Salt-activation of nonhydrolase enzymes for use in organic solvents

Enzymatic reactions are important for the synthesis of chiral molecules. One factor limiting synthetic applications of enzymes is the poor aqueous solubility of numerous substrates. To overcome this limitation, enzymes can be used directly in organic solvents; however, in nonaqueous media enzymes usually exhibit only a fraction of their aqueous-level activity. Salt-activation, a technique previously demonstrated to substantially increase the transesterification activity of hydrolytic enzymes in organic solvents, was applied to horse liver alcohol dehydrogenase, soybean peroxidase, galactose oxidase, and xanthine oxidase, which are oxidoreductase and oxygenase enzymes. Assays of the lyophilized enzyme preparations demonstrated that the presence of salt protected enzymes from irreversible inactivation. In organic solvents, there were significant increases in activity for the salt-activated enzymes compared to nonsalt-activated controls for every enzyme tested. The increased enzymatic activity in organic solvents was shown to result from a combination of protection against inactivation during the freeze-drying process and other as-yet undetermined factors.     

Recent advances of enzymatic reactions in ionic liquids

The tremendous potential of room temperature ionic liquids as an alternative to environmentally harmful ordinary organic solvents is well recognized. Ionic liquids, having no measurable vapor pressure, are an interesting class of tunable and designer solvents, and they have been used extensively in a wide range of applications including enzymatic biotransformation. In fact, ionic liquids can be designed with different cation and anion combinations, which allow the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ionic liquids provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. As compared to those observed in conventional organic solvents, the use of enzymes in ionic liquids has presented many advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. To date, a wide range of pronounced approaches have been taken to further improve the performance of enzymes in ionic liquids. This review presents the recent technological developments in which the advantages of ionic liquids as a medium for enzymes have been gradually realized.     

微生物嗜盐酶的研究进展

嗜盐酶一般来自于嗜盐菌,它的主要特点是严格依赖体系中一定的盐离子浓度,可以在高盐环境中维持其结构稳定,并且能够抵抗高温、p H和有机溶剂存在下的变性,因此在高盐、水/有机和非水介质环境的催化中具有重要的应用价值。本综述从盐对嗜盐酶活性和稳定性的影响、金属离子和有机溶剂对嗜盐酶的影响几个方面介绍了嗜盐酶的特点。在总结蛋白质数据库(PDB)中已有嗜盐酶的结构和特点的基础上,对嗜盐酶的嗜盐机制进行了分析,认为嗜盐酶不同于非嗜盐酶的特点在于盐桥和氢键明显增多,含有一些特殊的盐离子结合位点并且常以低聚体的形式存在,表面酸性氨基酸含量明显增多。最后对嗜盐酶的分子改造和应用进行了简要的介绍。     

Industrial potential of organic solvent tolerant bacteria

Most bacteria and their enzymes are destroyed or inactivated in the presence of organic solvents. Organic solvent tolerant bacteria are a relatively novel group of extremophilic microorganisms that combat these destructive effects and thrive in the presence of high concentrations of organic solvents as a result of various adaptations. These bacteria are being explored for their potential in industrial and environmental biotechnology, since their enzymes retain activity in the presence of toxic solvents. This property could be exploited to carry out bioremediation and biocatalysis in the presence of an organic phase. Because a large number of substrates used in industrial chemistry, such as steroids, are water-insoluble, their bioconversion rates are affected by poor dissolution in water. This problem can be overcome by carrying out the process in a biphasic organic-aqueous fermentation system, wherein the substrate is dissolved in the organic phase and provided to cells present in the aqueous phase. In bioprocessing of fine chemicals such as cis-diols and epoxides using such cultures, organic solvents can be used to extract a toxic product from the aqueous phase, thereby improving the efficiency of the process. Bacterial strains reported to grow on and utilize saturated concentrations of organic solvents such as toluene can revolutionize the removal of such pollutants. It is now known that enzymes display striking new properties in the presence of organic solvents. The role of solvent-stable enzymes in nonaqueous biocatalysis needs to be explored and could result in novel applications.     

