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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.88, #1-3)

α-chymotrypsin superactivity in cetyltrialkylammonium bromide-rich media by Francesco Alfani; Maria Cantarella; Nicoletta Spreti; Raimondo Germani; Gianfranco Savelli (pp. 1-15).

α-Chymotrypsin (α-CT) activity was tested with N-glutaryl-l-phenylalanine p-nitroanilide in buffered media with added cationic surfactants. The effect of the commercial cetyltrimethylammonium bromide (CTABr) was compared with that of three other surfactants with ethyl (CTEABr), propyl (CTPABr), and butyl (CTBABr) head groups. These were synthesized and purified in this laboratory. Surfactant head groups provided distinct environments that largely modulated the catalytic performance. Larger alkyl head group hydrophobicity led to a marked enhancement of α-CT activity. CTBABr-rich media induced the highest superactivity.Kinetic measurements were performed in Tris-HCl buffer at a surfactant concentration either below or above CMC, and α-CT superactivity occurred in both media. Positive interactions between the enzyme and surfactants happened independently of thesupramolecular organization of the medium. The reaction followed the Michaelis-Menten kinetics. The substrate to micelle aggregates binding constant was used to calculate the substrate concentration available for catalysis. The k _cat to k _m ratio was in CTBABr-rich media always higher than in pure buffer and depended on the surfactant concentration. α-CT superactivity depended on the pH value of buffer solution. Enzyme inactivation followed a single-step mechanism in pure buffer and a series mechanism in the presence of a surfactant. The rate of activity decay obeyed a first-order kinetics.

Keywords: α-Chymotrypsin; superactivity; stability; cationic surfactants; micellar aggregates

Enzymatic preparation of optically active silicon-containing amino acids and their application by Takuo Kawamoto; Hayato Yamanaka; Atsuo Tanaka (pp. 17-22).

Optically active 3-trimethyl silylalanine (TMS-Ala) was prepared by hydrolysis of N-acetyl-dl-TMS-Ala catalyzed by acylase I (aminoacylase; N-acylamino-acid amidohydrolase, EC3.5.1.14). Acylase I from porcine kidney (PKA) was found to be more effective than that from Aspergillus melleus in the preparation of l-TMS-Ala. Under the optimized conditions, optically pure l-TMS-Ala (>99% enantiomeric excess, ee) was obtained with a 72% yield. Furthermore, a highly optically pure d-TMS-Ala (96% ee) could also be obtained with a 76% yield by chemical hydrolysis of the residual substrate. Enzymatic synthesis of peptides containing TMS-Ala was also attempted in ethyl acetate. Benzyloxycarbonyl (Z)-l-TMS-Ala served as the substrate for thermolysin, whereas l-TMS-Ala-OMe was inactive as the amino component. In the case of inhibitory activity of dipeptides toward thermolysin, l-Leu-(l-TMS-Ala) was found to be a more potent inhibitor than l-Leu-l-Leu, which is known to be one of the most effective inhibitors of thermolysin among the dipeptides consisting of natural aminoacids.

Keywords: Enzymatic preparation; silicon-containing amino acid; trimethylsilylalanine; acylase I; dipeptide; thermolysin; inhibitor

Expression and characterization of a recombinant Fusarium spp. galactose oxidase by Feng Xu; Elizabeth J. Golightly; Palle Schneider; Randy M. Berka; Kimberly M. Brown; James A. Johnstone; Denise H. Baker; Claus C. Fuglsang; Stephen H. Brown; Allan Svendsen; Alan V. Klotz (pp. 23-32).

The Fusarium spp. (Dactylium dendroides) galactose oxidase was expressed in Aspergillus oryzae and Fusarium venenatum hosts. Under the control of an A. niger α-amylase or a Fusarium trypsin promoter, high level galactose oxidase expression was achieved. The recombinant oxidase expressed in the A. oryzae host was purified and characterized. The purified enzyme had a molecular weight of 66 k Da on sodium dodecyl sulfate-polymerase gel electrophoresis (SDS-PAGE) and 0.4 mol copper atom per mole protein. The stoichiometry increased to 1.2 after a Cu saturation. Based on a peroxidase-coupled assay, the enzyme preparation showed an activity of 440 turnover per second toward d-galactose (0.1 M) at pH7 and 20°C. The enzyme had an optimal temperature of 60°C at pH 6.0 and an activation free Gibbs energy of 33 kJ/mol. A series of d-galactose derivatives was tested as the reducing substrate for the oxidase. The difference in activity was interpreted by the stereospecificity of the oxidase toward the substituents in the pyranose substrate, particularly on the C5 and the cyclic hemiacetal O sites. The recombinan toxidase could act on some galactose-containing polysaccharides, such as guar gum, but was not able to oxidize several common redox compounds that lacked a primary alcohol functional group.

Keywords: Galactose oxidase; expression; specificity; Fusarium

Determination of arachidonic acid based on the prostaglandin H synthase catalyzed reaction by Anna D. Ilyina; José L. Martínez Hernández; César Estrada Badillo; María G. Sena Maldonado; Sara Carrillo Galindo; María Hernández González; Jesús Rodríguez Martínez (pp. 33-44).

