Immobilization of tyrosinase for use in nonaqueous media: Enzyme deactivation phenomena

Summary We have investigated features for minimizing the inactivation of tyrosinase (E.C. 1.14.18.1) that is caused by immobilization on glass beads and Celite^®. The degree of inactivation is dependent on the enzyme loading and the carrier's surface area. Addition of a sacrificial protein during the immobilization procedure offers a protective effect with increased residual activity on the basis of comparable enzyme loading.     

Remarkable activation of enzymes in nonaqueous media by denaturing organic cosolvents

The rates of transesterification reactions catalyzed by the protease subtilisin Carlsberg suspended in various anhydrous solvents at 30 degrees C can be increased more than 100-fold by the addition of denaturing organic cosolvents (dimethyl sulfoxide or formamide); in water, the same cosolvents exert no enzyme activation. At 4 degrees C, the activation effect on the lyophilized protease is even higher, reaching 1000-fold. Marked enhancement of enzymatic activity in anhydrous solvents by formamide is also observed for two other enzymes, alpha-chymotrypsin and Rhizomucor miehei lipase, and is manifested in two transesterification reactions. In addition to lyophilized subtilisin, crosslinked crystals of subtilisin are also amenable to the dramatic activation by the denaturing cosolvents. In contrast, subtilisin solubilized in anhydrous media by covalent modification with poly(ethylene glycol) exhibits only modest activation. These observations are rationalized in terms of a mechanistic hypothesis based on an enhanced protein flexibility in anhydrous millieu brought about by the denaturing organic cosolvents. The latter exert their lubricating effect largely at the interfaces between enzyme molecules in a solid preparation, thus easing the flexibility constraints imposed by protein-protein contacts. (c) 1996 John Wiley & Sons, Inc.     

On the issue of interfacial activation of lipase in nonaqueous media

The question of whether lipases can be activated by adsorption onto an interface in organic solvents was addressed using Rhizomucor miehei lipase as a model. In aqueous solution, this enzyme was shown to undergo a marked interfacial activation. However, lipase (either lyophilized or precipitated from water with acetone) suspended in ethanol or 2-(2-ethoxyethoxy)ethanol containing triolein exhibited no jump in catalytic activity when the concentration of triolein exceeded its solubility in these solvents, thereby resulting in formation of an interface. To test whether the lack of interfacial activation was due to the insolubility of the enzyme in organic media, lipase was covalently modified with poly(ethylene glycol). The modified lipase, although soluble in nonaqueous media, was still unable to undergo interfacial activation, regardless of the hydrophobicity of the interface. This inability was found to be caused by the absence of adsorption of lipase onto interfaces in organic solvents, presumably because of the absence of the hydrophobic effect (the driving force of lipase adsorption onto hydrophobic interfaces in water) in such media. The uncovered lack of interfacial adsorption and activation suggests that the short alpha-helical "lid" covering the active center of the lipase remains predominantly closed in nonaqueous media, thus contributing to diminished enzymatic activity. (c) 1996 John Wiley & Sons, Inc.     

非水酶学和非水相生物催化研究进展

近年来工业生物技术飞速发展,酶学和生物催化领域也取得突破性进展,特别在酶在非水相中活性及稳定性研究,耐溶剂生物催化剂的筛选、构建、修饰和改造,生物相容性和环境相容性好的绿色介质等方面取得了较大的进展。最近的研究热点和未来几年的研究方向主要为:基于基因组信息的耐溶剂酶的虚拟筛选和构建;基于自然界筛选新酶基因的耐溶剂酶重构和改造;离子液体等环境友好的绿色介质系统等几个方面。     

Silicone-immobilized biocatalysts effective for bioconversions in nonaqueous media.

