Nucleic‐Acid‐Templated Enzyme Cascades

Cellular metabolism involves complex sequences of organized enzymatic reactions, known as metabolic pathways, that convert substrates into readily usable materials. In nature, these enzymatic complexes are organized in a well‐defined manner so that the cascade reactions are more rapid and efficient than they would be if the enzymes were randomly distributed in the cytosol. Development of artificial enzyme cascades that resemble nature's organization of sequentially assembled enzymes is of current interest due to its potential applications, from diagnostics to the production of high‐value chemicals. Nucleic acids and their nanostructures have been used to organize enzyme cascades and have been shown to enhance the efficiencies and rates of sequential reactions. Here we summarize the recent progress in the development of artificial enzyme cascades and sequential reactions by arranging enzymes on various DNA/RNA templates and discuss the future directions of this research endeavour.     

Photochemical,Electrochemical, and Hydrogen-Driven Enzymatic Reductions in Reversed Micelles

A few examples of (multi)enzymatic reactions in reversed micellar media are described and discussed. The key enzymes in these examples are 20β-hydroxy-steroid dehydrogenase, enoate reductase, and an NAD⁺-independent hydrogenase, catalyzing the reactions of interest, viz., the site- and stereo-specific reduction of 20-ketosteroids to their corresponding 20β-hydroxy form, the chiral hydrogenation of α,β-unsaturated carboxylates, and the production of hydrogen, respectively. These cofactor-requiring enzymes are coupled to various in situ cofactor-regenerating systems, powered either by light, electricity, or hydrogen. The performance of all these systems is shown.     

乳液膜技术及其在酶化工的应用

本文评述了乳液膜技术在酶反应中的应用情况，着重从增强液膜稳定性，改进传质效果以及减少酶活损失３个方面进行了分析，并对其应用前景作了讨论。     

提高微藻培养过程的光能转化率：工程途径及研究进展

分析了光能转化率的研究现状及影响光能转化率的工程因素,综述了提高光能转化率的工程途径及研究进展. 从工程角度提高光能转化率的核心是改善光在反应器中的时空分布及反应器内的流体力学特征,具体方法包括优化入射光能量、根据培养阶段调整光源条件、人工导入光源、优化反应器几何形式和结构、流动内构件的设计和应用、通气量和通气区域优化等. 进一步提高光能转化率,需结合光的时空分布和流体流动,将工程和工艺因素相结合,开发反应器和优化工艺条件.     

The response of linear cascades to variations of holding times

An arbitrary system of isothermal first order reactions with no volume change on reaction is considered. A comparison is made between the response of the system in a cascade of ideal continuous stirred tank reactors and an ideal tubular reactor to variations of holding times.The performance criterion chosen is a linear combination of the exit concentrations and, contrary to what has been published previously, examples are given to show that for an m tank cascade non-equal tank cascades can be locally stationary and even localy extreme. In spite of this (except for certain trivial and degenerate cases) it can be shown that the global extrema occur for equal tank cascades. Furthermore, these extrema are dominated by those for an m + 1 tank cascade and these are all dominated by those of the turbular reactor.Cases where the performance criterion is augmented by a cost associated with the total holding time or where the total holding time is constrained (equality or inequality) are also discussed. In the first case the above results are essentially unchanged, while in the constrained cases examples are given to show that non-equal tank cascades can be optimal.     

Copolymer reactor operation for uniform product composition — analysis of continuous stirred-tank reactors

This paper deals with the basic mass balance calculations for the analysis and operation of steady state continuous stirred-tank reactor (CSTR) cascades used in copolymer production. After a brief introduction on copolymer theory, a Skeist-type graphical analysis is presented where the relations between copolymer composition, feed composition and reactor conversion are studied. To do this, an equilibrium-stage approach is used, much in the same manner as in the study of separation processes. The graphical procedure is then extended to CSTR cascades. Two cases are considered. In the first, copolymer compositions occurring in each tank as well as the final copolymer composition distribution at the end of the cascade are calculated. In the second, an original method is developed to calculate the interstage feeds intended to compensate tank-to-tank drift in composition. After discussing the applicability and limitations in practical cases, two examples are given for copolymer emulsions.     