This article presents a novel method of arachidonic acid (AA) determination based on the reaction catalyzed by prostaglandin H synthase (PGHs). The deoxygenated and nondeoxygenated (as control) buffers were used to obtain the PGHs preparations from bovine vesicular glands by two different methods. The higher specific activity was observed for solubilized preparations obtained by ultracentrifugation and deoxygenated buffers. The preparations obtained by Ca²⁺ treatment demonstrated higher stability of PGHs during its storage at −15°C. To record the initial rate of AA transformation, a spectrophotometric assay of PGHs cyclo-oxygenase and peroxidase activities was developed using adrenaline and ABTS as electron donors. No oxidation of A BTS was observed in the reaction of AA transformation catalyzed by the PGHs from bovinevesicular glands. However, this electron donor was successfully used in the reaction catalyzed by PGHs from sheep vesicular glands. No chemilum inescence was recorded in the reaction of AA transformation catalyzed by PGHs from bovine vesicular glands in the presence of luminol. The chemiluminescent intensity was measured after addition of hydrogen peroxide allowing quantitative assay of AA to be performed.

Keywords: Prostaglandin H synthase; arachidonic acid; adrenaline; A BTS; luminol

Water quality monitoring using an enhanced chemiluminescent assay based on peroxidase-catalyzed peroxidation of luminol by Anna D. Ilyina; José L. Martínez Hernández; Blanca H. López Luján; Juan E. Mauricio Benavides; Jorge Romero García; Jesús Rodríguez Martínez (pp. 45-58).

Enhanced chemiluminescence (ECL) describes the phenomenon of the light output increase in the reaction of oxidation of luminol catalyzed by horseradish peroxidase (HRP) in the presence of certain phenolic compounds. This work summarizes the effects of preincubation of certain substances with HRP on the chemiluminescent reaction intensity. Preincubation of herbicide, detergent, surfactants (Brij-96 and Tween-20), phenol, metal ions (mercury, cobalt, and nickel), and bactericide with HRP had an inhibitory effect on the enzyme activity. HRP-preincubation with metal ions (cadmium, magnesium, and zinc), as well as with some insecticides, stimulated the chemiluminescent intensity. Calibration graphs were obtained to demonstrate the possibility to determine the pollutant concentration. Light emission from the peroxidase catalyzed enhanced chemilum inescence is affected by a wide number of chemicals and, therefore, the method can beused for on-site monitoring of water quality. A rapid and simple assay to detect water contamination has been developed.

Keywords: Horseradish peroxidase; luminol; water pollutants

The role of biodegradation during bioventing of soil contaminated with jet fuel by G. Malina; J. T. C. Grotenhuis (pp. 59-76).

The enhancing rem oval of kerosene (jet fuel) from contaminated soil during bioventing resulting from biodegradation was compared to the physical removal by evaporation only on bench-scale columns at the controlled temperature of 20°C (±2.5°C). Carbon dioxide-free air and nitrogen were used as flushing gases, at the constant continuous flow rate of 1 dm³/h. Kerosene concentrations in soil up to 35000 mg/kg were not toxic for indigenous microbial population. Much slower kerosene biodegradation rates observed for soil from a contaminated site, as compared to soil artificialy contaminated with kerosene, were the result of a lower bioavailability of “aged” kerosene, and the presence of compounds that might be persistent or toxic to kerosene-specific degraders. The inhibitory effect of toluene to indigenous microorganisms was found at above 75% of the toluene saturation concentrations in the gas phase.After 29 d, the overall bioventing efficiency was 17–23%, depending on whether CO₂ production or O₂ uptake wasused for caculations, as compared to the removal of 10% when biodegradation was excluded. The increase in efficiency by 50–100% owing to biodegradation would be more spectacular at lowr kerosene concentrations during the “tailing” phase, with diffusionlimited desorption, and much lower evaporation of less volatile constituents. Limitation of bioventing as a result of low bioavailability related to intraparticle sorption of residual contamination is discussed.

Keywords: Kerosene; Biodegradation; evaporation; bench-scale columns; unsaturated zone; efficiency; bioavailability; sorption

Structural determinants for the efficient and specific interaction of thioredoxin with 2-oxoacid dehydrogenase complexes by Guenter Raddatz; Volker Kruft; Victoria Bunik (pp. 77-96).

Specificity and efficiency of thiored oxin action upon the 2-oxoacid dehydrogenase complexes are studied by using a number of thiored oxins and complexes. Bacterial and mammalian pyruvate and 2-oxoglutarate dehydrogenase systems display similar row of preference to thioredoxins that may result from thioredoxin binding to the homologous or common dihydrolipoamide dehydrogenase components of the complexes. The most sensitive tothioredoxin is the complex whose component exhibits the highest sequence similarity to eukaryotic thioredoxin reductase. Hence, thioredoxin binding to the complexes may be related to that in the thioredoxin reductase, a dihydrolipoamide dehydrogenase homolog. The highest potency of mitochondrial thioredoxin to affect the mitochondiral complexes is revealed. A 96–100% conservation of the mitochondrial thioredoxin structure is shown within the four known sequences and the N-terminus of the pigheart protein determined. Eleven thioredoxins tested biochemically are analyzed by multiple sequence alignment and homology modeling. Their effects correlate with the residues at the contact between the α 3/3₁₀ and α 1 helices, the length of the α 1 helix and charges in the α2–β3 and β4–β5 linkers. Polarization of the thioredoxin molecule and its active site surroundings are characterized. Thioredoxins with a highly polarized surface around the essential disulfide bridge (mitochondiral, pea f, and Arabidopsis thaliana h3) show low cross-reactivity as compared to the species with a decreased polarization of this area (e.g., from Escherichia coli). The strongest polarization of the whole molecule results in the highest magnitude of the electrostatic dipole vector of mitochondrial thioredoxin. Thiored oxins with the dipole orientation similar to that of the latter have the affinities for the 2-oxoacid dehydrogenase complexes, proportional to the dipole magnitudes. Thioredoxin with an opposite dipole orientation shows no effect. Activating and inhibitory thioredoxin disulfides are distinguished by the charges of the residues 13/14 (α1 helix(, 51 (α2–β3 linker), and 83/85 (β4–β5 linker), changing the dipole direction. The results show that the thioredoxin-target interplay may be controlled by the long-range interactions between the electrostatic dipole vectors of the proteins and the degree of their interface polarization.

