[Enzyme regeneration during hydrolysis of steam-pretreated willow and requirement for cellulase complex composition]. / Regeneratsiia fermenta pri gidrolize predobrabotannoi parovmy vzyvom drevesiny ivy i trebovaniia k sostavu tselliulaznogo kompleksa.

In order to reduce the total enzyme consumption in high-solids static hydrolysis of nonwashed steam-exploded willow Salix caprea by mixed cellulase of Trichoderma reesei + Aspergillus foetidus, two different approaches were proposed. In the first case, the enzyme activity adsorbed on residual solids after extended hydrolysis was used for hydrolysis of the newly added substrate. The initial mixing of fresh and hydrolyzed substrates was sufficient for the adsorbed enzyme redistribution and conversion of the new substrate portion, and permanent mechanical stirring was not required. Feeding of two additional portions of the exploded hardwood adjusted to pH 4 with dry caustic into the reactor with simultaneous replacement of accumulated sugars with fresh buffer (pH 4.5) resulted, on average, in a 90% conversion of cellulose at the final enzyme loading 8 IFPU per g ODM substrate, an average sugar concentration of 12%, and a glucose/xylose ratio of 5:1. In the second approach, weakly adsorbed cellulase fractions were used for static high-solids hydrolysis followed by their ultrafiltration recovery from the resultant sugar syrup. In contrast to the initial cellulase mixture whose residual activity in a syrup did not exceed 5-10% at the end of hydrolysis (48 h), up to 60% of weakly adsorbed enzyme fraction could be separated from sugar syrups by ultrafiltration and then reused. Weakly adsorbed enzymes displayed a hydrolysis efficiency of not less than 80% per IFPU enzyme consumed in extended hydrolysis of pretreated willow as compared to the original enzyme mixture. An electrophoretic study of the weakly adsorbed enzyme fraction identified T. reesei cellobiohydrolase II as the predominant component, whereas clear domination of T. reesei cellobiohydrolase I was found by electrophoresis of proteins tightly bound to hydrolysis residual solids.

[Enzymatic hydrolysis of willow treated with a steam burst without preliminary water extraction with a high concentration of substrate]. / Fermentativnyi gidroliz obrabotannoi parovym vzryvom drevesiny ivy bez predvaritel'noi vodnoi ékstraktsii pri vysokoi kontsentratsii substrata.

A laboratory reactor equipped with a screw press was used for hydrolysis of steam-SO2 exploded willow Salix caprea by a composition of Trichoderma reesei and Aspergillus foetidus enzyme preparations at high substrate concentrations. Optimal conditions providing the maximal volume of hydrolysis syrup with maximal sugar concentrations were determined. Two different hydrolysis procedures were developed in order to exclude initial washing of steam-pretreated plant raw material by large volumes of water, which is necessary to eliminate the inhibitory effect of explosion by-products on enzymatic hydrolysis. The first procedure included a one-hour-long enzymatic prehydrolysis of the substrate, then separation of sugar syrup containing 40-60 g/l of glucose, 20-25 g/l of xylose, and up to 10% of disaccharides, as well as up to 35% of the initial enzymatic activity, then addition of a diluted acetate buffer (pH 4.5), and subsequent hydrolysis of the substrate by the adsorbed enzymes leading to the final accumulation of up to 140 g/l glucose and up to 15 g/l xylose. In the second scenario, the exploded willow was initially adjusted by alkali to pH 4.5 and then hydrolyzed directly by added enzymes for 24 hours. This procedure resulted in a nearly total polysaccharide hydrolysis and accumulation of up to 170 g/l glucose and 20 g/l xylose. The reasons of inhibition of enzymatic hydrolysis are discussed.

A new approach to preparative enzymatic synthesis. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 9, Pages 1351-1361.

A new approach to preparative organic synthesis in aqueous-organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system "water-water-immiscible organic solvent." Thereby the enzyme is localized in the aqueous phase-this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations "water-water-miscible organic solvent," in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L-tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L-tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.

