Polyethylene glycol-modified enzymes trap water on their surface and exert enzymic activity in organic solvents

Summary Polyethylene glycol-modified enzymes dissolved and had high enzymic activity in organic solvents. A trace amount of water was found to be necessary for the activity. It was reasoned that the amphipathic polymer covalently attached to enzymes kept water molecules around them. This was supported by findings that : (1) high enzymic activity was found in water- immiscible solvents, whereas activity was never observed in water-miscible solvents; (2) enzymic activity was inhibited by increasing the concentration of dimethyl sulfoxide in benzene; (3) activity of lipase was inhibited by a water-miscible alcohol substrate, but was steadily elevated by increasing the concentration of a water-immiscible alcohol substrate; (4) water was not absorbed from benzene solution containing a modified enzyme by molecular sieves, while it was easily absorbed in the presence of a water-miscible organic solvent, dimethyl sulfoxide.     

Polyethylene Glycol-Modified Lipase Soluble and Active in Organic Solvents

A new approach in biotechnological processes is to use lipase modified with polyethylene glycol(PEG) which has both hydrophilic and hydrophobic properties. The PEG-lipase is soluble in organic solvents such as benzene and chlorinated hydrocarbons and exhibits high enzymic activity in organic solvents. The PEG-lipase catalyses the reverse reaction of hydrolysis in organic solvents; ester synthesis and ester exchange reactions. The PEG-lipase can also be conjugated to magnetite (Fe₃O₄). The magnetic lipase catalyses ester synthesis in organic solvents and can be readily recovered by magnetic force without loss of enzymic activity.     

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.     

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.     

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.     

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.     

Chemical modification of enzymes for enhanced functionality.

The explosion in commercial and synthetic applications of enzymes has stimulated much of the interest in enhancing enzyme functionality and stability. Covalent chemical modification, the original method available for altering protein properties, has now re-emerged as a powerful complementary approach to site-directed mutagenesis and directed evolution for tailoring proteins and enzymes. Glutaraldehyde crosslinking of enzyme crystals and polyethylene glycol (PEG) modification of enzyme surface amino groups are practical methods to enhance biocatalyst stability. Whereas crosslinking of enzyme crystals generates easily recoverable insoluble biocatalysts, PEGylation increases solubility in organic solvents. Chemical modification has been exploited for the incorporation of cofactors onto protein templates and for atom replacement in order to generate new functionality, such as the conversion of a hydrolase into a peroxidase. Despite the breadth of applicability of chemically modified enzymes, a difficulty that has previously impeded their implementation is the lack of chemo- or regio-specificity of chemical modifications, which can yield heterogeneous and irreproducible product mixtures. This challenge has recently been addressed by the introduction of a unique position for modification by a site-directed mutation that can subsequently be chemically modified to introduce an unnatural amino acid sidechain in a highly chemo- and regio-specific manner.     

Thermodynamic analysis of solvent effect on substrate specificity of lyophilized enzymes suspended in organic media

A simple methodology has been successfully employed to explain the solvent dependence of the substrate specificity of enzymes in organic media. This methodology, which does not require the knowledge of the enzyme structure and is thus applicable to lyophilized and other noncrystalline enzyme preparations, predicts that the k(cat)/K(M) ratio for two substrates should be proportional to their Raoult's law activity coefficients. This approach has been validated for two enzymes, subtilisin Carlsberg and alpha-chymotrypsin, catalyzing the propanolysis of unnatural (in addition to natural) ester substrates in a variety of anhydrous solvents. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 340-344, 1997.     

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.     

Designing enzymes for use in organic solvents.

Enzymes are routinely used in organic solvents where numerous reactions of interest to synthetic and polymer chemists can be performed with high selectivity. Recently, it has become apparent that the catalytic properties of an enzyme can be tailored to a specific catalytic requirement by the use of solvent and protein engineering. The former involves altering the polarity, hydrophobicity, water content, etc., of the organic milieu, while the later applies site-directed mutagenesis to alter the physicochemical properties of the biocatalyst. The dominant effects of organic solvents on enzyme structure and function, and the potential of solvent and protein engineering to design enzymes to function optimally in organic media, are the major foci of this review.     

Microbial enzymes for oxidation of organic molecules

Enzymatic systems employed by microorganisms for oxidative transformation of various organic molecules include laccases, ligninases, tyrosinases, monooxygenases, and dioxygenases. Reactions performed by these enzymes play a significant role in maintaining the global carbon cycle through either transformation or complete mineralization of organic molecules. Additionally, oxidative enzymes are instrumental in modification or degradation of the ever-increasing man-made chemicals constantly released into our environment. Due to their inherent stereo- and regioselectivity and high efficiency, oxidative enzymes have attracted attention as potential biocatalysts for various biotechnological processes. Successful commercial application of these enzymes will be possible through employing new methodologies, such as use of organic solvents in the reaction mixtures, immobilization of either the intact microorganisms or isolated enzyme preparations on various supports, and genetic engineering technology.     