Keywords: 2-Oxoacid dehydrogenase complex; thioredoxin; protein-protein interaction; electrostatic dipole vector; structural homology

Enantioselective hydrolysis of a Schiff's base of D,L-Phenylalanine ethyl ester in water-poor media via the reaction catalyzed with α-chymotrypsin immobilized on hydrophilic macroporous gel support by Yury N. Belokon; Konstantin A. Kochetkov; Fatima M. Plieva; Nikolai S. Ikonnikov; Viktor I. Maleev; Virinder S. Parmar; Rajesh Kumar; Vladimir I. Lozinsky (pp. 97-106).

The application of immobilized α-chymotrypsin for the purpose of enantioselective hydrolysis of a Schiff's base of D,L-Phe-OEt (D,L-SBPH) in the mixed water-acetonitrile media with the different content of water is described. The immobilized biocatalyst was prepared by the chemical coupling of the enzyme to poly(vinyl alcohol) (PVA) cryogel—the macroporous hydrogel prepared by means of the freezing-thawing techniques. SBPH is water insoluble, and, therefore, acetonitrile (MeCN) with minor water additives was used as a solvent for the reaction of enantics elective hydrolysis of the racemic substrate. The process was conducted for 96–200 h, and L-Phe with the purity up to 98% e.e. precipitated in both the reaction medium and gel-carrier bulk. Theproduct wasrecovered by washing the organo-insoluble sediment with aqueous ammonia. D-Phe with the purity up to 85% e.e. was recovered from the organic solution of D-ester after its acidic hydrolysis. The PVA-cryogel-attached enzyme was effective in SBPH hydrolysis in MeCN/water mixtures. The immobilized biocatalyst was active for more than 1 mo of application and could be successfully used after another 4 mo storage at +10°C.

Keywords: Biocatalysis in MeCN water-poor media; immobilized α-chymotrypsin; macroporous poly(vinyl alcohol) cryogel carrier; L-Phe; D-Phe

Genetic transformation of immobilized competent cells by Elena Efremenko; Teimur Aliev; Anna Panina; Irina Badalian; Sergey Varfolomeyev (pp. 107-117).

This study describes the investigation of the possibility of genetic transformation of already immobilized competent cells by plasmids. The preliminary prepared competent cells were entrapped into granules of an insoluble carrier, a cryogel of poly(vinyl alcohol). The specific activity of organophosphate hydrolase and ampicillin resistance conferred by pOPf1 plasmid were used as markers of successful transformation of the immobilized competent cells. The effect of main experimental conditions of transformation usually used for free cells, i.e., time of incubation of cells with DNA solution, temperature, and time of heat shock, on the transformation efficiency of the immobilized competent cells has been studied. A num ber of important factors of preparation of immobilized transformed cells, i.e., the concentration of im mobilized competent cells inside the granules, the concentration of DNA solution used for transformation, have been shown to affect the OPH-activity of the final immobilized transformants. The possibility of transformation of the immobilized competent cells by both single- and double-stranded plasmid DNA molecules has been demonstrated.

Keywords: Immobilized cells; cryogel of poly(vinyl alcohol); genetic transformation; organ ophosphatehydrolase

A comparative study of different cellulase preparations in the enzymatic treatment of cotton fabrics by Alexander V. Gusakov; Alejandro G. Berlin; Nonna N. Popova; Oleg N. Okunev; Olga A. Sinitsyna; Arkady P. Sinitsyn (pp. 119-126).

Twenty-nine cellulase preparations from different sources were compared interms of their abrasive activities (the ability to remove Indigo from denim) and their ability tosaccharify cellulose. Nodirectrelationship could be found between these two abilities. The preparations were divided into three groups: (1) with a high yield of reducing sugars after 24 h hydrolysis of Avicel cellulose but low abrasive activity; (2) universal cellulases that could both effectively hydrolyze cellulose and remove Indigo from denim; and (3) cellulase samples with high abrasive activity but low saccharification ability.Cellobiohydrolase (CBH) and xylanase were purified from different fungi by chromatofocusing on a Mono P column and subjected to limited proteolysis with papain yielding cellulose-binding and core (catalytic) domains. The adsorption ability and backstaining index of both CBH and xylanase core proteins were notably lower than the respective parameters for the in itial nondigested enzymes indicating that protein adsorption on the surface of cotton fibers is a crucial factor causing Indigo backstaining during the enzymatic denim washing procedure.