Application of polyethylene glycol-modified enzymes in biotechnological processes: organic solvent-soluble enzymes

A new approach in biotechnological processes is to use enzymes modified with polyethylene glycol which has both hydrophilic and hydrophobic properties. The modified enzymes are soluble in organic solvents such as benzene, toluene and chlorinated hydrocarbons and exhibit high enzymic activities in these organic solvents. Modified hydrolytic enzymes catalysed the reverse reaction of hydrolysis in organic solvents: formation of acid—amide bonds by modified chymotrypsin, and ester synthesis and ester exchange reactions by modified lipase. Modified catalase and modified peroxidase efficiently catalyse their respective reactions in organic solvents. The results of this research indicate great potential for applications in the fields of biotechnology and enzymology.     

超声对酶的影响

超声对酶的影响与超声的强度和介质的性质有关。适宜的超声可提高酶促反应速度,较低强度的超声可提高酶的活性,而较高强度的超声会降低酶促反应速度甚至使酶失活。介质性质不同时,超声对酶活力的促进效果与稳定性不同。非极性介质中酶对于超声的抗性要优于水溶液。在较低强度的超声作用下,超声增加了底物的传质作用,并可导致酶分子的构象发生变化。     

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.     

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.     

Enzymes in non-aqueous solvents

For a variety of reasons including increased recognition of the large degree of association, by non-polar interaction, of enzymes with other cellular components such as membranes, enzymes are increasingly being investigated in mixed solvents less polar than water. Such solvents may be quite relevant because their polarity more nearly resembles the natural cellular microenvironment than does pure water. The single most important criterion in selecting a non-aqueous solvent is its compatibility with the maintenance of the enzyme's catalytic activity, which must be determined experimentally for each enzyme. Non-aqueous solvents have a variety of effects on enzymes: they may bind specifically, compete with substrate binding, dissociate multimers, shift an equilibrium between two enzyme conformations, alter the amount of helix, react with the enzyme, stabilize or destabilize the enzyme, and affect the rate of the catalytic reaction in several different ways. Typically, modest concentrations of hydroxylic solvents have little effect on rates, and may even enhance the rate significantly. Higher concentrations give lower rates, in a solvent-specific and enzyme-specific manner. Hydroxylic solvents may replace water as acceptor of a phosphoryl, glycosyl, or acyl group produced by a hydrolytic enzyme. Non-aqueous solvents also make it possible to run hydrolytic reactions in the reverse direction, forming a condensation product and water as a by-product. Non-aqueous solvents are being extensively used in cryoenzymology as antifreeze agents, in solubilizing and purifying enzymes, and to a lesser degree in two-phase systems in which the non-polar substrate is dissolved in the non-aqueous phase. Liquefied aqueous phenol is an extraordinary solvent for enzymes and other proteins. It is a powerful denaturant which rapidly and irreversibly extracts the enzyme into the phenol-rich phase of a phenol-water system. This property makes phenol useful for removing protein contaminants, and for detecting labile enzyme-substrate intermediates, by extracting substrate covalently bound to enzyme into the phenol-rich phase away from all other substrate, which generally remains in the aqueous phase.     

Biocatalysis in semi-aqueous and nearly anhydrous conditions

In the past few years there have been prolific advances in activating enzymes for nonaqueous biocatalysis. Molecular dynamics simulations complement recent experimental results and offer new insights into the deleterious effects of organic solvents, such as water stripping and active-site penetration. Methods for activating enzymes in semi-aqueous or nonaqueous media include protein engineering, chemical modification, and co-lyophilization with non-buffer salts. Enzyme immobilization on novel polymeric supports and the use of zeolite molecular sieves can also increase solvent tolerance, enhance activity, and improve enantioselectivity. The recent implementation of enzymes in ionic liquids has also led to better long-term stability relative to traditional organic solvents and the simultaneous solubilization of enzymes, cofactors and substrates.     