A hydrophobic silicone polymer could be effectively applied to immobilization of two kinds of biocatalysts operating in organic media. Horse liver alcohol dehydrogenase, which was solubilized in a small amount of water, or deposited on water-filled hydrophilic particles, was immobilized in this material. This configuration of the preparation involving finely dispersed aqueous phase permitted a simple packed-bed operation for the enzymatic oxidation of alcohol and reduction of aldehyde with a coupled-substrate NAD(H) recycling in n-hexane. Another example was the immobilization of Nocardia corallina which catalysed epoxidation of liquid alkenes such as 1-tetradecene, 1-octene, and styrene in the presence of n-hexadecane. In order to adjust the hydrophobicity-hydrophilicity balance of the support, it was effective to immobilize the cells in a mixed matrix composed of silicone polymer and Ca-alginate gel. The optimum composition of the mixed matrix, which yielded the highest productivity of epoxide, was 80-90% silicone + 20-10% alginate for the production of 1,2-epoxytetradecane, 40-50% silicone + 60-50% alginate for 1,2-epoxyoctane, and almost 0% silicone + 100% alginate for styrene oxide. This significant change of the optimum composition was primarily associated with the degree of substrate inhibition.     

Catalysis by amine oxidases in nonaqueous media

The synthetic potential of amine oxidases was examined in different reaction systems, ranging from aqueous solutions to organic solvents with low water content. Substantial conversion was achieved in biphasic systems, which eliminated the product inhibition observed in the aqueous system. The conversion was particularly high in the more hydrophobic solvents. The use of low water systems was studied using amine oxidase immobilized on celite and pre-equilibrated in a salt hydrate environment to reach a constant water activity. Addition of water in the solvent was shown to be unnecessary, with significant conversion being attained through the water supplied by pre-equilibration of the immobilized enzyme at a_w=0.55. The use of organic solvent-containing reaction systems thus presents a convenient method for oxidising poorly water-soluble amines using amine oxidases.     

Enzyme stabilization by covalent binding in nanoporous sol-gel glass for nonaqueous biocatalysis

A unique nanoporous sol-gel glass possessing a highly ordered porous structure (with a pore size of 153 A in diameter) was examined for use as a support material for enzyme immobilization. A model enzyme, alpha-chymotrypsin, was efficiently bound onto the glass via a bifunctional ligand, trimethoxysilylpropanal, with an active enzyme loading of 0.54 wt%. The glass-bound chymotrypsin exhibited greatly enhanced stability both in aqueous solution and organic solvents. The half-life of the glass-bound alpha-chymotrypsin was >1000-fold higher than that of the native enzyme, as measured either in aqueous buffer or anhydrous methanol. The enhanced stability in methanol, which excludes the possibility of enzyme autolysis, particularly reflected that the covalent binding provides effective protection against enzyme inactivation caused by structural denaturation. In addition, the activity of the immobilized alpha-chymotrypsin was also much higher than that of the native enzyme in various organic solvents. From these results, it appears that the glass-enzyme complex developed in the present work can be used as a high-performance biocatalyst for various chemical processing applications, particularly in organic media. Published by John Wiley & Sons     

Organosoluble enzyme-polymer complexes: A novel type of biocatalyst for nonaqueous media

Summary A novel type of organosoluble biocatalyst, representing a non-covalent complex of enzyme with a sugar-based amphiphilic polymer is described. The subtilisin Carlsbergpalmitoyl poly(sucrose acrylate) complex was found to be soluble and catalytically active in a number of organic solvents of different nature.     

Enzyme catalysis in the presence of nonaqueous solvents using chloroperoxidase

Studies exploring the effect of two nonaqueous solvents on enzyme activity were done using chloroperoxidase as a model system. Chloroperoxidase produced by Caldariomyces fumago is a bifunctional enzyme with halogenating activity at pH 3 and peroxidation activity at pH 5 to 6. Methanol affected both of these activities similarly. Furthermore, methanol and the halogen acceptor, monochlorodimedon, competitively inhibit the reaction. These results are discussed in terms of the site of action of methanol. At 10% methanol concentration, the enzyme retained up to 33% of its activity depending on the monochlorodimedon concentration. Dimethylsulfoxide at 10% concentration permitted up to 47% retention of activity. Its effects on the enzyme are more complex than methanol and are discussed in terms of a transitory inactivation of the enzyme.     

