Alexander K. Gladilin

Enzyme-polyelectrolyte complexes in water-ethanol mixtures: Negatively charged groups artificially introduced into α-chymotrypsin provide additional activation and stabilization effects

Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

LysK, the enzyme lysing Staphylococcus aureus cells: specific kinetic features and approaches towards stabilization.

LysK, the enzyme lysing cells of Staphylococcus aureus, can be considered as perspective antimicrobial agent. Knowledge of LysK properties and behavior would allow optimizing conditions of its storage as well as formulating strategy towards its stabilization. Reaction of LysK with substrate (suspension of autoclaved Staphylococcus aureus cells) has been found to be adequately described by the two-stage Michaelis-Menten kinetic scheme. Ionization of the enzyme and enzyme-substrate complex is important for revealing catalytic activity, which is controlled by two ionogenic groups with pK 6.0 and 9.6. Ionization energy of the group with pK 6.0 is of 30 kJ/mol, thus, pointing out on His residue; pK 9.6 might be attributed to metal ion or metal-bound water molecule. At temperatures lower than 40 degrees C, LysK stability depends on its concentration, pH and presence of low molecular weight additives. Results of electrophoresis under native and denaturing conditions as well as sedimentation analysis strongly suggest that aggregation is behind LysK inactivation. Decrease in the enzyme concentration, as well as addition of low molecular mass polyols (glycerol, sorbitol, sucrose, trehalose) and Ca(2+) cations resulted in an enhanced (more than 100 times) stability of LysK. Dramatic stability decline observed in a narrow temperature range (40-42 degrees C) was accompanied by changes in LysK secondary structure as confirmed by CD spectroscopy studies. According to computer modeling data, Cys and His residues and metal cation might play a crucial role for LysK catalytic activity. Our data on the enzyme activity in the presence of ethylenediaminetetraacetic acid and different metal cations confirmed the importance of metal cation in LysK catalysis.

Dry enzyme-polymer complexes: Stable organosoluble biocatalysts for nonaqueous enzymology

SummaryComplexes of chymotrypsin and laccase with amphiphilic alkylated poly(ethyleneimine) (CEPEI) were obtained in a dry state by evaporation of nonaqueous solutions of the polymer containing solubilized enzymes. Enzyme-polymer complexes could be readily redissolved in organic solvent with full restoration of catalytic activity and showed remarkably high storage stability in the dry state.

Addition of Polybrene improves stability of organophosphate hydrolase immobilized in poly(vinyl alcohol) cryogel carrier

Organophosphate hydrolase, covalently attached to the beads of poly(vinyl alcohol) cryogel in the presence of Polybrene, was fivefold more stable in 15% (v/v) ethanol solution than the free enzyme. Immobilized biocatalyst, prepared with an addition of Polybrene, retained a half of its initial activity in 50% (v/v) aqueous ethanol solution, 90% of activity during 10 working cycles of Paraoxon hydrolysis and 85% of activity after storage in the 50 mM CHES buffer (pH 9.0) at room temperature for 2 months.

Enzyme-polyelectrolyte noncovalent complexes as catalysts for reactions in binary mixtures of polar organic solvents with water

SummaryThe formation of non-covalent complexes with polyelectrolytes has been suggested to enhance the resistance of enzymes towards inactivation by organic solvents in their homogeneous mixtures with water. Existence of such complexes in water-cosolvent media was proved by experiments with a fluorescence dye, eosin. In the case of catalysis by α-chymotrypsin, formation of the complex with polyelectrolytes produced two major eflects: i) considerable increase in enzyme activity at concentrations of ethanol and N,N-dimethylformamide of 10–30 % v/v; ii) conservation of the enzymatic activity at cosolvent concentrations of more than 40% v/v, where the native enzyme is completely inactive. General character of the observed activation and stabilization phenomena was shown by example of several experimental systems.

Formation of quasi-regular compact structure of poly(methacrylic acid) upon an interaction with α-chymotrypsin

Structure and dynamic properties of free poly(methacrylic acid) (PMA) and PMA complexed with alpha-chymotrypsin (CT) were studied using the time resolved fluorescence anisotropy technique. We have found that the interaction of PMA with CT induces the formation of a quasi-regular structure of PMA. At a CT/PMA weight ratio of 4:1 the interaction with CT leads to formation of approximately four equal segments of polyelectrolyte, each binding one CT molecule and characterized by an independent rotational mobility. Increase of the CT/PMA weight ratio above 8:1 gives rise to the overall rotation of the whole enzyme-polyelectrolyte complex. In water-ethanol mixtures the mobility of PMA segments containing CT decreases and the structure of the complex becomes even more rigid due to enhancement of the electrostatic interaction between CT and PMA. Formation of the compact and quasi-regular structure of the complex is perhaps the main reason behind the enhancement of enzyme stability and suppression of enzyme aggregation in water-organic cosolvent mixtures.

Activity and stability of native and modified subtilisins in various media.

The activity and stability of native subtilisin 72, its complex with poly(acrylic acid), and subtilisin covalently attached to poly(vinyl alcohol) cryogel were studied in aqueous and organic media by hydrolysis of specific chromogenic peptide substrates. Kinetic parameters of the hydrolysis of Glp-Ala-Ala-Leu-pNA by native subtilisin and its complex with poly(acrylic acid) were determined. Based on the comparative study of stability of native and modified subtilisins in media of various compositions, it was established that covalent immobilization of subtilisin on poly(vinyl alcohol) cryogel is the most effective approach to improve enzyme stability in water as well as in mixtures with low water content.

Kinetic behavior of phosphotriesterase and its non-covalent complexes with polyelectrolyte in systems with polar and non-polar organic solvents

Soluble phosphotriesterase from E. coli DH5α together with E. coli DH5α cells with the phosphotriesterase activity were co-immobilized into poly(vinyl alcohol) (PVA) cryogel and studied in water/organic systems with polar and non-polar organic solvents. The phosphotriesterase activity was competitively inhibited by polar organic solvents. The inhibition constant correlated with the dielectric constant (e) of the solvent. The rate of the enzyme-catalyzed reaction in biphasic non-polar solvent/water systems was independent of water/organic ratio and the hydrophobicity of the solvent. Formation of the non-covalent complexes with polyelectrolytes was suggested to enhance the resistance of the phosphotriesterase towards inactivation by organic solvents in their homogeneous mixtures with water.

Peptide synthesis in dimethylformamide-based organic media catalysed by non-covalent chymotrypsin-polyelectrolyte complexes

Chymotrypsin-catalysed kinetically-controlled peptide synthesis was studied in mixtures of dimethylformamide (DMF) and acetonitrile containing 6% water. The free enzyme showed no detectable activity with 60% or more DMF. However, by pre-forming a complex with poly-acrylate (optimum 50 carboxyl groups to 1 enzyme molecule), activity and stability were good at 60% DMF, and reasonable at 70% DMF. In 60% DMF, the peptide Ac-Tyr-Lys-NH2 could be made in 95% yield using this catalyst.

Polycation Stabilization of the Enzyme LysK Lysing Staphylococcus Aureus Cells

The influence of polycation (polybrene) on the activity and stability of LysK enzyme lysing Staphylococcus Aureus cells was studied and a tenfold increase in stability at 37°C was found. It was demonstrated that the stabilization effect is determined by the number of single-point Coulomb interactions of oppositely charged groups of the enzyme and polycation.