Extraction of mitochondrial membrane proteins into organic solvents in a functional state

Protein-lipid complexes in organic solvents can be used as the starting material in the reassembly of functional planar and spherical bilayers (Montal, M., Darszon, A. and Schindler, H. (1981) Q. Rev. Biophys. 14, 1-79). The transfer of three enzymes of the inner mitochondrial membrane into organic solvents as protein-lipid complexes has been studied to understand better the extraction process. The enzymes studied were cytochrome c oxidase, ATPase and succinate dehydrogenase. These enzymes were transferred into hexane and diethyl ether in an active state, however, the activities extracted varied quantitatively, depending on the amount of protein of the starting preparation, the concentration of phospholipids and the cation employed. In all conditions cytochrome c oxidase was extracted with the highest yield and specific activity, and it was actually enriched in the organic extract. The values for succinate dehydrogenase and ATPase were lower, but their specific activities were similar to those of the starting material. This indicates that some membrane proteins are preferentially extracted into organic solvents in a functional state. The enzymes, as protein-lipid complexes, are fairly stable in organic solvents; in a month of storage at 4 degrees C in hexane some enzymes loose less than 50% of their activity.     

Surface-modified polymeric nanogranules containing entrapped enzymes: A novel biocatalyst for use in organic media

Summary A novel biocatalytic system for use in organic solvents, based on enzymes entrapped into surface-modified polymeric nanogranules, is suggested. Nanogranulated biocatalysts are soluble in organic solvents of different polarity, possess high stability and catalytic activity, and can be used continuously in membrane reactors.     

Inactivation and stabilization of stabilisins in neat organic solvents

The stability of the serine proteases from Bacillus amyloliquefaciens (subtillisin BPN') and Bacillus licheniformis (subtilisin Carlsberg) was investigated in various anhydrous solvents at 45 degrees C. The half-life of subtilisin BPN' in dimethyl-formamide dramatically depends on the pH of the aqueous solutions from which the enzyme was lyophilized, increasing from 48 min to 20 h when the pH is raised from 6.0 to 7.9. Both subtilisins exhibited substantial inactivation during multihour incubations in tert-amyl alcohol and acetonitrile when enzymatic activities were also measured in these solvents; however, when the enzymes were assayed in water instead, hardly any loss of activity was detected. This surprising difference appears to stem from the partitioning of the bound water essential for catalytic activity from the enzymes into the solvents. When assayed in organic solvents, this time-dependent stripping of water results in decay of enzymatic activity; however, when assayed in water, where the dehydrated subtilisins can undergo rehydration thereby recovering catalytic activity, little inactivation is observed. In agreement with this hypothesis, the addition of small quantities of water tert-amyl alcohol stabilized the subtilisins in it even when enzymatic activity was measured in the nonaqueous solvent. Ester substrates (vinyl butyrate and trichloroethyl butyrate) greatly enhanced the stability of both subtilisins in organic solvents possibly because of the formation of the acyl-enzymes.     

Immobilization of enzymes with polyaziridines.

A novel method of enzyme immobilization using a low molecular weight prepolymer of tri-functional aziridines which can immobilize enzymes both by covalent attachment and entrapment within a gel matrix is described. The enzymes are immobilized on a solid support and exhibit an excellent retention of enzymatic activity. The immobilization procedure is essentially a single step process which can be easily performed at room temperature or 4 degrees C in either aqueous solution or in an inert organic solvent. The polyaziridines used in the immobilization are nontoxic, available in bulk at low cost and completely miscible with water and many organic solvents, thus providing one of the most satisfactory methods of immobilization available.     