Enzymatic transition state poise and transition state analogues

The development of kinetic isotope effect methods for enzymatic reactions has resulted in the systematic determination of enzymatic transition state structure for several distinct chemical reaction mechanisms. Although it is early in the experimental development of the method, examples of concerted nucleophilic displacements (A(N)D(N) or S(N)2), aromatic nucleophilic displacements (A(N)D(N) or S(N)Ar), and both concerted and stepwise dissociative nucleophilic displacements (D(N)A(N) and D(N)A(N); S(N)1 reactions) have been exemplified. The transition state for each reaction exhibits a characteristic extent of bond-breaking and bond-making, defined here as transition state poise. Thus, concerted nucleophilic displacements (S(N)2 or D(N)A(N)) exhibit various extents of residual bond order to the leaving group and to the attacking nucleophile at the transition state. Aromatic nucleophilic displacements reach their rate-limiting transition states before or after formation of the tetrahedral intermediate. Several concerted, symmetric nucleophilic displacements at carbon have been described. Enzymatic transition state poise is summarized in a single diagram of bond orders using the terminology of Jencks. The analysis reveals enzymatic contributions to transition state poise, provides precedent for assignment of reaction types, and summarizes the current status of the experimental characterization of enzymatic transition states. Binding strengths of transition state analogues are readily correlated with transition state poise.     

Oxidation of PCE with a UV LED Photocatalytic Reactor

This paper is the first to investigate photocatalysis using a cutting‐edge and energy‐efficient solid‐state light source: Ultraviolet (UV) Light Emitting Diodes (LED's). UV LED's do not involve mercury vapor, can be driven with direct current (DC), and have a long lifetime of 100 000 hours. UV LED's with a peak wavelength of 375 nm were tested for perchloroethylene (PCE) photocatalytic oxidation over Degussa P–25 TiO₂. At a UV light output of only 49 μW/cm², the designed reactor delivers a PCE conversion of up to 43 %. If the UV LED price continues to drop, it is very likely that UV LED's will replace UV lamps as the favored light source in photocatalysis applications.     

Two-Stage Ozonation – Adsorption Purification of Ground Water from Trichloroethylene and Tetrachloroethylene with Application of Commercial Carbon Adsorbents

ABSTRACT

The aim of the research is to choose the most efficient adsorbent for two-stage ozone-sorption purification of groundwater containing both trichloroethylene (TCE) and tetrachloroethylene (PCE) between three carbon sorbents produced in Russia (AUT-M, CAUSORB-221, and AG-3). Sorption isotherms of TCE and PCE on AUT-M and CAUSORB-221 at 296 K were fitted by the Freundlich equation. The better TCE and PCE sorption ability of AUT-M in comparison with CAUSORB – 221 and AG-3 was demonstrated.

The optimum parameters for ozonation and sorption stages of groundwater purification from TCE and PCE are elucidated using laboratory and pilot-plant scales. Prolonged test of this technology for purification of ground demonstrated that the higher achievable efficiency of destruction with ozone is 94% for TCE and 38% for PCE. Ozonation-sorption treatment of groundwater allows one to achieve TCE and PCE removal efficiency of 96-97% and 92-94% correspondingly. The most efficient carbon sorbent is microporous carbon fiber AUT-M. Using this sorbent, TCE and PCE concentrations in treated water decrease below the MPC level (5 μg/L) adopted in Russia. It is concluded that the combination of ozonation with sorption of residual contaminants by carbon sorbents is a promising way for the purification of waters containing chlorinated contaminants.     