Stability of hydrolytic enzymes in water-organic solvent systems

The effects of organic solvents on the stabilities of bovine pancreas trypsin, chymotrypsin, carboxypeptidase A and porcine pancreas lipase were studied. Water-miscible solvents (ethanol, acetonitrile, 1,4-dioxane and dimethyl sulfoxide) and water-immiscible solvents (ethyl acetate and toluene) were used in 100 mM phosphate buffer (pH 7.0) or 100 mM Tris/HCl buffer (pH 7.0) in concentrations of 20–80% (v/v). All hydrolytic enzymes studied were inactivated by mixtures containing dimethyl sulfoxide at higher concentrations. Trypsin and carboxypeptidase A resisted solvent mixtures containing acetonitrile, 1,4-dioxane and ethanol. They preserved more than 80% of their starting activities during 20-min incubations. The activities of lipase and chymotrypsin decreased with increasing concentration of water-miscible polar organic solvents, but at higher concentrations (80%) 70–90% of the activity remained. In mixtures with water-immiscible solvents, the decrease in activity of carboxypeptidase A was pronounced. Trypsin and chymotrypsin underwent practically no loss in activity in the presence of toluene or ethyl acetate. In respect of stability, the polar solvent proved to be more favorable for lipase. These results suggest that the conformational stabilities of hydrolytic enzymes are highly dependent on the solvent-protein interactions and the enzyme structure.     

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.     

Protease-catalyzed synthetic reactions and immobilization-activation of the enzymes in hydrophilic organic solvents.

The catalytic feature of serine proteases for synthetic reactions in hydrophilic organic solvents and effects of immobilization by complexation with polysaccharides are described. Free alpha-chymotrypsin and subtilisin Carlsberg catalyze esterification, transesterification, and peptide synthesis in hydro-organic cosolvents with less than 10% water. Subtilisin BPN' is catalytically less active. The medium effects on the reaction kinetics and product yield were investigated in terms of the nature of solvent and water content in the reaction systems. The substrate- and stereo-specificities of the enzymes suggest that the enzymes maintain their native conformations in these low-water organic solvents. The catalytic activities of the proteases markedly increase by immobilization or complexation with polysaccharides, such as chitin or chitosan. The results of the rate measurements suggest that the primary role of the support materials is the activation of the enzymes and the increase in substrate concentration at reaction sites.     

Sorption of enzymes on ion exchange resins of various structure]

The isothermic adsorption of microbial and animal enzymes on carboxyl and sulpha-cation exchange resins was studied. The adsorption isotherms are curves with a maximum. The adsorption of alpha-amylase was studied in the presence of organic solvents. It was found that organic solvents influenced the isothermic adsorption of alpha-amylase, which is associated with changes in the interactions between protein molecules in solution. The adsorption system was in equilibrium in all the cases.     

Ester synthesis in benzene by polyethylene glycol-modified lipase from Pseudomonas fragi 22.39B

Lipase (EC 3.1.1.3) from Pseudomonas fragi 22.39B was modified with polyethylene glycol. The modified lipase was soluble in organic solvents such as benzene and chlorinated hydrocarbons, and catalyzed the synthesis of esters from fatty acids and alcohols in these solvents. The longer the chain length of fatty acid, the higher the ester synthesis activity. A similar specificity was not observed with other substrates like alcohol. Values of K_m and V_max were revealed by kinetic study on the ester synthesis reaction with the modified lipase in benzene. Fatty acids with branched carbon chain at the position neighboring the carboxyl group did not serve as substrates of ester synthesis.     

Peptide synthesis catalyzed by polyethylene glycol-modified chymotrypsin in organic solvents

Chymotrypsin modified with polyethylene glycol was successfully used for peptide synthesis in organic solvents. The benzene-soluble modified enzyme readily catalyzed both aminolysis of N-benzoyl-L-tyrosine p-nitroanilide and synthesis of N-benzoyl-L-tyrosine butylamide in the presence of trace amounts of water. A quantitative reaction was obtained when either hydrophobic or bulky amides of L- as well as D-amino acids were used as acceptor nucleophiles, while almost no reaction occurred with free amino acids or ester derivatives. The acceptor nucleophile specificity of modified chymotrypsin as a catalyst in the formation of both amide and peptide bonds in organic solvents was quite comparable to that in aqueous solution as well as to that of the leaving group in hydrolysis reactions. By contrast, the substrate specificity of modified chymotrypsin in organic solvents was different from that in water since arginine and lysine esters were found to be as effective as aromatic amino acids to form the acyl-enzyme with subsequent synthesis of a peptide bond.     

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.     

Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media

Subtilisin Carlsberg, an alkaline protease from Bacillus licheniformis, was modified with polyoxyethylene (PEG) or aerosol-OT (AOT), and the solubility, conformation, and catalytic activity of the modified subtilisins in some organic media were compared under the same conditions. The solubility of modified subtilisins depended on the solubility of the modifier. On the other hand, the conformational changes depended on the solubility, rather than the property, of the modifier. When the modified subtilisin was dissolved in water-miscible polar solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran, significant conformational changes occurred. When modified subtilisin was dissolved in water-immiscible organic solvents, such as isooctane and benzene, the solvent did not induce significant conformational changes. The catalytic activity in the transesterification reaction of the N-acetyl-L-phenylalanine ethylester of the modified subtilisin in organic solvents was higher than that of native subtilisin. The high activity of modified subtilisin was thought to be due to a homogeneous reaction by the dissolved enzymes.