Patents and literature biocatalysis in nonaqueous media

Biocatalysis in nonaqueous media is being used in increasing regularity both in academic and industrial research. A variety of biocatalysts have been used in organic media including enzymes, multi-enzyme systems, and whole cells. In addition, the nonaqueous media has encompassed both monophasic and biphasic solvent systems, enzymes and whole cells in reversed micelles, enzymes and cells in nearly anhydrous (no added water) solvents, and enzymes catalytically active in supercritical fluids and the gas phase. Recent US and overseas patents and scientific literature on biocatalysis in nonaqueous media are surveyed. Patent abstracts are summarized individually, and literature references are divided into major subheadings.     

Inhibitory Effect of DMSO on Halohydrin Dehalogenase: Experimental and Computational Insights into the Influence of an Organic Co-solvent on the Structural and Catalytic Properties of a Biocatalyst

Although the application of organic solvents in biocatalysis is well explored, in-depth understanding of the interactions of solvent with proteins, in particular oligomeric ones, is still scant. Understanding these interactions is essential in tailoring enzymes for industrially relevant catalysis in nonaqueous media. In our study, the homotetrameric enzyme halohydrin dehalogenase (HHDH) from Agrobacterium radiobacter AD1 (HheC) was investigated, as a model system, in DMSO/water solvent mixtures. DMSO, the most commonly used co-solvent for biocatalytic transformations, was found to act as a mixed-type inhibitor with a prevalent competitive contribution. Even 5 % (v/v) DMSO inhibits the activity of HheC by half. Molecular dynamics (MD) simulations showed that DMSO keeps close to Ser-Tyr catalytic residues forming alternate H-bonds with them. Stability measurements paired with differential scanning calorimetry, dynamic light scattering methods and MD studies revealed that HheC maintains its structural integrity with as much as 30 % (v/v) DMSO.     

Enzyme catalysis in organic solvents: a promising field for optical biosensing

The ability of enzymes to work in non-aqueous media offers new and almost unexploited possibilities for the development of new optical biosensors. The advantages of performing biocatalytic reactions in non-aqueous media are discussed in relation to their possible application in optical biosensor design. Attention is focused on the factors that influence enzymatic catalysis in organic solvents, including the role of enzyme-associated water, criteria for solvent selection and the alteration of enzyme specificity. Recent examples of relevant applications and future prospects of organic-phase optical biosensing are discussed.     

有机介质中酶催化的基本原理

酶在有机溶剂中催化作用的研究日益受到重视,其应用范围也越来越广.本文就有机介质中酶催化的基本原理进行了讨论,包括酶的结构和催化机理,以及溶剂和水对酶的结构和催化功能的影响.同时,本文归纳出提高酶活性的一系列方法,其中不少方法简便易行,能使酶活性提高10²-10⁵倍.     

离子液体的制备及其在酶催化反应中的应用

离子液体，尤其是非水溶性离子液体可以作为一种溶剂或酶的载体用于非水相酶促反应中，也可以用于双相体系中的酶促反应。本文概括性介绍了常见离子液体的制备，总结和讨论了离子液体中酶的活性、稳定性、反应选择性以及各类酶在离子液体中的催化反应行为。离子液体的物性及其与酶的相容性对酶本身及酶促反应都有很大的影响。在非水相酶促反应中，离子液体的极性作用不遵从通常用来判别大多数有机物溶剂行为的规则，比如lgP规则。     

Entrapment of enzyme in water-restricted microenvironment for enzyme-mediated catalysis under microemulsion-based organogels

Nonaqueous enzymology has emerged as a major area of biotechnology research and development. Enzymes in organic solvents offer great potential for the biocatalysis of a wide range of chemical processes that cannot occur in water. One of the most commonly used methods for carrying out enzymatic conversions in organic solvents is enzymes solubilized in water-in-oil (w/o) microemulsions or water containing reverse micelles. In reverse micelles, enzyme molecules are solubilized in discrete hydrated micelles formed by surfactants within a continuous phase, i.e., nonpolar organic solvent. Under appropriate conditions, these solutions are homogeneous, thermodynamically stable, and optically transparent. However, there are very few examples of preparative-scale enzyme-catalyzed synthesis in water-in-oil microemulsion. One reason is that despite the advantages offered by microemulsion media, product isolation and enzyme reuse from such singlephase liquid medium is more complex than in competing methodologies in which the catalyst is present as a separate solid phase. Therefore, the approach simplifying product isolation, and enzyme reuse from microemulsion-based media, has been the use of a gelled microemulsion system, especially gelatin silica nanocomposite.     

