Enzymes in organic media. Forms, functions and applications.

Enzyme catalysis in low water containing organic solvents is finding an increasing number of applications in diverse areas. This review focuses on some aspects which have not been reviewed elsewhere. Different strategies for obtaining higher activity and stability in such media are described. In this context, the damaging role of lyophilization and the means of overcoming such effects are discussed. Ultrasonication and microwave assistance are two emerging approaches for enhancing reaction rates in low water media. Control of water activity and medium engineering are two crucial approaches in optimization of catalytic behaviour in nonaqueous enzymology. Organometallics and synthesis/modification of polymers are two areas where nonaqueous enzymology can play a greater role in the coming years. The greater understanding of enzyme behaviour in nonaqueous media is expected to lead to larger and even more diverse kinds of applications.     

Activity and dynamics of an enzyme, pig liver esterase, in near-anhydrous conditions

Water is widely assumed to be essential for life, although the exact molecular basis of this requirement is unclear. Water facilitates protein motions, and although enzyme activity has been demonstrated at low hydrations in organic solvents, such nonaqueous solvents may allow the necessary motions for catalysis. To examine enzyme function in the absence of solvation and bypass diffusional constraints we have tested the ability of an enzyme, pig liver esterase, to catalyze alcoholysis as an anhydrous powder, in a reaction system of defined water content and where the substrates and products are gaseous. At hydrations of 3 (±2) molecules of water per molecule of enzyme, activity is several orders-of-magnitude greater than nonenzymatic catalysis. Neutron spectroscopy indicates that the fast (≤nanosecond) global anharmonic dynamics of the anhydrous functional enzyme are suppressed. This indicates that neither hydration water nor fast anharmonic dynamics are required for catalysis by this enzyme, implying that one of the biological requirements of water may lie with its role as a diffusion medium rather than any of its more specific properties.     

交联蛋白晶体技术及其应用

作为蛋白工程的一项突破性技术,交联蛋白晶体技术正受到广泛重视。蛋白晶体的交联形式不仅保留了蛋白的生物活性,而且显示了对热、极端pH和温度、有机溶剂和蛋白水解酶的更高稳定性。本文介绍交联蛋白晶体的制备和特性及其在生物医学、有机合成、环境催化和色谱分析中的应用 。     

Engineering enzymes for non-aqueous solvents.

Enzymes may be redesigned to permit catalysis in non-aqueous solvents by engineering their amino acid sequences, thereby altering their physical and chemical properties to suit the new solvent environment. The interactions that contribute to protein stability in non-aqueous solvents are discussed in the context of attempting to identify possible approaches to constructing enzymes which exhibit enhanced stability in non-aqueous media. These approaches are illustrated by several examples where protein engineering has resulted in enzymes that are better suited for catalysis in organic solvents.     

Homogeneous Biocatalysis in Organic Solvents and Water-Organic Mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Homogeneous biocatalysis in organic solvents and water-organic mixtures

Biocatalysis in non-aqueous media has undergone tremendous development during the last decade, and numerous reactions have been introduced and optimized for synthetic applications. In contrast to aqueous enzymology, biotransformations in organic solvents offer unique industrially attractive advantages, such as: drastic changes in the enantioselectivity of the reaction, the reversal of the thermodynamic equilibrium of hydrolysis reactions, suppression of water-dependent side reactions, and resistance to bacterial contamination. Currently, the field is dominated by heterogeneous biocatalysis based primarily on lyophilized enzyme powders, cross-linked crystals, and enzymes immobilized on inert supports that are mainly applied in enantioselective synthesis. However, low reaction rates are an inherent problem of the heterogeneous biocatalysis, while the homogeneous systems have the advantage that the elimination of diffusional barriers of substrates and products between organic and water phases results in an increase in the reaction rate. Here the discussion is focused on the correlation between activity and structure of the intact enzymes dissolved in neat organic solvents, as well as modifications of natural enzymes, which make them soluble and catalytically active in non-aqueous environment. Factors that influence conformation and stability of the enzymes are also discussed. Current developments in non-aqueous biocatalysts that combine advantages of protein modification and immobilization, i.e., HIP plastics, enzyme chips, ionic liquids, are introduced. Finally, engineering enzymes for biotransformations in non-conventional media by directed evolution is summarized.     