Keywords: Cellulase; limited proteolysis; adsorption; textile; denim fabrics; stone washing; Indigo; backstaining

Recombinant firefly luciferase in Escherichia coli by Irina Lundovskikh; Ekaterina Dementieva; Natalya Ugarova (pp. 127-136).

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.

Keywords: Recombinant firefly luciferase; bioluminescence; immobilization; ATP assay

Prolonged plasma clot lysis induced by acyl-derivatives of urokinase in vitro by Roza B. Aisina; Liliya I. Moukhametova; Ekaterina F. Firsova; Sergei D. Varfolomeyev (pp. 137-143).

Two acyl-derivatives of urokinase, p-trimethylam inocinnamoyl-urokinase (TMAC-Uk) and p-guanidinobenzoyl-urokinase (GB-Uk), reactivating with different rates (k _reac were 6×10⁻⁴/s and 6×10⁻⁵/s, respectively) were prepared. In comparison with free urokinase, acyl-activators were more stable in human plasma, and their stability increased with the decrease in the reactivation rate. Plasma clotlysis induced by all three agents was time- and dose-dependent, but acyl-activators caused a more prolonged fibrinolysis and lengthened lag-phase than free urokinase. Slowly reactivating GB-Uk induced the most long-lasting clotlysis, whereas free urokinase was more effective for the first 3 h. A combination of GB-Uk with low dose urokinase promoted the long-lasting clotlysis with the shortened lag-phase.

Keywords: Urokinase; acyl-urokinase; plasma stability; fibrinolysis kinetics; combined fibrinolysis

Immobilized enzymes and cells in poly(N-vinyl caprolactam)-based hydrogels by Elena A. Markvicheva; Svetlana V. Kuptsova; Tatyana Yu. Mareeva; Alexander A. Vikhrov; Tamara N. Dugina; Svetlana M. Strukova; Yury N. Belokon; Konstantin A. Kochetkov; Ekaterine N. Baranova; Vitali P. Zubov; Denis Poncelet; Virinder S. Parmar; Rajesh Kumar; Lev D. Rumsh (pp. 145-157).

A one-step mild method for entrapping animal cells and enzymes in macroporous composite poly (N-vinyl caprolactam)-calcium alginate (PVCL-CaAlg) hydrogels is described. Some properties of immobilized enzymes, such as thermal and storage stabilities and stability in water/organic media were investigated. Composite PVCL-CaAlg gels were successfully applied to immobilize a number of proteases, namely, trypsin, α-chymotrypsin, carboxypeptidase B, and thrombin. Thermal stability of the immobilized preparations obtained by entrapment in hydrogel beads allowed us to use them at 65–80†C, while the native enzymes were completely inactivated at 50–55°C. Various applications of enzymes and cells immobilized in beads weredemonstrated. Immobilized trypsin and carboxypeptidase B were applied to prepare human insulin from recombinant proinsulin. The hydrogel beads with entrapped α-chymotrypsin were used in enantioselective hydrolysis of Shiff's base of D,L-phenylalanine ethyl ester (SBPH) in acetonitrile/water medium. Thrombin immobilized in PVCL-based hydrogel films was shown to be a promising compound for wound treatment. To prepare pure preparations of monoclonal antibodies (MAb) several hybridoma cell lines, including hybridoma cell lines producing MAb to interleukin-2, were successfully cultivated in the hydrogel beads.

Keywords: Poly (N-vinyl caprolactam; composite macroporous hydrogel; gel-entrapped proteases; biocatalysis in water-organic media; enantioselective hydrolysis; thrombin; wound healing; immobilized animal cells

Enteropeptidase by Anna G. Mikhailova; Lev D. Rumsh (pp. 159-174).

A preparative method for purification of enteropeptidase (enterokinase) (EC 3. 4. 21.9) is developed. A highly purified form of this enzyme is stabilized by calcium ions and does not contain any other proteolytic enzyme contaminations. These enteropeptidase preparations were successfully used for cleavage of a variety of fusion proteins containing the tetraaspartyl-lysyl sequence in an arbitrary position on the polypeptide chain. A series of substrates was methodically studied, which resulted in the suggestion that the peptide and fusion protein substrates (K m-200μM and 125μM, respectively) were bound to the enzyme through the linker (Asp)₄ Lys at the binding site on the light chain of enteropeptidase. Much more efficient hydrolysis of the natural substrate trypsinogen (K _m=2.4μM) testifies to a significant contribution of other sites of the substrate and the enzyme in productive binding Avariation in the enzyme's uniquespecificity wasshown to be a result of the autolysis caused by the loss of calcium ions; the cleavage sites were determined. The truncated enzyme containing the C-terminal fragment 466–800 of its heavy chain and the intact light chain does not distinguish the natural substrate trypsinogen, fusion protein, or peptide substrates. These results suggest that the N-terminal fragment 118–465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.

Keywords: Enteropeptidase (enterokinase); fusion (recombinant) proteins; autolysis; specificity; purification; affinity chromatography; inhibitors

Preparation of proteolytic enzymes from kamchatka crab Paralithodes camchatica hepatopancreas and their application by G. N. Rudenskaya; V. A. Isaev; A. M. Shmoylov; M. A. Karabasova; S. V. Shvets; A. I. Miroshnikov; A. B. Brusov (pp. 175-183).