Silver (I)-Mediated Separations by Nonaqueous Capillary Electrophoresis: Nonaqueous Argentation Electrophoresis

This study represents the first application of Ag(I) charge transfer complexation in nonaqueous capillary electrophoresis. This method applies the principles of argentation chromatography to nonaqueous electrophoretic separations and is termed “nonaqueous argentation electrophoresis”. Since the separations are performed in 100% nonaqueous media, the advantages of nonaqueous solvents, such as enhanced solubility and flexibility in selectivity enhancement, compared to an aqueous or mixed hydroorganic solvent, are realized. A variety of compounds were separated. Qualitatively, the separation of eleven sulfonamides in 100% acetonitrile is shown to improve greatly upon the addition of Ag(I). These results also show that nonaqueous argentation electrophoresis provides fast, well-resolved separations of compounds, such as N-containing heterocyclics, that can selectively complex with Ag(I). Migration data and separation selectivities of these compounds by nonaqueous argentation electrophoresis were compared to previous aqueous argentation electrophoresis results. Selectivities were found to be significantly different for the two separation media. Ag(I) complexation provides an effective means of manipulating selectivity in nonaqueous capillary electrophoresis.     

生物催化产业化进展

生物催化吸引人的特征包括高效性、多样性、底物专一性、区域选择性、化学 选择性、对映选择性以及温和的反应条件．总结了一些化学公司当前在生物催化产 业化方面所取得的成就．预计在今后的十年间，将会有更多优于传统化学路线的生 物催化方法被广泛地应用于各种精细化学品的工业生产中.     

Analyzing the molecular basis of enzyme stability in ethanol/water mixtures using molecular dynamics simulations

One of the drawbacks of nonaqueous enzymology is the fact that enzymes tend to be less stable in organic solvents than in water. There are, however, some enzymes that display very high stabilities in nonaqueous media. In order to take full advantage of the use of nonaqueous solvents in enzyme catalysis, it is essential to elucidate the molecular basis of enzyme stability in these media. Toward this end, we performed μs-long molecular dynamics simulations using two homologous proteases, pseudolysin, and thermolysin, which are known to have considerably different stabilities in solutions containing ethanol. The analysis of the simulations indicates that pseudolysin is more stable than thermolysin in ethanol/water mixtures and that the disulfide bridge between C30 and C58 is important for the stability of the former enzyme, which is consistent with previous experimental observations. Our results indicate that thermolysin has a higher tendency to interact with ethanol molecules (especially through van der Waals contacts) than pseudolysin, which can lead to the disruption of intraprotein hydrophobic interactions and ultimately result in protein unfolding. In the absence of the C30-C58 disulfide bridge, pseudolysin undergoes larger conformational changes, becoming more open and more permeable to ethanol molecules which accumulate in its interior and form hydrophobic interactions with the enzyme, destroying its structure. Our observations are not only in good agreement with several previous experimental findings on the stability of the enzymes studied in ethanol/water mixtures but also give an insight on the molecular determinants of this stability. Our findings may, therefore, be useful in the rational development of enzymes with increased stability in these media.     

Exploitation of reversed micelles as membrane mimetic reagents

The aqueous micelle model and its relation to the various models proposed for aggregation of surfactants in nonaqueous solvents, and the controversy surrounding the applicability of the critical micelle concentration to these aggregates, have been discussed. The effect of the addition of water to, and the nature of acid-base interactions in, surfactants solutions in apolar solvents have been included in a review of the kinetics of the reactivity of esters and other organic sustrates, inoragatic reaction mechanisms, catalysis by solubilized enzymes and electron and proton transfer reactions in these media. Where possible, analogies have been made with membrance mediated processes.     