Chemo‐enzymatic synthesis of poly(2,2,2‐trichloroethyl 10‐hydroxydecanate)‐block‐polystyrene combining enzymatic self‐condensation polymerization and ATRP

Enzymatic polymerization in a non‐natural environment is of interest as an environmentally friendly methodology as an alternative to the use of conventional chemical organometallic catalysts. Chemo‐enzymatic synthesis of the AB‐type diblock copolymer poly(2,2,2‐trichloroethyl 10‐hydroxydecanate)‐block‐polystyrene (PHD‐b‐PSt) was carried out by combining enzymatic self‐condensation polymerization (eSCP) and atom‐transfer radical polymerization (ATRP). Biocatalyst Novozyme 435 was successful in catalyzing the eSCP of a novel ω‐hydroxyester, i.e. 2,2,2‐trichloroethyl 10‐hydroxydecanate. The resulting ? CCl₃‐terminated PHD initiated the ATRP of styrene, a ‘living’/controlled radical polymerization. The analysis of the hydrolysate from the copolymer proved the presence of a block copolymer structure. In addition, the well‐defined diblock copolymer PHD‐b‐PSt self‐assembled into nanoscale micelles in aqueous solution. The chemo‐enzymatic synthesis of diblock copolymer PHD‐b‐PSt was achieved by the combination of eSCP and ATRP. The structures and composition of the block copolymer were characterized by means of NMR, infrared and gel permeation chromatography measurements. Differential scanning calorimetry analysis showed that a microphase‐separation structure was formed in the copolymer, which was caused by the crystallization of the PHD segments. As investigated with atomic force microscopy and dynamic light scattering, these micelles had a mean diameter and a spherical shape. To our knowledge, this is the first example of a chemo‐enzymatic synthesis based on eSCP and ATRP. Copyright © 2007 Society of Chemical Industry     

Adhesively bonded connections in the context of timber engineering – A Review

If there is one domain of civil engineering in which adhesives are currently booming, then it is timber engineering. Natural adhesives have been used for centuries to structurally connect timber elements, a trend that culminated in the beginning of the 20th century with the introduction of glue-laminated beams. With the introduction of synthetic adhesives, and their increasing economic success after World War II, a wide range of products is now available that have the potential to free timber engineering from most of its structural and size limitations. This review article is intended to shed some light on the current state-of-the-art regarding adhesively bonded connections in the context of timber engineering. First, the relevant properties of timber as an adherend are discussed, then different – including several hybrid – approaches for structurally jointing timber are illustrated and finally, different design approaches are presented.     

Enzymatic Redox Cascade for One‐Pot Synthesis of Uridine 5′‐Diphosphate Xylose from Uridine 5′‐Diphosphate Glucose

Synthetic ways towards uridine 5′‐diphosphate (UDP)‐xylose are scarce and not well established, although this compound plays an important role in the glycobiology of various organisms and cell types. We show here how UDP‐glucose 6‐dehydrogenase (hUGDH) and UDP‐xylose synthase 1 (hUXS) from Homo sapiens can be used for the efficient production of pure UDP‐α‐xylose from UDP‐glucose. In a mimic of the natural biosynthetic route, UDP‐glucose is converted to UDP‐glucuronic acid by hUGDH, followed by subsequent formation of UDP‐xylose by hUXS. The nicotinamide adenine dinucleotide (NAD⁺) required in the hUGDH reaction is continuously regenerated in a three‐step chemo‐enzymatic cascade. In the first step, reduced NAD⁺ (NADH) is recycled by xylose reductase from Candida tenuis via reduction of 9,10‐phenanthrenequinone (PQ). Radical chemical re‐oxidation of this mediator in the second step reduces molecular oxygen to hydrogen peroxide (H₂O₂) that is cleaved by bovine liver catalase in the last step. A comprehensive analysis of the coupled chemo‐enzymatic reactions revealed pronounced inhibition of hUGDH by NADH and UDP‐xylose as well as an adequate oxygen supply for PQ re‐oxidation as major bottlenecks of effective performance of the overall multi‐step reaction system. Net oxidation of UDP‐glucose to UDP‐xylose by hydrogen peroxide (H₂O₂) could thus be achieved when using an in situ oxygen supply through periodic external feed of H₂O₂ during the reaction. Engineering of the interrelated reaction parameters finally enabled production of 19.5 mM (10.5 g L ⁻¹) UDP‐α‐xylose. After two‐step chromatographic purification the compound was obtained in high purity (>98%) and good overall yield (46%). The results provide a strong case for application of multi‐step redox cascades in the synthesis of nucleotide sugar products.