A method was elaborated to obtain “Moricrase”, a complex of collageno and fibrinolytic proteinases from Paralithodes camchatica hepatopancreas. Moricrol, a Moricrase-containing ointment, was shown to exhibit wound-healing activity in the treatment of burns and trophic ulceration as well as postoperative scars. The following homogeneous proteinases were isolated from Moricrase: collagenolytic serine proteinase, trypsin, metalloproteinase, carboxypeptidase, and aminopeptidase. An efficient collagenolytic activity of Moricrase is a result of cooperative action of these enzymes.

Keywords: Collagenolytic complex of proteases; king crab Paralithodes camchatica ; fibrinolytic properties; enzymatic débridement; purulent wounds; scarry tissue

Mechanism of mercurial perturbation in proteins by Anatolii E. Myshkin; Vera S. Konyaeva (pp. 185-194).

Kinetics of the S-mercurized horse oxyhemoglobin coagulation has been turbidimetrically studied in the presence of ligands with different affinity for the mercury(II) atom. Coordination of the mercaptide mercury with ligands results in a marked inhibition of the coagulation process, which supports the previously suggested mechanism according to which the mercaptide mercury affects the protein conformation via its additional coordination with one of the neighboring mercury-binding sites, supposedly carboxylate group of an aspartic acid residue. A new insight into the mechanism of the currently practiced mercury-intoxication therapy is discussed.

Keywords: Hemoglobin; oxyhemoglobin; mercury; aggregation; coagulation; higher oligomers

The influence of serum fatty acid binding proteins on arachidonic acid uptake by macrophages by Mikhail L. Strokin; Marina G. Sergeeva; Alevtina T. Mevkh (pp. 195-200).

The role of serum fatty acid binding proteins (FABPs) in arachidonic acid (AA) uptake by murine peritoneal macrophages has been studied. The kinetics of [³H]arachidonic acid uptake by the cells was investigated over a wide range of AA concentration (10⁻¹⁰–10⁻⁵ M). It was shown that these putative fatty acid transporters dramatically change the uptake processes. In the presence of FABPs, the time-course curves of AA uptake exhibited two distinct periods: one with a rapid AA uptake during the first hour with an equilibrium in 1–2.5 h and another with an equilibrium reached in 20 h, whereas in the absence of FABPs the uptake curves were smooth without kinks and with the equilibrium reached in 10 h. In addition, it was shown that the amount of incorporated AA was linearly dependent on the concentration of AA over the range of 10⁻¹⁰–10⁻⁶ M in the presence of serum FABPs and 10⁻¹⁰–10⁻⁷ M in their absence.We assume that the changes in the character of AA uptake by macrophages in the presence of FABP soccur due to the interaction of FABPs with the cell plasma membrane.

Keywords: Archidonic acid; fatty acid metabolism; fatty acid binding proteins; macrophages

Electrochemical and ESR studies of au-protein from Micrococcus luteus by Lidiya A. Levchenko; Svetlana I. Kulakovskaya; Alexander V. Kulikov; Anatolii P. Sadkov; Alexander E. Shilov (pp. 201-210).

Au-protein from Micrococcus luteus, with and without Au inactive center, and chloroauric acid (HAu ^IIICl₄·4H₂O) with the addition of rutin, catechol, and riboflavin have been studied by means of electrochemistry and ESR. The redox potentials for Au-protein, as well as for the complexes Au-rutin and Au-catechol, have been measured, and ESR spectra of complexes Au-rutin and Au-catechol have been recorded. It has been shown that the Au atom binds to Au-protein via OH-groups of rutin. Flavin does not participatein gold binding. Au-protein is characterized by two peaks of cyclic voltammogram, −0.37 and −0.54 V. Au-protein with these potentials is able to function in the electron-transport chain of membranes between flavoproteins and quinones.

Keywords: Gold-accumulating bacteria; Au-protein; NADH-oxidase; cyclic voltammogram; ESR

A new mechanism of acyl group transfer in the reaction catalyzed by D-glyceraldehyde-3-phosphate dehydrogenase by Elena V. Schmalhausen; Vladimir I. Muronetz; Natalya K. Nagradova (pp. 211-220).

D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) catalyzes the oxidative phosphorylation of its substrate in a two-step reaction. Asa result of the first, oxidativestep, the covalent intermediate where in 3-phosphoglyceroyl moiety is bound to Cys149 of the active center is subjected to nucleophilic attack by inorganic phosphate, but remains resistant to hydrolytic decomposition. This ensures tight coupling of oxidation with phosphorylation in glycolysis. In this article, we present the experimental evidence for the conversion of GAPDH into a form capable of performing the reaction in the absence of inorganic phosphate. The structural basis for this conversion is the oxidation of a cysteine residue (probably Cys 153) into a sulfenic acid derivative under mild conditions to affect the integrity of the essential Cys 149. As a result, an intram olecular transfer of 3-phosphoglyceroyl group from the active center Cys 149 to Cys 153 becomes possible with subsequent hydrolysis of the sulfenyl carboxylate intermediate.

