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.     

Catalysis by enzymes entrapped into reversed micelles of surfactants in organic solvents. Peroxidase in the aerosol OT-water-octane system

Spectral and catalytic parameters of peroxidase solubilized in the aerosol OT-water-octane system have been studied. The spectrum of peroxidase solubilized in octane with AOT reversed micelles, a degree of surfactant hydration being above 12, is actually identical to that of the enzyme aqueous solution. On the other hand, significant spectral changes have been detected when transferring the enzyme from water to the reversed micelle medium at low degrees of surfactant hydration, precisely [H2O]/[AOT] less than 12. The reversed micelle-entrapped peroxidase catalyses the oxidation of pyrogallol with hydrogen peroxide much more actively (at [H2O]/[surfactant] approximately 13) than that in aqueous solution. The entrapment of peroxidase into surfactant reversed micelles increases precisely the catalytic constant of the reaction, i.e. the virtual reactivity of the enzyme increases ten and hundred times depending on degrees of surfactant hydration and concentration. The systems of reversed micelles may be considered as models of biomembranes. Our findings hence show that enzymes in vivo can be much more catalytically active then it appears possible to reveal in conventional experiments in vitro in aqueous solutions.     

Kinetic analysis of deactivation of immobilized alpha-chymotrypsin by water-miscible organic solvent in kyotorphin synthesis.

Two different immobilized chymotrypsin derivatives were used to synthesize kyotorphin, using N-benzoyl-L-tyrosine ethyl ester and L-arginine ethyl ester as substrates, in water-DMF media. The first was adsorbed onto Celite particles and the second was multipoint covalently attached into polyacrylamide gel. In all cases, the conversion of the carboxyl substrate was carried out in first-order reaction conditions. For the adsorbed enzyme, the reaction kinetics deviated from first-order likely due to a fast irreversible inactivation of enzyme during the reaction time even at low DMF concentration (15-20% v/v). The covalent attachment of enzyme resulted in elimination of irreversible activity loss by organic solvent up to 60% (v/v) of DMF. The catalytic activity of the covalent derivative was conserved as appropriate for performing a synthetic reaction up to 60% v/v of DMF (in comparison to 30% v/v for the adsorbed derivative), showing a clear improvement in its stability against reversible denaturation by this solvent. The selectivity of the synthetic reaction was slightly enhanced (from 40-50%) with the increase in DMF concentration to 80% v/v, but it was significantly improved (to 80%) when L-argininamide was used as nucleophile.     

The principles of enzymes stabilization: VI. Catalysis by water-soluble enzymes entrapped into reversed micelles of surfactants in organic solvents

• 1.1.|The possibility of stabilizing water-soluble enzymes againsts the inactivating action of organic solvents by means of surfactants has been studied. Several enzymes (α-chymotrypsin (EC 3.4.21.1), trypsin (EC 3.4.21.4), pyrophosphatase (EC 3.6.1.1), peroxidase (EC 1.11.1.7), lactate dehydrogenase (EC 1.1.1.27) and pyruvate kinase (EC 2.7.1.40)) were used to demonstrate that enzymes can be entrapped into reversed micelles formed by surfactants (Aerosol OT, cetyltrimethylammonium bromide, Brij 56) in an organic solvent (benzene, chloroform, octane, cyclohexane). The enzymes solubilized in this way retain their catalytic activity and substrate specificity.
• 2.2.|A kinetic theory has been put forward that describes enzymatic reactions occurring in a micelle-solvent pseudobiphasic system. In terms of this theory, an explanation is given for the experimental dependence of the Michaelis-Menten equation parameters on the concentrations of the components of a medium (water, organic solvent, surfactant) and also on the combination of the signs of the charges in the substrate molecules and on interphase (++, +−, −−).
• 3.3.|The results obtained by us may prove important for applications of enzymes in organic synthesis and for studying the state and role of water in the structure of biomembranes and active centres of enzymes.

     

Enzymatic peptide synthesis in organic solvent mediated by gels of copolymerized acrylic derivatives of alpha-chymotrypsin and polyoxyethylene.