Enzymatic catalysis in nonaqueous solvents

Subtilisin and alpha-chymotrypsin vigorously act as catalysts in a variety of dry organic solvents. Enzymatic transesterifications in organic solvents follow Michaelis-Menten kinetics, and the values of V/Km roughly correlate with solvent's hydrophobicity. The amount of water required by chymotrypsin and subtilisin for catalysis in organic solvents is much less than needed to form a monolayer on its surface. The vastly different catalytic activities of chymotrypsin in various organic solvents are partly due to stripping of the essential water from the enzyme by more hydrophilic solvents and partly due to the solvent directly affecting the enzymatic process. The rate enhancements afforded by chymotrypsin and subtilisin in the transesterification reaction in octane are of the order of 100 billion-fold; covalent modification of the active center of the enzymes by a site-specific reagent renders them catalytically inactive in organic solvents. Upon replacement of water with octane as the reaction medium, the specificity of chymotrypsin toward competitive inhibitors reverses. Both thermal and storage stabilities of chymotrypsin are greatly enhanced in nonaqueous solvents compared to water. The phenomenon of enzymatic catalysis in organic solvents appears to be due to the structural rigidity of proteins in organic solvents resulting in high kinetic barriers that prevent the native-like conformation from unfolding.     

Operational concept for the improved synthesis of (R)-3,3'-furoin and related hydrophobic compounds with benzaldehyde lyase

Biphasic reaction systems for enzyme catalysis are an elegant way to overcome limited solubility and stability of reactants and facilitate continuous processes. However, many synthetically useful enzymes are not stable in biphasic systems of water and organic solvent. The entrapment in polymer beads of polyvinyl alcohol has been shown to enable the stable operation of enzymes unstable in conventional biphasic reaction systems. We report the extension of this concept to continuous operation in a fluidised bed reactor. The enzyme benzaldehyde lyase was used for the continuous synthesis of enantiopure (R)-3,3'-furoin. The results show enhanced stability with half-life times under operation conditions of more than 100 h, as well as superior enzyme utilisation in terms of productivity. Furthermore, racemisation and oxidation of the product could be successfully prevented under the non-aqueous and inert reaction conditions.     

Aspects Of Biocatalyst Stability In Organic Solvents

The stability of biocatalysis in systems containing organic solvents is reviewed. Among the examples presented are homogeneous mixtures of water and water-miscible organic solvents, aqueous/organic two-phase systems, solid biocatalysts suspended in organic solvents, enzymes in reverse micelles and modified enzymes soluble in water immiscible solvents. The stability of biocatalysts in organic solvents depends very much on the conditions. The hydrophobicity or the polarity of the solvent is clearly of great importance. More hydrophobic solvents (higher log P values) are less harmful to enzymes than less hydrophobic solvents. The water content of the system is a very important parameter. Some water is essential for enzymatic activity; however, the stability of enzymes decreases with increasing water content. Mechanisms of enzyme inactivation are discussed.     

Enzymes in Low Water Systems

Water is fundamental for enzyme action and for formation of the three-dimensional structure of proteins. Hence, it may be assumed that studies on the interplay between water and enzymes can yield insight into enzyme function and formation. This has proven correct, because the numerous studies that have been made on the behavior of water-soluble and membrane enzymes in systems with a low water content (reverse micelles or enzymes suspended in nonpolar organic solvents) have revealed properties of enzymes that are not easily appreciated in aqueous solutions. In the low water systems, it has been possible to probe the relation between solvent and enzyme kinetics, as well as some of the factors that affect enzyme thermostability and catalysis. Furthermore, the studies show that low water environments can be used to stabilize conformers that exhibit unsuspected catalytic properties, as well as intermediates of enzyme function and formation that in aqueous media have relatively short life-times. The structure of enzymes in these unnatural conditions is actively being explored.     