Keywords: D-glyceraldehyde-3-phosphate dehydrogenase; catalytic mechanism; acyltransfer; phosphorolysis; hydrolysis; sulfenic acid; hydrogen peroxide

Methodological approach to development of enzymatic technologies for semisynthetic betalactam antibiotic production by Pauline S. Nys; Valentina B. Kurochkina (pp. 221-229).

The algorithm for the research and design of optimized processes for synthesis of semisynthetic betalactam antibiotics with the use of peptidases with mechanism of action based on the acylenzyme interm ediate formation as a catalyst was formulated.The applicability of the proposed approach to the development of the processes for enzymatic synthesis of cefoxitin and cefazolin, semisynthetic cephalosporins, as an example, was demonstrated.

Keywords: Enzymatic synthesis; direct synthesis; acyltransfer synthesis; betalactams; cephalosporins; cefoxitin; cefazolin

Nitrile hydratase of Rhodococcus by Tatyana E. Leonova (Pogorelova); Olga B. Astaurova; Ludmila E. Ryabchenko; Alexander S. Yanenko (pp. 231-241).

Strain Rhodococcus rhodochrous M8 exhibiting high nitrile hydratase (NHase) activity is a promising catalyst for commercial production of acrylamide. With the aim to enhance the biocatalyst activity, regulation of NHase synthesis in this strain has been investigated. The synthesis of NHase and amidase, which also participated in nitriles metabolism in this strain, was induced by isobutyronitrile, acetamide, or urea. At the same time, the addition of glucose, fructose (rapidly metabolizable carbon sources), or ammonium ions to a medium decreased NHase and amidase synthesis. Using Northern blot analysis we demonstrated that these compounds control the expression of NHase at the transcriptional level. The observed effects of glucose and fructose are not related to the decrease in the intracellular concentration of inducer (acetamide), since these compounds cause neither repression nor inhibition of an inducible active system of acetamide uptake The synthesis of NHase in R. rhodochrous M8 is proposed to be subjected to different regulatory circuits, i.e., in duction, carbon, and nitrogen catabolite regulation, operating at the transcription level. We have optimized the growth conditions for R. rhodochrous M8 on the basis of the obtained data. Application of the improved biocatalyst for acrylamide production allows us to obtain the concentrated (38%) solution of acrylamide.

Keywords: Rhodococcus rhodochrous ; nitrile hydratase; induction; catabolite repression; nitrogen regulation; acrylamide production

Enzyme activation and inactivation induced by low doses of irradiation by Marina A. Orlova; Olga A. Kost; Vladimir A. Gribkov; Irina G. Gazaryan; Aleksandr V. Dubrovsky; Valeriy A. Egorov; Nina N. Troshina (pp. 243-255).

Activation phenomenon has been observed with two sets of enzymes under the conditions of low dosage irradiation. Activation was registered for angiotensin-converting enzyme under in vitro γ-irradiation (0.662 MeV, pulse duration approx 10s) at dose levels of 1–3 Gy and under X-ray irradiation (approx 9 keV, pulseduration approx 10⁻⁹s) at dose levels of 2×10⁻⁵ Gy. An activation effect has also occurred for native and recombinant horseradish peroxidase and tobacco peroxidase under γ-irradiation. The phenomenon observed is rationalized in terms of a kinetic model suggesting the existence of at least one activated enzyme conformation induced by radiolysis. The activity oscillations registered in dense plasma focus experiments were rationalized using the same model with the corresponding kinetic equation converted into the form describing the decaying oscillations caused by exciting force. The model analysis is presented.

Keywords: Radiation activation; angiotensin-converting enzyme; peroxidase; dense plasma focus; γ-irradiation; kinetic model; activity oscillation; periodic process

Preparation of γ-aminobutyric acid using E. coli cells with high activity of glutamate decarboxylase by A. Yu. Plokhov; M. M. Gusyatiner; T. A. Yampolskaya; V. E. Kaluzhsky; B. S. Sukhareva; A. A. Schulga (pp. 257-265).

γ-Aminobutyric acid (GABA) is a major inhibitory neurotransmitter synthesized in the central nervous system from glutamate by glutamate decarboxylase (GAD). It has applications in the production of many drugs. The technology of GABA synthesis by treating L-glutamic acid with the cells of the gene-engineered GAD superproducer strain of Escherichia coli GAD K10 was developed. Cell growing in the presence of 0.02 mM pyridoxal phosphate (PLP) causes the 2- to 2.5-fold increase of total productivity of the cells. The best way to prepare the cells for the reaction was their thermal activation by pretreatment for 1 h at 53°C. The optimal conditions for this reaction were 37°C and pH 4.6. The rate of the enzymatic reaction is the function of acetate concentration with the maximum at 0.5 M acetate. The total amount of GABA synthesized using 1 g of wet cells reached 23–25 g. The final concentration of GABA in the reaction medium was 280–300 g/L. The yield of the product was about 99%.

Keywords: γ-Am inobutyric acid; glutamate decarboxylase; Escherichia coli ; pyridoxal 5-phosphate

Anticancer enzyme L-lysine α-oxidase by Helena M. Treshalina; Elena V. Lukasheva; Ludmila A. Sedakova; Galina A. Firsova; Galina K. Guerassimova; Natalia V. Gogichaeva; Temir T. Berezov (pp. 267-273).