Copolymers of acrylated derivatives of alpha-chymotrypsin and polyethylene glycol (PEG) have been prepared and used as biocatalysts for the synthesis of model peptides in organic solvent containing a low quantity of water. Other peptide couplings have been tried to point out the chemico- and stereoselectivity and examples of segment couplings are given.     

Catalysis by enzymes entrapped into hydrated surfactant aggregates having lamellar or cylindrical (hexagonal) or ball-shaped (cubic) structure in organic solvents

Instead of aqueous solutions, universally recognized in enzymology, ternary systems of the water/organic solvent/surfactant type are suggested as liquid-crystalline media for enzymatic reactions. Two systems, water/octane/Aerosol OT and water/cyclohexane/Brij 96, have been used to solubilize acid and alkaline phosphatases and peroxidase. The enzymes under study do function in liquid-crystalline mesophases having lamellar, cylindrical (reversed hexagonal) and ball-shaped (reversed cubic) packing of the surfactant molecules. A significant result is that the phase transition from one liquid-crystalline structure to another entails, as a rule, a reversible change in the catalytic activity of the solubilized enzyme.     

Effect of organic solvent on biotransformation of benzaldehyde to benzyl alcohol by free and silicone-alginate entrapped cells

Summary Biotransformations of benzaldehyde to benzyl alcohol by whole cells of baker's yeast immobilised in a 50%:50% silicone-alginate mixed hydrophobic/hydrophilic matrix were successfully carried out in apolar solvents. In more polar solvents, the biotransformation was ineffective when either free or entrapped cells were used.     

Stabilization and activation of alpha-chymotrypsin in water-organic solvent systems by complex formation with oligoamines

Formation of enzyme-oligoamine complexes was suggested as an approach to obtain biocatalysts with enhanced resistance towards inactivation in water-organic media. Complex formation results in broadening (by 20-40% v/v ethanol) of the range of cosolvent concentrations where the enzyme retains its catalytic activity (stabilization effect). At moderate cosolvent concentrations (20-40% v/v) complex formation activates the enzyme (by 3-6 times). The magnitude of activation and stabilization effects increases with the number of possible electrostatic contacts between the protein surface and the molecules of oligoamines (OA). Circular dichroism spectra in the far-UV region show that complex formation stabilizes protein conformation and prevents aggregation in water-organic solvent mixtures. Two populations of the complexes with different thermodynamic stabilities were found in alpha-chymotrypsin (CT)-OA systems depending on the CT/OA ratio. The average dissociation constants and stoichiometries of both low- and high-affinity populations of the complexes were estimated. It appears that it is the low-affinity sites on the CT surface that are responsible for the activation effect.     

Doubly entrapped baker's yeast survives during the long-term stereoselective reduction of ethyl 3-oxobutanoate in an organic solvent

To attain long-term bioreaction in organic solvents with living microorganisms, we tried to protect the microorganisms from the toxicity of the solvent by immobilization. In this study, baker's yeast, which is not tolerant to organic solvents such as isooctane, was selected as a model microorganism and the immobilized living yeast cells were examined for activity in the steroselective reduction of ethyl 3-oxobutanoate to ethyl (S)-3-hydroxybutanoate in isooctane; an activity that correlated well with the viability of the yeast cells. It was found that double entrapment, that is, further entrapment of calcium-alginate-gel-entrapped cells with a urethane prepolymer, made it possible for the yeast to remain viable in isooctane, although other conventional immobilization methods, such as single entrapment using polysaccharide or synthetic resin prepolymers, were insufficient for its protection. Furthermore, doubly entrapped living yeast cells could carry out the stereoselective reduction in isooctane repeatedly for a long period (more than 1200 h) with occasional cultivation. Thus, double entrapment enabled a microorganism sensitive to organic solvents to survive over long-term bioreaction in an organic solvent. Received: 29 August 1997 / Received last revision: 24 December 1997 / Accepted: 13 January 1998     

Asymmetric bioreduction of keto esters by immobilized baker's yeast entrapped in calcium alginate beads in both water and organic/water solvent systems

Immobilized baker's yeast entrapped in calcium alginate beads, ca 1.5 mm diameter, was used more than 10 times in water and reduced ethyl 3-oxobutanoate and ethyl benzoylformate to the corresponding chiral hydroxy esters in good chemical yields and in high enantioselectivities. The biocatalyst was also successfully used in organic/water solvent systems such as hexane/water and acetonitrile/water, and in other systems, particularly in hexane, converted keto esters into their individual chiral hydroxy esters.     