     

Enzymatic hydrogelation of small molecules

Enzymes, a class of highly efficient and specific catalysts in Nature, dictate a myriad of reactions that constitute various cascades in biological systems. Self-assembly, a process prevalent in Nature, also plays important roles in biology, from maintaining the integrity of cells to performing cellular functions and inducing abnormalities that cause disease. To explore enzyme-regulated molecular self-assembly in an aqueous medium will help to understand and control those important biological processes. On the other hand, certain small organic molecules self-assemble in water to form molecular nanofibers and result in a hydrogel, which is referred to as a "supramolecular hydrogel" (and the small molecules are referred to as "supramolecular hydrogelators"). Supramolecular hydrogelators share common features, such as amphiphilicity and supramolecular interactions (pi-pi interactions, hydrogen bonding, and charge interactions among the molecules, among others) that result in nanostructures and form the three-dimensional networks as the matrices of hydrogels. In this Account, we discuss the use of enzymes to trigger and control the self-assembly of small molecules for hydrogelation, which takes place in vitro or in vivo, extra- or intracellularly. Using phosphatase, thermolysin, beta-lactamase, and phosphatase/kinase as examples, we illustrate the design and application of enzyme-catalyzed or -regulated formation of supramolecular hydrogels that offer a new strategy for detecting the activity of enzymes, screening for enzyme inhibitors, typing bacteria, drug delivery systems, and controlling the fate of cells. Since the expression and distribution of enzymes differ by the types and states of cells, tissues, and organs, using an enzymatic reaction to convert precursors into hydrogelators that self-assemble into nanofibers as the matrices of the hydrogel, one can control the delivery, function, and response of a hydrogel according to a specific biological condition or environment, thus providing an accessible route to create sophisticated materials for biomedicine. Particularly, intracellular enzymatic hydrogelation of small molecules offers a unique means for scientists to integrate molecular self-assembly with inherent enzymatic reactions inside cells for developing new biomaterials and therapeutics at the supramolecular level and improving the basic understanding of dynamic molecular self-assembly in water.     

Adsorption of tetrachloroethylene (PCE) in gas phase on zeolites of faujasite type: Influence of water vapour and of Si/Al ratio

This study is focused on the adsorption of a chlorinated volatile organic compound, the tetrachloroethylene (PCE), on dealuminated faujasite type zeolites with framework Si/Al ratio between 5 and 100. PCE dynamic adsorption experiments with and without water vapour (relative humidity of, respectively, 50% and 0%) were carried out in a fixed bed reactor at 50 °C. Breakthrough curves were fitted by a model using the integral of a Gauss distribution. PCE adsorption capacities depend on the adsorbent microporous volume. However, in presence of water vapour, PCE adsorption is favoured on hydrophobic zeolites but also depends on the diffusional limitations inside the porous system. In order to have a better understanding of water molecules adsorption, isotherms were measured using thermogravimetric method at 25 °C. The presence of water vapour generally decreases PCE uptake but its influence decreases as the Si/Al ratio of the adsorbent increases. Experiments with various gases hourly space velocity (GHSV) and inlet PCE concentrations were also performed. PCE complete desorption was obtained on HFAU(Si/Al = 17) at 180 °C. This easy regeneration of the sample permitted adsorption/regeneration cycles maintaining good adsorption properties.     