Microbial lipases: at the interface of aqueous and non-aqueous media. A review

In recent times, biotechnological applications of microbial lipases in synthesis of many organic molecules have rapidly increased in non-aqueous media. Microbial lipases are the 'working horses' in biocatalysis and have been extensively studied when their exceptionally high stability in non-aqueous media has been discovered. Stability of lipases in organic solvents makes them commercially feasibile in the enzymatic esterification reactions. Their stability is affected by temperature, reaction medium, water concentration and by the biocatalyst's preparation. An optimization process for ester synthesis from pilot scale to industrial scale in the reaction medium is discussed. The water released during the esterification process can be controlled over a wide range and has a profound effect on the activity of the lipases. Approaches to lipase catalysis like protein engineering, directed evolution and metagenome approach were studied. This review reports the recent development in the field ofnon-aqueous microbial lipase catalysis and factors controlling the esterification/transesterification processes in organic media.     

Enzymology in monophasic organic media.

Over the past year, an important area of research has been directed towards the fundamental aspects of enzymes and new applications of enzymology in monophasic organic media. Much of this research has focused on the factors that influence enzymatic catalysis in monophasic organic solvents, including the importance of enzyme-associated water, and the effect of organic solvents on enzyme structure and thermodynamic features. From an applications perspective, new advances in the use of enzymes in organic and polymer syntheses and optical resolutions have been made.     

The activity of human CYP2D6 in low water organic solvents

P450 enzymes are of high interest for synthetic applications due to their ability to catalyze hydroxylation reactions at inactivated C-H bonds. The low solubility of many substrates in buffer, however, is limiting the applications of P450s. Our recent demonstration that the P450 enzymes CYP2D6 and CYP3A4 can function very well in biphasic solvent systems is one step towards overcoming this drawback, but is not practical when substrates or products are unstable in water, or with water-soluble products. An alternative strategy, which also facilitates enzyme recycling, is to directly resuspend lyophilized enzyme into nearly anhydrous organic solvents. Interestingly, we report here that CYP2D6 colyophilized with trehalose and suspended in n-decane shows higher activity than in aqueous buffer. This study demonstrates the unexpected high tolerance of CYP2D6 to some low water organic solvents and provides an alternative strategy to facilitate the use of this enzyme in synthesis.     

Stability and activity of a thermostable malic enzyme in denaturants and water-miscible organic solvents

A study was made of the effects of common protein denaturants and water-miscible organic solvents on both the stability and activity of the malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40] from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. At 25 degrees C, the enzyme was not inactivated in 4 M urea or 0.05% SDS over 24 h, while the half-life was 30 min in 6 M guanidine hydrochloride and 5 h in 0.075% SDS. The enzyme stability in water-miscible organic solvents at 25 degrees C is somewhat surprising: after a 24-h incubation, the enzyme was completely active in 50% dimethylformamide; it lost 15% of its initial activity in 50% methanol or 15% ethanol. However, the resistance to organic solvents was greatly reduced at higher temperatures. The enzyme was able to catalyze the malate conversion even in the presence of 1.5% Triton X-100 or sodium deoxycholate. A number of solvents were found to stimulate the malic activity independent of time. Studies with 50% methanol revealed that the activation was reversible and inversely related to the temperature; moreover, the solvent was demonstrated to exclusively affect the maximal velocity of catalysis, the Km values for both substrates being unchanged. Investigation was made to find out whether there was a correlation between enzyme stability, as well as activation, and hydrophobicity of the organic medium. The residual malic activity after incubation in the water/organic medium correlated inversely with the logarithm of the partition coefficient in octanol/H2O of the mixture used as a hydrophobicity index. On the other hand, the extent of activation depended directly on the logarithm of the molar concentration of the organic solvent required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents and protein denaturants in general, the malic enzyme from Sulfolobus solfataricus can be considered suitable for biotechnological applications.     

Enzymes from solvent-tolerant microbes: Useful biocatalysts for non-aqueous enzymology