Synthesis of N-protected peptide alcohols catalyzed by subtilisin or alpha-chymotrypsin in organic solvents

A series of N-protected peptide alcohols were synthesized using amino alcohols with unprotected hydroxy groups as amino components by the catalysis of subtilisin or alpha-chymotrypsin in organic solvents. N-protected aromatic amino acid esters were more suitable as acyl donors for subtilisin. The influences of different N-protecting groups, organic solvents, and content of water on synthesis of N-protected peptide alcohols were systematically studied.     

Direct electrochemical characterization of Vitreoscilla sp. hemoglobin entrapped in organic films

The redox properties of a prokaryotic, Vitreoscilla sp. hemoglobin (VHb) in fuzzy organic films are studied with electrochemistry. This VHb exhibits irreversible electrochemical response at bare pyrolytic graphite (PG) electrode surfaces. However, upon being entrapped in organic films, the heterogeneous electron transfer rate of VHb will be sufficiently high to produce a quasi-reversible electrochemical response. The observation of electrocatalysis (reduction of O2) by hemes suggests that the protein can retain its biological activity under these conditions.     

Cellular uptake of ribonucleic acids entrapped into liposomes.

Ribonucleic acids were entrapped into phospholipid vesicles (liposomes). After incubation of the liposomes containing RNA (L- RNA), the RNA was introduced into the cells. The kinetics of L- RNA uptake by the cells in culture were studied. The uptake of L- RNA is linear over a broad vesicle concentration range depending on temperature, and at 37 degrees C uptake levels reach a plateau after 3 hours. Inhibitors of cellular energy metabolism have little effect on the uptake, and thus fusion, as the main mechanism of uptake, is proposed.     

Effects of polyethylene glycol on stability of alpha-chymotrypsin in aqueous ethanol solvent

The effects of polyethylene glycol (PEG) of different molecular weights (400, 2000, 6000, 12,000, 20,000, and 35,000) on the conformational stability and catalytic activity of alpha-chymotrypsin in 60% ethanol were studied. The inactivation caused by the organic solvent was not influenced by PEG 400. However, the PEGs with higher molecular weights up to 35,000 increased the stability of the enzyme, but this alpha-chymotrypsin stabilizing effect was molecular weight-independent. With increase of the molecular weight of PEG, a more stable tertiary structure of the enzyme was observed.     

Peptide bond synthesis catalyzed by alpha-chymotrypsin.

alpha-Chymotrypsin [EC 3.4.21.1] catalyzed the syntheses of peptide bonds with various N-acylated amino acids or peptides having aromatic or hydrophobic amino acid residues at the C-terminal position as carboxyl components, and amino acid derivatives, peptides or their derivatives as amine components. A neutral pH was most efficient and quite high concentrations of alpha-chymotrypsin and starting materials were required for synthesis. Four amine components, hydrophobic or bulky amino acid residues were useful at the N-terminal position. Stereospecificity was also observed at the N-terminal position of amine components. Peptide synthesis was not usually seen when the products were soluble in the reaction mixture. This could be partly overcome by increasing the concentration of either the carboxyl or the amine component to more than ten times that of the other.     

Preparation of highly active alpha-chymotrypsin for catalysis in organic media

A simple one step process for the preparation of free alpha-chymotrypsin, using an organic solvent to precipitate the enzyme from a buffered solution, followed by washing with organic solvents, is described. This preparation gave 132 times greater esterification activity than lyophilized powder.     

Peptide synthesis in aqueous-organic solvent mixtures with alpha-chymotrypsin immobilized to tresyl chloride-activated agarose

alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.