Dramatic solvent and hydration effects on the transition state of soybean peroxidase

Enzymes are shown to function in nonaqueous media; however, relatively little information is available on the influence of the organic solvent as well as its associated water content on the properties of the enzymatic transition states. A better understanding of these effects will be useful in developing kinetic models that can then be used to predict optimal solvent and substrate choices for enzymatic reactions in organic media. The influence of the reaction media on soybean peroxidase-catalyzed oxidation of para-substituted phenols was studied using Hammett analysis for several organic solvent systems. The catalytic activity and substrate specificity of the enzyme are influenced by the nature of the solvent and its associated hydration. These findings may allow one to draw conclusions about the reaction mechanism and the roles of solvent and solvent hydration on enzyme function.     

General Aspects and Optimization of Enantioselective Biocatalysis in Organic Solvents: The Use of Lipases [New Synthetic Methods (76)]

Enantioselective biocatalysis in nonaqueous media is becoming increasingly important in preparative synthetic chemistry. This article discusses (1) the general catalytic properties of enzymes in nonaqueous environments, (2) the basic principles that govern lipase-catalyzed enantioselective esterification and transesterification reactions in organic media for the preparation of optically active acids and alcohols, (3) the determination of kinetic and thermodynamic parameters, and (4) the quantitative analysis of published data.     

Wasser als Lösungsmittel: Koordinationskatalyse

Water is a favoured solvent in inorganic-analytical reactions and also in biocatalysis. With the development of the homogeneous coordination catalysis and its transfer into multiphase systems water became interesting as polar phase. Prerequisite was the synthesis of water-soluble catalytically active complexes which can be recycled by simple phase separation. Some advantages with respect to activity and selectivity could be observed for aqueous organic syntheses. Moreover, water gives the possibility for supramolecular aggregations of amphiphilic molecules and is a good medium for supported catalysts. Supercritical water shows a rather different behaviour as solvent and as reactant. An advantage of water in industrial application is the easy accessibility and also the environmental conservation.     

Modified enzymes for reactions in organic solvents

Recent studies on biocatalysis in water—organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a _w ) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.     

Catalytic reactions in ionic liquids

The chemical industry is under considerable pressure to replace many of the volatile organic compounds (VOCs) that are currently used as solvents in organic synthesis. The toxic and/or hazardous properties of many solvents, notably chlorinated hydrocarbons, combined with serious environmental issues, such as atmospheric emissions and contamination of aqueous effluents is making their use prohibitive. This is an important driving force in the quest for novel reaction media. Curzons and coworkers, for example, recently noted that rigorous management of solvent use is likely to result in the greatest improvement towards greener processes for the manufacture of pharmaceutical intermediates. The current emphasis on novel reaction media is also motivated by the need for efficient methods for recycling homogeneous catalysts. The key to waste minimisation in chemicals manufacture is the widespread substitution of classical 'stoichiometric' syntheses by atom efficient, catalytic alternatives. In the context of homogeneous catalysis, efficient recycling of the catalyst is a conditio sine qua non for economically and environmentally attractive processes. Motivated by one or both of the above issues much attention has been devoted to homogeneous catalysis in aqueous biphasic and fluorous biphasic systems as well as in supercritical carbon dioxide. Similarly, the use of ionic liquids as novel reaction media may offer a convenient solution to both the solvent emission and the catalyst recycling problem.     

有机溶剂对酶催化活性和选择性的影响

有机溶剂中酶的结构与功能与在水中有很大的不同, 通过调整反应介质可系统地改善酶针对目标反应的活性和选择性。重点阐述了溶剂对酶催化反应的活性和选择性的影响及其控制策略, 给出了酶催化选择性的热力学预测模型。     

Nonaqueous versus aqueous capillary electrophoresis of alpha-helical polypeptides: effect of secondary structure on separation selectivity

The CE separation of alpha-helical polypeptides composed of 14-31 amino acid residues has been investigated using aqueous and nonaqueous BGEs. The running buffers were optimized with respect to pH. Generally, higher separation selectivities were observed in nonaqueous electrolytes. This may be explained by a change in the secondary structure when changing from water to organic solvents. Circular dichroism spectra revealed a significant increase in helical structures in methanol-based buffers compared to aqueous buffers. This change in secondary structure of the polypeptides contributed primarily to the different separation selectivity observed in aqueous CE and NACE. For small oligopeptides of two to five amino acid residues no significant effect of the solvent was observed in some cases while in other cases a reversal of the migration order occurred when changing from aqueous to nonaqueous buffers. As these peptides cannot adopt secondary structures the effect may be attributed to a shift of the pKa values in organic solvents compared to water.     

