjugates make them challenging targets for synthesis. Although progress in chemical synthesis and engineering is impressive, there is still a need to selectively introduce and remove protecting groups in the total synthesis of target molecules of increasing complexity. Multiple hydroxyl-groups with similar reactivities have to be differentiated in order to form the desired glycosidic bonds in a regio- and stereospecific way. To complement the existing chemical tools and ingredients, biocatalysts for selective carbon–carbon bond formation and glycosylation reactions have been developed. The availability of auxiliary ingredients like transfer reagents is a prerequisite for the development of viable biocatalytic process steps. In the case of dihydroxyacetone-phosphate-dependent aldolases, e.g. fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), the large-scale availability of dihydroxyacetone-phosphate (DHAP) eliminates the need to synthesize the donor DHAP. For the pyruvate-dependent aldolases, e.g. the N-acetylneuraminic acid aldolase (EC 4.1.3.3) and acetaldehyde-dependent aldolases like the 2-deoxy-d-ribose-5-phosphate aldolase (4.2.1.4), the donors pyruvate and acetaldehyde are also available on a large scale. A broad range of natural and recombinant aldolases have been produced in stable lyophilized form. Recombinant transketolase together with a new synthesis of hydroxypyruvates has provided a platform technology for the preparation of monosaccharides, whereby the carbon backbone is extended by a two-carbon unit (C2-elongation). Natural and recombinant glycosyltransferases have been prepared on a large-scale to establish biocatalytic glycosylations in water as highly regio- and stereospecific reaction methodologies without the need for laborious protecting group manipulations, solubility adaptations and complex synthetic schemes. In order to simplify the synthetic manipulations for specific glycosylations, toolkits for β-1,4-galactosylations, α-1,3-galactosylations and α-1,3-fucosylations have been developed for rapid quantitative conversions. The introduction of matched pairs of biocatalysts and transfer reagents as ingredients together with the optimized reaction methodology as tool provide an important starting point for biocatalytic glycomics.     

Engineered biosynthetic pathways and biocatalytic cascades for sustainable synthesis

Nature exploits biosynthetic cascades to construct numerous molecules from a limited set of starting materials. A deeper understanding of biosynthesis and extraordinary developments in gene technology has allowed the manipulation of natural pathways and construction of artificial cascades for the preparation of a range of molecules, which would be challenging to access using traditional synthetic chemical approaches. Alongside these metabolic engineering strategies, there has been continued interest in developing in vivo and in vitro biocatalytic cascades. Advancements in both metabolic engineering and biocatalysis are complementary, and this article aims to highlight some of the most exciting developments in these two areas with a particular focus on exploring those that have the potential to advance both pathway engineering and more traditional biocatalytic cascade development.     

Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer-Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.     

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.     

Rational improvement of simvastatin synthase solubility in Escherichia coli leads to higher whole-cell biocatalytic activity

Simvastatin is the active pharmaceutical ingredient of the blockbuster cholesterol lowering drug Zocor. We have previously developed an Escherichia coli based whole-cell biocatalytic platform towards the synthesis of simvastatin sodium salt (SS) starting from the precursor monacolin J sodium salt (MJSS). The centerpiece of the biocatalytic approach is the simvastatin synthase LovD, which is highly prone to misfolding and aggregation when overexpressed from E. coli. Increasing the solubility of LovD without decreasing its catalytic activity can therefore elevate the performance of the whole-cell biocatalyst. Using a combination of homology structural prediction and site-directed mutagenesis, we identified two cysteine residues in LovD that are responsible for nonspecific intermolecular crosslinking, which leads to oligomer formation and protein aggregation. Replacement of Cys40 and Cys60 with alanine residues resulted in marked gain in both protein solubility and whole-cell biocatalytic activities. Further mutagenesis experiments converting these two residues to small or polar natural amino acids showed that C40A and C60N are the most beneficial, affording 27% and 26% increase in whole cell activities, respectively. The double mutant C40A/C60N combines the individual improvements and displayed approximately 50% increase in protein solubility and whole-cell activity. Optimized fed-batch high-cell-density fermentation of the double mutant in an E. coli strain engineered for simvastatin production quantitatively (>99%) converted 45 mM MJSS to SS within 18 h, which represents a significant improvement over the performance of wild-type LovD under identical conditions. The high efficiency of the improved whole-cell platform renders the biocatalytic synthesis of SS an attractive substitute over the existing semisynthetic routes.