Solvent-tolerant microbes are a newly emerging class that possesses the unique ability to thrive in the presence of organic solvents. Their enzymes adapted to mediate cellular and metabolic processes in a solvent-rich environment and are logically stable in the presence of organic solvents. Enzyme catalysis in non-aqueous/low-water media is finding increasing applications for the synthesis of industrially important products, namely peptides, esters, and other trans-esterification products. Solvent stability, however, remains a prerequisite for employing enzymes in non-aqueous systems. Enzymes, in general, get inactivated or give very low rates of reaction in non-aqueous media. Thus, early efforts, and even some recent ones, have aimed at stabilization of enzymes in organic media by immobilization, surface modifications, mutagenesis, and protein engineering. Enzymes from solvent-tolerant microbes appear to be the choicest source for studying solvent-stable enzymes because of their unique ability to survive in the presence of a range of organic solvents. These bacteria circumvent the solvent’s toxic effects by virtue of various adaptations, e.g. at the level of the cytoplasmic membrane, by degradation and transformation of solvents, and by active excretion of solvents. The recent screening of these exotic microbes has generated some naturally solvent-stable proteases, lipases, cholesterol oxidase, cholesterol esterase, cyclodextrin glucanotransferase, and other important enzymes. The unique properties of these novel biocatalysts have great potential for applications in non-aqueous enzymology for a range of industrial processes.     

Activity and stability of cross-linked tyrosinase aggregates in aqueous and nonaqueous media

Cross-linked tyrosinase aggregates were prepared by precipitating the enzyme with ammonium sulfate and subsequent cross-linking with glutaraldehyde. Both activity and stability of these cross-linked enzyme aggregates (CLEAs) in aqueous solution, organic solvents, and ionic liquids have been investigated. Immobilization effectively improved the stability of the enzyme in aqueous solution against various deactivating conditions such as pH, temperature, denaturants, inhibitors, and organic solvents. The stability of the CLEAs in various organic solvents such as tert-butanol (t(1/2)=326.7h at 40°C) was significantly enhanced relative to that in aqueous solution (t(1/2)=5.5h). The effect of thermodynamic water activity (a(w)) on the CLEA activity in organic media was examined, demonstrating that the enzyme incorporated into CLEAs required an extensive hydration (with an a(w) approaching 1.0) for optimizing its activity. The impact of ionic liquids on the CLEA activity in aqueous solution was also assessed.     

细胞表面展示技术在环境修复方面的研究进展*

人类社会工业化导致各种有毒物质被排放到环境中造成严重的污染。除了自然降解外,传统的处理方法包括化学转化、物理吸附、离子交换和电化学方法等,但存在二次污染、能源需求高、投资成本高、再生效率低、低浓度废水处理效率低等缺点。细胞表面展示技术是一种通过表面锚定蛋白在细胞表面连接功能肽的新型、高效的生物技术。与细胞内和分泌物表达系统相比,微生物表面展示的蛋白质对有机溶剂、蛋白酶、温度和pH的变化表现出更强的稳定性。通过细胞培养就可以获得固定在细胞表面的蛋白酶,避免了蛋白质纯化、浓缩等繁琐的程序。此外,细胞表面展示技术是良好的单细胞水平突变体文库高通量筛选平台。综述细胞表面展示技术在环境生物修复方面的研究进展,重点介绍该技术的应用和未来发展前景。     

Pressure affects enzyme function in organic media

Pressure affects enzyme function in nonaqueous media. Activation volumes have been determined and provide evidence that the primary effect of pressure is to enhance the stripping of water off an enzyme in polar organic solvents and leads to decreased enzymatic activity. Activation volumes of subtilisin Carlsberg in organic solvents, particularly with the enzyme hydrated, have a larger magnitude than activation volumes determined in aqueous solutions. This study provides further evidence that enzymatic activity in polar organic solvents is dominated by the interaction of enzyme-bound water with the solvent. From a practical standpoint, however, the results of this study suggest that enzymatic catalysis in organic solvents may be controlled by the combined effects of pressure and enzyme hydration. (c) 1993 John Wiley & Sons, Inc.     

Micellar enzymology: potentialities in fundamental and applied areas

Micellar enzymology, a new trend in molecular biology, studies catalysis by enzymes entrapped in hydrated reversed micelles of surfactants (phospholipids, detergents) in organic solvents. In this review, the key research problems of micellar enzymology are formulated and examples of biocatalysis in microheterogeneous media are discussed. In particular, new applications are presented of micellar enzymology in fine organic syntheses, in clinical and chemical analyses (bioluminescence and enzyme immunoassays), in bioconversion of energy and mass, in therapy (engineering of new drugs capable of targeted penetration into cells), as well as in biotechnology (processes using nanogranulated or nanocapsulated enzymes).