Synthesis and Characterization of Pyridine Compounds for Amperometric Measurements of Leukocyte Esterase

We introduce a new class of substrates (compounds I – III ) for leukocyte esterase (LE) that react with LE yielding anodic current in direct proportion to LE activity. The kinetic constants K_m and k_cat for the enzymatic reactions were determined by amperometry at a glassy carbon electrode. The binding affinity of I – III for LE was two orders of magnitude better than that of existing optical LE substrates. The specificity constant k_cat/K_m was equal to 2.7, 3.8, and 5.8×10⁵ m ^?1 s^?1 for compounds containing the pyridine ( I ), methoxypyridine ( II ), and (methoxycarbonyl)pyridine ( III ), respectively, thus showing an increase in catalytic efficiency in this order. Compound III had the lowest octanol/water partition coefficient (log p=0.33) along with the highest topological surface area (tPSA=222 Ų) and the best aqueous solubility (4.0 mg mL^?1). The average enzymatic activity of LE released from a single leukocyte was equal to 4.5 nU when measured with compound III .     

Towards Copper(I) Clusters for Photo-Induced Oxidation of Biological Thiols in Living Cells**

The cell redox balance can be disrupted by the oxidation of biological peptides, eventually leading to cell death, which provides opportunities to develop cytotoxic drugs. With the aim of developing compounds capable of specifically inducing fatal redox reactions upon light irradiation, we have developed a library of copper compounds. This metal is abundant and considered essential for human health, making it particularly attractive for the development of new anticancer drugs. Copper(I) clusters with thiol ligands (including 5 novel ones) have been synthesized and characterized. Structures were elucidated by X-ray diffraction and showed that the compounds are oligomeric clusters. The clusters display high photooxidation capacity towards cysteine – an essential amino acid – upon light irradiation in the visible range (450 nm), while remaining completely inactive in the dark. This photoredox activity against a biological thiol is very encouraging for the development of anticancer photoredox drugs.The in vitro assay on murine colorectal cancer cells (CT26) did not show any toxicity – whether in the dark or when exposed to 450 nm light, likely because of the poor solubility of the complexes in biological medium.     

Industrially Relevant Enzyme Cascades for Drug Synthesis and Their Ecological Assessment

Environmentally friendly and sustainable processes for the production of active pharmaceutical ingredients (APIs) gain increasing attention. Biocatalytic synthesis routes with enzyme cascades support many stated green production principles, for example, the reduced need for solvents or the biodegradability of enzymes. Multi-enzyme reactions have even more advantages such as the shift of the equilibrium towards the product side, no intermediate isolation, and the synthesis of complex molecules in one reaction pot. Despite the intriguing benefits, only a few enzyme cascades have been applied in the pharmaceutical industry so far. However, several new enzyme cascades are currently being developed in research that could be of great importance to the pharmaceutical industry. Here, we present multi-enzymatic reactions for API synthesis that are close to an industrial application. Their performances are comparable or exceed their chemical counterparts. A few enzyme cascades that are still in development are also introduced in this review. Economic and ecological considerations are made for some example cascades to assess their environmental friendliness and applicability.     

Mechanistic aspects of beta-bond-cleavage reactions of aromatic radical cations

The mesolytic cleavage of a beta-C-X bond (ArCR(2)-X(*+) --> ArCR(2)(*/+) + X(+/*)) is one of the most important reactions of alkylaromatic radical cations. In this Account, our group's results concerning some fundamental aspects of this process (cleavage mode, structural and stereoelectronic effects, competitive breaking of different beta-bonds, nucleophilic assistance, possible stereochemistry, carbon vs oxygen acidity in arylalkanol radical cations) are presented and critically discussed for reactions where X = H, CR(3), SR, and SiR(3). Several examples illustrating how this information was exploited as a tool to detect electron-transfer mechanisms in chemical and enzymatic oxidations are also reported.     

Recent Developments and Strategies for Mutually Orthogonal Bioorthogonal Reactions

Over the past decade, several different metal-free bioorthogonal reactions have been developed to enable simultaneous double-click labeling with minimal-to-no competing cross-reactivities; such transformations are termed ‘mutually orthogonal’. More recently, several examples of successful triple ligation strategies have also been described. In this minireview, we discuss selected aspects of the development of orthogonal bioorthogonal reactions over the past decade, including general strategies to drive future innovations to achieve simultaneous, mutually orthogonal click reactions in one pot.