Polymerization of phenols catalyzed by peroxidase in nonaqueous media

Polymers produced by horseradish-peroxidase-catalyzed coupling of phenols have been explored as potential substitutes for phenol-formaldehyde resins. To overcome low substrate solubilities and product molecular weights in water, enzymatic polymerizations in aqueous-organic mixtures have been examined. Peroxidase vigorously polymerizes a number of phenols in mixtures of water with water-miscible solvents such as dioxane, acetone, di-methylformamide, and methyl formate with the solvent content up to 95%. As a result, various phenolic polymers with average molecular weights from 400 to 2.6 x 10(4) D were obtained depending on the reaction medium composition and the nature of the phenol. Peroxidase-catalyzed copolymerization of different phenols in 85% dioxane was demonstrated. Poly(p-phenylphenol) and poly(p-cresol) were enzymatically prepared on a gram scale. They had much higher melting points, and in addition, poly(p-phenylphenol) was found to have a much higher electrical conductivity than phenol-formaldehyde resins.     

Solid-phase peptide synthesis by ion-paired alpha-chymotrypsin in nonaqueous media

Solid-phase synthesis of dipeptides in low-water media was achieved using AOT ion-paired alpha-chymotrypsin solubilized in organic solvents. Multiple solvents and systematic variation of water activity, a(w), were used to examine the rate of coupling between N-alpha-benzyloxycarbonyl-L-phenylalanine methyl ester (Z-Phe-OMe) and leucine as a function of the reaction medium for both solid-phase and solution-phase reactions. In solution, the observed maximum reaction rate in a given solvent generally correlated with measures of hydrophobicity such as the log of the 1-octanol/water partitioning coefficient (log P) and the Hildebrand solubility parameter. The maximum rate for solution-phase synthesis (13 mmol/h g-enzyme) was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.30. For the synthesis of dipeptides from solid-phase leucine residues, the highest synthetic rates (0.14-1.3 mmol/h g-enzyme) were confined to solvent environments that fell inside abruptly defined regions of solvent parameter space (e.g., log P > 2.3 and normalized electron acceptance index <0.13). The maximum rate for solid-phase synthesis was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.14. In 90/10 and 70/30 (v/v) isooctane/tetrahydrofuran environments with a(w) set to 0.14, seven different N-protected dipeptides were synthesized on commercially available Tentagel support with yields of 74-98% in 24 h.     

Enzyme activation for organic solvents made easy

Enzymes are highly selective catalysts that perform intricate chemistries at ambient temperatures and pressures. Although water is the solvent of life, it is a poor solvent for most synthetic organic reactions and, therefore, most chemists avoid aqueous solutions for synthetic applications. However, when removed from the aqueous environment and placed in an organic solvent, enzyme activity is reduced greatly. Here, we present a general overview of recent efforts to activate enzymes for use in nonaqueous media, giving particular focus to the use of simple salts as additives that result in significant biocatalytic improvements.     

Preparation and enzymatic behavior of surfactant-enveloped enzymes for glycosynthesis in nonaqueous aprotic media

Surfactant-enveloped enzymes (SEEs) were prepared from pure cellulases, cellobiohydrolase I and endoglucanase I (Cel7A and Cel7B, respectively), via simply freeze-drying water-in-oil emulsions, wherein the aqueous phase containing each cellulase was stabilized with the nonionic surfactant, dioleyl-N-d-glucona-l-glutamate. The enzymatic tolerance of SEEs to various nonaqueous solvents was investigated, aiming at a novel synthetic approach in biocatalytic glycoengineering. SEE-Cel7A preserved ca. 67% of the original activity after 3 h incubation in lithium chloride (LiCl)/dimethylacetamide (DMAc) that is a good solvent for carbohydrates but completely deactivates intact enzymes. This excellent enzymatic durability depended on the preparation conditions of SEEs, e.g. pH and salt species of the aqueous phase during SEE preparation. SEE-Cel7A or SEE-Cel7B was applied as a biocatalyst to synthesize cellulose, a sugar polymer which is insoluble in common solvents but dissolves in LiCl/DMAc. Both SEEs could catalyze the direct dehydration of cellobiose without any activation of the anomeric carbon, a property that is indispensable for conventional chemo-enzymatic synthesis. The SEE-Cel7A provided short-chain cellulose with the degree of polymerization (DP) ca. 20, and longer-chain cellulose with DP ca. 60 was preferentially obtained by the SEE-Cel7B, possibly through preferential reverse hydrolysis instead of inherent hydrolysis. Nonaqueous SEE-mediated biocatalysis using inexpensive glycohydrolases and sugars that do not need to be chemically modified beforehand would have potentially wide applications in glycoengineering.     

Enzyme engineering for nonaqueous solvents. II. Additive effects of mutations on the stability and activity of subtilisin E in polar organic media.