[Fluorescent immunoanalysis. Synthesis, spectro-fluorescent and immunologic properties of conjugates of coproporphyrin I with antibodies]. / Fluorestsentni immunoanaliz. Sintez, spektral'no-fluorestsentnye i immunologicheskie svoistva kon''iugatov koproporfirina I s antitelami

The synthesis of protein conjugates with the new high-efficient fluorescent labile coproporphyrin-I was optimized. A number of conjugates of monoclonal antibodies with different coproporphyrin-I content were synthesized, and their spectral properties were studied in water and micellar solutions, i.e. adsorption, excitation and emission spectra, fluorescence quantum yields, fluorescence pH-dependences. The binding constants of coproporphyrin-I and its protein conjugates with serum albumin were determined. The antibodies labelled with coproporphyrin-I retain the functional activity and photochemically stable in water solutions. The sensitivity of fluorometric detection of coproporphyrin-I and its conjugates with proteins is more than 10 times greater than in case of FITC.

[Beta-galactosidase from E. coli as a marker in immunoenzyme analysis]. / Beta-galaktozidaza E. coli kak marker v immunofermentnom analize.

In the present review the authors analyze factors influencing sensitivity of the enzyme immunoassay (EIA). beta-Galactosidase from E. coli was chosen as a marker enzyme. Physico-chemical properties of the enzyme, detection methods and various ways of obtaining chemical conjugates with antigens and antibodies are discussed. Some examples of using beta-galactosidase as a label in homogeneous and heterogeneous EIA are given which enables different compounds to be analyzed with a high sensitivity. New approaches employing gene engineering for constructing "fusions" between beta-galactosidase and antigens (alternative to chemical conjugates) are discussed that can be used in the near future.

Structure-stability relationship in proteins: fundamental tasks and strategy for the development of stabilized enzyme catalysts for biotechnology.

The problem of relationships between the protein structure and its stability comprises two major questions. First, how to elucidate the peculiarities of the protein structure responsible for its stability. Second, knowing the general molecular basis of protein stability, how to change the structure of a given protein in order to increase its stability. This review is an attempt to show the modern state of the first (fundamental) and the second (applied) aspects of the problem.

Kinetics of the inactivation of the protein-lipid complex, firefly luciferase, by sodium deoxycholate and its reactivation by phosphatidylcholine.

Firefly luciferase has been shown to be a protein-lipid complex. Phospholipids and neutral lipids bound to luciferase have been identified. Sodium deoxycholate rapidly inactivated the enzyme, but an excess of phosphatidylcholine recovered luciferase activity. From the kinetics of inactivation and reactivation, a mechanism for interaction of the enzyme with detergents and phospholipids has been proposed. The substrates ATP and Mg2+ stabilized luciferase during delipidation.

[Kinetic study of 3,4-dihydroxyphenyl-L-alanine synthesis in Citrobacter freundii cells immobilized in carrageenan]. / Kineticheskoe izuchenie sinteza 3,4-dioksifenil-L-alanina kletkami Citrobacter freundii, immobilizovannymi v karraginan.

A kinetic study was carried out of the enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine (DOPA) by the Citrobacter freundii 62 cells, possessing tyrosine-phenol-lyase (TPL) activity, immobilized in carrageenan, and optimum conditions of the reaction were found. The dependence of the TPL activity and its stability on the conditions of the DOPA synthesis was investigated. The TPL activity was higher and more stable in the immobilized cells as compared to free ones.

NAD-dependent hydrogenase from Alcaligenes eutrophus Z1: does it have a regulatory centre?

Evidence is presented for the existence of a relatively high-potential regulatory centre in the NAD-dependent hydrogenase from the hydrogen oxidizing bacterium Alcaligenes eutrophus Z1. Reduction of the hydrogenase to the redox potentials lower than -100 mV converts the enzyme into a catalytically active state that is remarkably stable to oxidants. Once activated, the enzyme does not loose its activity on intensive oxygenation for at least 3 hours. A novel hydrogenase ESR signal with a wide temperature optimum and a approximately -100 mV midpoint redox potential was detected. We suggest that the reduction of this redox centre trigger conformational changes in the inactive oxidized enzyme molecule, thus reorganizing the latter into the active one.