Fungal L-lysine α-oxidase (1.4.3.14) (LO) from Trichodermaharzianum Rifai presents an oxidoreductase with a firmly attached coenzyme—FAD. This stable enzyme catalyzes an oxidative deamination of L-lysine yielding hydrogen peroxide, ammonia, and α-ketoacid. LO exhibits antitumor activity toward 5 of 12 tested transplantable tumors. The sensitive tumors were ascitichepatoma 22 (T/C=201%, CR=66%); mammary adenocarcinoma Ca-755 (TGI=96%); melanoma B-16 (TGI=81%); AKATOL (TGI=75%); RSHM 5 (TGI=79%). LO therapeutic activity was observed within a wide range of doses, 35–350 U/kg, by intraperitoneal daily injections for 5 d.Contrary to Escherichia coli L-asparaginase, LO demonstrates its antitumor activity by the low therapeutic doses in vivo within a wide range of optimal doses and through an other antitumor spectrum. Fisher lymphadenosis L-5178y highly sensitive toward L-asparaginase appeared to be LO resistant.The possible mechanisms of LO antitumor activity through the key biochemical processes are discussed.

Keywords: L-lysine α-oxidase; L-amino acid oxidase; antitumor enzyme therapy; anticancer testing in vivo; schedule of L-lysine α-oxidase anticancer therapy

An attempt to create stable biomimetic sensors for express analysis of hydrogen peroxide in water solutions by Tofik M. Nagiev; Malahat T. Abbasova; Salim M. Baba-Zade; Sadig A. Kuliev; Elena B. Stepanova; Lala M. Agamamedova (pp. 275-284).

The physico-chemical properties of a new type catalase sensor, the so-called biomimetic sensors, modulating some of the catalase biosensor functions were investigated. These sensors have technological advantages over their biological analogs because of properties usually attributed to chemical sensors. The developed electrochemical system lies between bio- and chemical sensors.

Keywords: Hematin; biomimic; biomimetic electrode; catalase fragment; alumina; Pattex; hydrogen peroxide; electrochemical potential; autooscillation; diffusion

Antiinfluenza virus activity of a bacteriocin produced by Lactobacillus delbrueckii by Julia Serkedjieva; Svetla Danova; Iskra Ivanova (pp. 285-298).

A novel antibacterial substance produced by Lactobacillus delbrueckii has been isolated and characterized (1). The inhibitory agent corresponded to the criteria for bacteriocins. It was active against lactic acid bacteria (LAB) species and several food-borne pathogens. The cell-free supernatant was purified by HPLC gel-filtration. Three preparations at different purification steps were tested for activity on the reproduction of influenza virus A/chicken/Germany, strain Weybridge (H7N7) and strain Rostock (H7N1) in cell cultures of chicken embryo fibroblasts (CEF). The inhibitory effect was shown to be highly selective and specific. Expression of viral glycoproteins hemagglutinin, neuraminidase, and nucleoprotein on the surface of infected cells, virus-induced cytopathic effect, infectious virus yield, and hemagglutinin production were all reduced at nontoxic concentrations of the crude preparation (B1). B1 did not protect cells from infection, did not affect adsorption, and slightly inhibited viral penetration into infected cells. The purification did not enhance the cellular toxicity and increased about 870-fold the virus-inhibitory activity. No inactivating effect on extracellular virus was found.

Keywords: Bacteriocin; Lactobacillus delbrueckii ; influenza virus; viral inhibition

Enzymes of Opuntia ficus-indica (L.) Miller with potential industrial applications-I by Grimaneza Teixeira; Ana R. Santana; M. Salomé Pais; Alda Clemente (pp. 299-312).

We report on the screening of different enzymes such as arylamidases, lipases, proteinases, and glucosidases in plant extracts of the Cactaceae family, genus Opuntia, as well as on a newly purified plant proteases from O. ficus-indica fruit extracts. These proteinases showed the maximum activity at pH 5.2 and 55°C and FTC-casein was the best of the escreened substrates. Proteolytic activities were activated by anti-oxidant compounds and by some divalent cations. These proteinases were efficiently inhibited by cystein proteinase inhibitors and by 1,10-phenanth roline. The estimated M _t for the main proteolytic activity was about 23.2 kDa. The results on milk clotting characteristics suggest a potential use of the fruit cystein enzymes of this plant in dairy industries.

Keywords: Proteinases; proteinase inhibitor; arylamidases; lipases; glucosidases; collagenase; FITC; FTC-protein; milk clotting

Structural studies of penicillin acylase by James A. Brannigan; Guy G. Dodson; Sarah H. Done; Lorraine Hewitt; Colin E. McVey; Keith S. Wilson (pp. 313-319).

Penicillin acylases are used in the pharmaceutical industry for the preparation of antibiotics. The 3-D structure of Penicillin Gacylase from Escherichia coli has been solved. Here, we present structural data that pertain to the unanswered questions that arose from the original strucutre. Specificity for the amide portion of substrate was probed by the structure determination of a range of complexes with substitutions around the phenylacetyl ring of the ligand. Altered substrate specificity mutations derived from an in vivo positive selection process have also been studied, revealing the structural consequences of mutation at position B71.Protein processing has been analyzed by the construction of site-directed mutants, which affect this reaction with two distinct phenotypes. Mutations that allow processing but yield inactive protein provide the structure of an ES complex with a true substrate, with implications for the enzymatic mechanism and stereospecificity of the reaction. Mutations that preclude processing have allowed the structure of the precursor, which includes the 54a mino acid linker region normally removed from between the A and B chains, to be visualized.