Single amino acid substitutions increase the activity and stability of subtilisin E in mixtures of organic solvents and water, and the effects of these mutations are additive. A variant of subtilisin E that exhibits higher activity in mixtures of dimethylformamide (DMF) and water (Q103R) was created by random mutagenesis combined with screening for improved activity (K. Chen and F. H. Arnold, in preparation). Another mutation, N218S, known to improve both the activity and stability of subtilisin BPN', also improves the activity and stability of subtilisin E in the presence of DMF. The effects of the two substitutions on transition-state stabilization are additive. Furthermore, the Q103R mutation that improves activity has no deleterious effect on subtilisin stability. The double mutant Q103R+N218S is 10 times more active than the wild-type enzyme in 20% (v/v) DMF and twice as stable in 40% DMF. Although the effects of single mutations can be impressive, a practical strategy for engineering enzymes that function in nonaqueous solvents will most likely require multiple changes in the amino acid sequence. These results demonstrate the excellent potential for engineering nonaqueous-solvent-compatible enzymes.     

Peroxidase activity and stability of surfactant-heme complex in nonaqueous media

A surfactant-heme complex was prepared from hemin using a water-in-oil emulsion with a synthetic nonionic surfactant. The heme complex was soluble in anhydrous benzene with peroxidase activity for the oxidation of o-phenylene-diamine using tert-butyl hydroperoxide as an oxidant. An absorption spectrum of the heme complex in benzene was distinct from that of free heme in an aqueous buffer solution owing to the different aggregation states in the respec-tive solution. Moreover, the heme complex could not be decomposed in benzene even in excess of the hydroper-oxide due to enhanced stability.     

Obtaining higher transesterification rates with subtilisin Carlsberg in nonaqueous media

Three phase partitioning (protein precipitate obtained as an interfacial layer between lower aqueous and upper t-butanol phases, formed by the addition of ammonium sulphate and t-butanol to the aqueous solution of protein) followed by lyophilization in the presence of two-component excipient resulted in 400-480x increases in transesterification activity of lyophilized powders of subtilisin Carlsberg, depending on the solvent. The three phase partitioned enzyme, 'dried' by washing with butanol, gave 3-4x higher rates (depending on the solvent used) than the enzyme preparation dried by lyophilization in the presence of two-component excipient system.     

Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media

A protein solubilization method has been developed to directly solubilize protein clusters into organic solvents containing small quantities of surfactant and trace amounts of water. Termed "direct solubilization," this technique was shown to solubilize three distinct proteins - subtilisin Carlsberg, lipase B from Candida antarctica, and soybean peroxidase - with much greater efficiencies than extraction of the protein from aqueous solution into surfactant-containing organic solvents (referred to as extraction). More significant, however, was the dramatic increase in directly solubilized enzyme activity relative to extracted enzyme activity, particularly for subtilisin and lipase in polar organic solvents. For example, in THF the initial rate towards bergenin transesterification was ca. 70 times higher for directly solubilized subtilisin than for the extracted enzyme. Furthermore, unlike their extracted counterparts, the directly solubilized enzymes yielded high product conversions across a spectrum of non-polar and polar solvents. Structural characterization of the solubilized enzymes via light scattering and atomic force microscopy revealed soluble proteins consisting of active enzyme aggregates containing approximately 60 and 100 protein molecules, respectively, for subtilisin and lipase. Formation of such clusters appears to provide a microenvironment conducive to catalysis and, in polar organic solvents at least, may protect the enzyme from solvent-induced inactivation.     

A method for obtaining equilibrium tautomeric mixtures of reducing sugars via glycosylamines using nonaqueous media

Equilibrium tautomeric mixtures of several mono- and disaccharides are obtained in anhydrous form, without the use of water, by reacting the commercially available reducing sugars with ammonia gas in dry methanol, followed by the concentration of the resultant solution to dryness. Mutarotation and hydrolysis of the initially formed glycosylamine in the resultant medium account for the transformation. Equilibrium anomeric mixtures enriched in the beta-form of commercially available sugars such as alpha-D-glucose and alpha-lactose have not only vastly increased solubility, but are also synthetically valuable as these can be readily converted to the methyl/benzyl/trimethylsilyl ether and other derivatives for further transformations.