Poly(3-hydroxybutyrate)-depolymerase from Pseudomonas lemoignei: catalysis of esterifications in organic media

Lipase catalysis in nonaqueous media is recognized as a powerful tool in organic and more recently polymer synthesis. Even though none of the currently known polyhydroxyalkanoate (PHA) depolymerases have lipase activity, they do have a catalytic center that resembles that of lipases. Motivated by the above, the potential of using the poly(3-hydroxybutyrate), PHB, depolymerase from Psuedomonas lemoignei in organic media to catalyze ester-forming reactions was investigated. The effect of different organic solvents (benzene-d(6), cyclohexane-d(12), and acetonitrile-d(3)) on the activity of the PHB-depolymerase toward propylation of L-lactide was studied. A significant difference in the catalytic rate was observed as a function of solvent polarity. The selectivity of the PHB-depolymerase (P. lemoignei) to catalyze the propylation of a series of different lactones including epsilon-caprolactone, delta-butyrolactone, gamma-butyrolactone, and D, L, meso, and racemic lactides has been studied with the PHB-depolymerase (P. lemoignei) in organic solvents. Important differences in the reactivity of these lactones, as well as selective hydrolysis of stereochemically different linear lactic acid dimers, were observed. Moreover, the ability of the PHB-depolymerase to catalyze the solventless polymerization of epsilon-caprolactone and trimethylene carbonate was investigated.     

Designing for sustainability with biocatalytic and chemoenzymatic cascade processes

Cascade reactions have been widely recognized to cut costs, decrease solvent usage, and reduce cycle times in chemical processes. Recently, biocatalytic cascades have altered how we design synthetic routes to complex molecules to achieve sustainable commercial processes for pharmaceutical, agricultural, and fine chemical industries. With advancements in protein engineering and an increase in the number of enzyme classes available to chemists, industrial and academic groups alike have endeavored to expand the scope of biocatalysis from single reactions to multi-enzyme cascades to rapidly build complex molecular structures. Recent reports have drawn inspiration from biosynthetic pathways and have applied engineered enzymes to in vitro enzymatic cascades. Furthermore, combining transition-metal catalysis and enzymes in one-pot chemoenzymatic cascades likewise serves to broaden the scope of biocatalysis, enabling traditional chemical reactions to be performed under mild aqueous conditions. In this article, we review recent biocatalytic and chemoenzymatic cascades from 2019 to 2021.     

Cycloaddition reactions in aqueous systems: A two-decade trend endeavor

Cycloaddition reactions are an integral and weighty part of organic chemistry in pedagogy and research as well. The wealthy literature on cycloaddition reactions from their birth up to now, unequivocally witnesses to their leading chemistry. The so-called “conventional solvents” are organic solvents that have indubitably promoted their success. Yet, the toxicity facet of these solvents impedes their use freely and with no fear. Not only is the operating chemist uncomfortable while experimenting, but also the environment is equally threatened. Working out the cycloaddition reactions and other organic ones in aqueous systems would certainly bring some relief to the chemist and to the environment as well. Unusual outcomes in terms of yield, reactivity and selectivity compared to those performed in organic solvents were commonly observed, and have overwhelmed the chemists with surprise indeed. In this review, homo Diels–Alder reactions in aqueous media include those involving the following dienophiles: maleimides, α,β-unsaturated esters, p-benzoquinones, vinyl ketones, phenyl-1-(2-pyridyl)-2-propen-1-one, α,β-unsaturated esters. A special case is the organocatalysis of Diels–Alder cycloaddition of α,β-unsaturated ketones (aldehydes). Of no less importance, some hetero Diels–Alder cycloaddition reactions in water systems are delineated. The impact of additives (salts, organic and inorganic chemicals), micellar catalysis and Lewis/Brønstëd acid catalysis on outcomes of such cycloaddition reactions is discussed. The 1,3-dipolar cycloaddition methodology applied to aqueous media has brought forth a number of heterocyclic compounds, usually with a regio- and stereoselectivity pecularity. These heterocycles include triazoles, tetrazoles, pyrazoles, isoxazoles, isoxazolidines, pyrroles and pyrrolidines. The superiority of copper(I) catalysis in the azide-alkyne cycloaddition (Huisgen cycloaddition) in water is endorsed by a number of examples.