Keywords: Penicillin acylase; 3-D structures; site-directed mutagenesis; substrate binding; autocatalytic processing; precursor

Direct and mediated electron transfer catalyzed by anionic tobacco peroxidase by Florentina-Daniela Munteanu; Lo Gorton; Annika Lindgren; Tautgirdas Ruzgas; Jenny Emnéus; Elisabeth Csöregi; Irina G. Gazaryan; Igor V. Ouporov; Elena A. Mareeva; L. Mark Lagrimini (pp. 321-333).

The properties of anionic tobacco peroxidase (TOP) adsorbed on graphite electrode have been studied in direct and mediated electron transfer in a wall-jet flow injection system. The percentage of tobacco peroxidase molecules active in directelectron transfer is about 83%, which is higher than that for horeradish peroxidase (40–50%). This observation is explained in terms of the lower degree of glycosylation of TOP compared with horseradish peroxidase and, therefore, a reduced in terference from the oligosaccharide chains with direct electron transfer. Calcium ions cause an 11% drop in the reaction rate constant toward hydrogen peroxide. The detection limit of calcium chloride has been estimated as 5 m M. The results obtained by means of bioeletrochemistry, stopped-flow kinetics, and structural modeling provide evidence for the interaction between calcium cations and negatively charged residues at the distal domain (Glu-141, heme propionates, Asp-79, Asp-80) blocking the activesite. The observation that both soluble and immobilized enzyme under go conformational changes resulting in the blockade of the active site indicates that the immobilized enzyme preserves conformational flexibility. An even stronger suppressing effect of calcium ions on the rate constant for mediated electron transfer was observed. In the case of direct electron transfer, this couldmean that there is nodirect contact between the electrode and the active site of TOP. The electrons are shuttled from the active site to the surface of the electrode through electron transfer pathways in the protein globule that are sensitive to protein conformational changes.

Keywords: Direct electron transfer; mediated electron transfer; orientation effect; conformational changes; model structure; tobacco peroxidase; biosensor; calcium effect; rate constant

Biosensors based on a light-addressable potentiometric sensor (LAPS) for analysis in both aqueous solutions and organic solvents by Elena G. Kondrat'eva; Tatiana A. Osipova; Vladimir I. Sklyar; Anatolyi N. Reshetilov (pp. 335-344).

Characteristics of a light-addressable potentiometric sensor (LAPS) based on silicon with Ta₂O₅ dielectric are reported. The pH sensitivity obtained is 45 mV/pH unit in the pH range from 4.01 to 7.5 (for measurements conducted in citrate buffer solutions) and 55 mV/pH unit over the pH range from 7.5 to 8.9 (for measurements conducted in Tris-HCl buffer solutions). The kinetic characteristics (K _m ³ V _m ⁵ pH-profile) of different enzymes, i.e., glucose oxidase, α-chymotrypsin, butyrylcholin esterase, and urease, have been measured under homogeneous conditions. The values of the Michaelis constant obtained are very close to analogous data described in the literature. Biosensors were prepared by immobilization of the enzymes on a pH-sensitive matrix. Two methods of immobilization were used: incorporation in to a hydrophilic matrix of bovine serum albumin (BSA) and incorporation into a hydrophobic matrix of modified polyethylenimine (PEl). It is demonstrated that LAPS can be used for recording the enzymatic reactions in organic media (mixture of the solvents).

Keywords: Light-addressable potentiometric sensor (LAPS); biosensors; immobilization; polymer matrix

Detection of major xylanase-containing cellulose-binding domain from Penicillium verruculosum by combination of chromatofocusing and limited proteolysis by Alejandro G. Berlin; Alexander V. Gusakov; Olga A. Sinitsyna; Arkady P. Sinitsyn (pp. 345-352).

Adsorption on microcrystalline cell ulose of enzyme components of cellulase complex from Penicillium verruculosum was studied by chromatofocusing on a Mono P column. The most strongly adsorbed and major component was identified as xylanase (XYN) with MW 65 k Da and pl 4.5. The high adsorption degree of XYN on cellulose indicated the possible presence of a cellulose-binding domain in the molecular sturcture. Limited proteolysis of XYN with papain was carried out. Kinetics of proteolysis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and measuring activities toward insoluble xylan and 4-methylumbelliferyl-β-d-lactoside (MUF-LAC). During the proteolysis, formation of two polypeptides with MW 51 and 14k Da was observed. No loss of activity toward thesolu blesubstrate was observed, wherease the activity toward xylan decreased rapidly. Adsorption distribution coefficient (K _d) of the core protein separated by gel-filtration was found to be 15 times lower than the K _d for the initial nondigested XYN (0.02 and 0.29 L/g, respectively). The activity of core protein toward insoluble xylan was close to zero, whereas the activity toward MUF-LAC was close to that exhibited by the original enzyme. The results presented indicate a bifunctional organization of XYN, where one domain acts as a binding anchor for insoluble substrates and the other, localized in the core protein, contains the active site.

Keywords: Penicillium verruculosum ; xylanase; cellulose-binding domain; limited proteolysis; papain; enzyme adsorption

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.88, #1-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.88, #1-3)