Do organic solvents affect the catalytic properties of lipase? Intrinsic kinetic parameters of lipases in ester hydrolysis and formation in various organic solvents

When it is assumed that organic solvents do not interfere with the binding process nor with the catalytic mechanism, the contribution of substrate-solvent interactions to enzyme kinetics can be accounted for by just replacing substrate concentrations in the equations by thermodynamic activities. It appears from the transformation that only the affinity parameters (K(m), K(sp)) are affected by this. Thus, in theory, the values of these corrected, intrinsic parameters (K(m) (int), k(sp) (int)) and the maximal rate (V(1)) should be equal for all media. This was tested for hydrolysis, transesterification, and esterification reactions catalyzed by pig pancreas lipase and Pseudomonas cepacia lipase in various organic solvents. Correction was carried out via experimentally determined activity coefficients for the substrates in these solvents or, if not feasible, from values in data bases. However, although the kinetic performances of each enzyme in the solvents became much more similar after correction, differences still remained. Analysis of the enzyme suspensions revealed massive particles, which explains the low activity of enzymes in organic solvents. However, no correlation was found between estimates of the amount of catalytically available enzyme (present at the surface of suspended particles or immobilized on beads) and the maximal rates observed. Moreover, the solvents had similar effects on the intrinsic parameters of suspended and immobilized enzyme. The possible causes for the effects of the solvents on the catalytic performance of the enzymes, remaining after correction for solvent-substrate interactions and the amount of participating enzyme, are discussed with respect to the premises on which the correction method is based. (c) 1995 John Wiley & Sons, Inc.     

Application of PEG-enzyme and magnetite-PEG-enzyme conjugates for biotechnological processes

Enzymes can be made soluble and active in organic solvents by chemical modification with an amphipathic macromolecule, polyethylene glycol (PEG). The PEG-enzyme conjugates can also be conjugated to magnetite (Fe₃O₄). The magnetic enzymes stably disperse in both organic solvents and aqueous solutions. When lipase is prepared as such a conjugate, it catalyses ester synthesis in organic solvents, and can be readily recovered by magnetic force without loss of enzymic activity. This approach could have a great practical potential.     

Organic solvents strip water off enzymes

Exchange of enzyme-bound H(2)O with T(2)O in aqueous solution followed by freeze drying provided tritiated water bound to chymotrypsin, subtilisin Carlsberg, and horseradish peroxidase. The desorption of T(2)O from these enzymes suspended in various organic solvents showed that all three enzymes lost enzyme-bound water with peroxidase losing the most T(2)O of the three in solvents of moderate to high polarity. Polar solvent resulted in the highest degree of T(2)O desorption (e.g., methanol desorbed from 56%-62% of the bound T(2)O), while nonpolar solvents resulted in the lowest degree of desorption (e.g., hexane desorbed from 0.4%-2% of the bound T(2)O). Desorption is nearly immediate with most of the desorbable T(2)O being released from the enzymes within the first 5 min. Both solvent dielectric and a measure of the saturated molar solubility of water in a given solvent provide accurate correlations between the properties of the organic solvents and the extent of T(2)O desorption. This investigation shows that water stripping from an enzyme into a nonaqueous medium does occur and can be significant in polar solvents.     

Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N-Ac-L-Phe-NH(2), their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand-induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramaticaliy enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient-induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its "regular" counterpart. (c) 1993 John Wiley & Sons, Inc.     

Phospholipid-based reverse micelles

Physicochemical investigations on the aggregation of phospholipids (mainly phosphatidylcholines) in organic solvents are reviewed and compared with the aggregation behaviour of phospholipids in aqueous medium. In particular we review the data showing that phosphatidylcholines (lecithins) form reverse micellar structures in certain apolar solvents. In these systems not only low molecular weight compounds but also catalytically active enzymes and entire cells can be solubilized. In addition, highly viscous phosphatidylcholine gels can be obtained in organic solvents upon solubilizing a critical amount of water. Generally, phospholipid-based reverse micelles can be regarded as thermodynamically stable models for inverted micellar lipid structures possibly occurring in biological membranes.