[Thermostabilities of plant phenol oxidase and peroxidase, determining the technology of their use in food industry].

Stabilities of phenol oxidase and peroxidase from tea plant (Camellia sinensis L.) clone Kolkhida leaves, apple (Malus domestica L.) cultivar Kekhura fruits, walnut (Juglans regia L.) green pericarp, and horseradish (Armoracia lapathifolia Gilib) roots were studied using different storage temperature modes and storage duration. It was demonstrated that both enzymes retained residual activities (approximately 10%) upon 20-min incubation at 80 degrees C. Phenol oxidases from tea, walnut, and, especially, apple, as well as tea peroxidase were stable during storage. A technology for treatment of plant oxidases was proposed, based on the use of a natural inhibitor phenol oxidase and peroxidase, isolated from tea leaves, which solving the problem of residual activities of these enzymes, arising during pasteurization and storage of beverages and juices. It was demonstrated that browning of apple juice during pasteurization and beer turbidity during storage could be efficiently prevented using the natural inhibitor of these enzymes.

[Plant potential for detoxification (review)]. / Detoksikatsionnyi potentsial rastenii (obzor).

Data on the uptake, excretion, and biodegradation of organic xenobiotics by plants are reviewed. Detoxification pathways operating in plants and their role in remediation of biosphere are described. Structure-, concentration, and time-dependent effects of xenobiotics on the ultrastructural organization of cells are analyzed.

[Isolation and physico-chemical characteristics of Staphylococcus hyaluronidase]. / Vydelenie i fiziko-khimicheskie svoi'stva Stafilokokkovoi' gialuronidazy.

A scheme for purification of hyaluronidase from Staphylococcus aureus 0-15 that allows to produce a 550-fold-purified preparation with a high specific activity was developed. Hyaluronidase was found to consist of two molecular forms with different molecular weights. Staphylococcus hyaluronidase referred to as a complex preparation and its molecular forms have action optimum at pH 5.0-7.2 and at a temperature between 30 and 37 degrees C. The enzymes were highly specific to their substrate: they catalyzed the depolimerization of hyaluronic acid but did not split the other glycosaminoglycans.

[Nitrogen enzymes and energy metabolism from dogfish liver and in a Catrex preparation]. / Fermenty azotnogo i énergeticheskogo obmenov iz pecheni katrana i v preparate katréks.

The activity of enzymes of nitrogen and energy metabolisms from dogfish liver and a commercial preparation Catrex manufactured in the Scientific-Industrial Association "Adaptogen" (Tbilisi) was studied. The liver homogenate contains active glutamate dehydrogenase (GD), malate dehydrogenase (MD) and lactate dehydrogenase (LD) catalysing in vitro the reaction in both directions, as well as active glutamine synthetase, aspartate transaminase and alanine transaminase. These enzymes are also present in Catrex, but their activities are less. After 10-day storage of the liver homogenate and the Catrex preparation the enzymes slightly inactivated. Two isozymes of MD and four isozymes of LD were detected in the liver homogenate by polyacrylamide gel electrophoresis. In Catrex the two MD isozymes and only three LD isozymes were found.

beta-Glucosidases from cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum as typical glycosidases.

By ethanol precipitation (v/v) and chromatography on Sephadex SP, DEAE (or DEAE-cellulose), and G-200 beta-glucosidases (EC 3.2.1.21) from the culture filtrates of cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum grown on the medium with cellulose containing materials were isolated. The enzymes were homogenous as shown by different techniques. The substrate specificities of the obtained enzymes were studied. beta-Glucosidases had higher affinity for p-nitrophenyl-beta-D-glucopyranoside than for cellobiose (Km 1.25, 0.34, 0.20 and 5.4, 2.0, 1.2 mM, respectively) and were able to hydrolyze both laminaribiose and gentiobiose; but they were unable to cleave cotton fiber, carboxymethylcellulose, and other glycans to reducing sugars. They showed transglycosylase activity. Ki values for arylglucosidase activity of beta-glucosidases from A. terreus, G. candidum, and T. longibrachiatum in the presence of either glucose or glucono-1,5-lactone were 12.2, 6.0, 2.1 and 0.20, 0.19, 0.07 mM, respectively. The Mr's were estimated by gel filtration and by sedimentation equilibrium centrifugation to 200,000, 200,000, 350,000, respectively. The isoelectric points of beta-glucosidases were 4.8, 5.9, and 4.2, respectively. The optimum temperatures and pH's were 60, 50, and 50 degrees C and at pH 4.5, 4.5, and 4.8-5.7, respectively. These properties appear to relate beta-glucosidases obtained in the present study to typical glycosidases.

[Purification and properties of staphylococcal hyaluronidase]. / Ochistka i svoistva stafilokokkovoi gialuronidazy.

Microbial hyaluronidase (EC 4.2.2.1) was isolated from the culture fluid of Staphylococcus aureus 0-15 with purification by precipitation with 1 volume of ethyl alcohol, chromatography on DEAE cellulose and ultrafiltration through DA type membranes with the pore size of 0.65 micron ("Millipore") and PM-10 membranes ("Amicon"). The specific activity of the enzyme averaged to 2700 turbidimetric units or 32130 IU. 6585-fold purification of the enzyme was performed. The optimum action on hyaluronic acid was observed at pH 5.0-6.5. Hyaluronidase was inhibited by Fe3+, Fe2+ and Cu2+, activated by Ca2+ and stabilized by 0.15 M NaCl. It was detected that the enzyme had two molecular forms with the isoelectric points of 5.4 and 6.5 and the molecular weights of 55 000 and 24 0000 D respectively. The glycoprotein nature of the enzyme was shown. The immobilized form of hyaluronidase on activated polyglucin, a soluble biocompatible polymer was prepared. The form is characterized by higher thermostability.

Dried calcium alginate/magnetite spheres: A new support for chromatographic separations and enzyme immobilization.

Dried spheres made from an alginate solution containing magnetite particles have excellent potential as a support for enzyme immobilization and chromatographic applications. The beads were found to be much stronger than gels such as polyacrylamide and dextran, indicating that high flow rates and pressures could be used in column separations. The support withstood not only temperatures of up to 120 degrees C, but also most pH values and common solvents. While some solutions, such as phosphate buffers, dissolved the spheres, stabilization with Tyzor TE(R) eliminated this problem. The physical properties of the beads include a glasslike density of 2.2 g/mL, excellent sphericity, low porosity, and a narrow size distribution. The magnetite present in the support allows the beads to be used for magnetic separations such as high gradient magnetic filtration. Their high degree of microroughness provides a large exposed surface area for enzyme and ligand binding. Mixed Actinomyces fradiae proteases and Aspergillus niger alpha-amylase, two enzymes representative of classes which attack large substrates, were immobilized on the bead's surface with high activity and stability. A cyanuric dye which can be used in chromatographic applications (Cibacron Blue F3GA(R)) was also readily coupled to the surface of this support with good yield. The support should have a wide range of applications in bioseparation and immobilized biochemical technology.

Immobilization of mold and bacterial amylases on silica carriers.

The immobilization of alpha-amylase and glucoamylase was investigated by several coupling methods on silica carriers, different types of Silokhroms, and silica gels. The most active immobilized mold and bacterial alpha-amylases and mold glucoamylase were obtained with titanium salts. These activities were twice the value of that obtained by glutaraldehyde or azo coupling. The half-lives of A. oryzae alpha-amylase, B. subtilis alpha-amylase, and A. niger glucoamylase, immobilized to silica carriers at 45 degrees C and under continuous operation at a high concentration of substrate, were 14, 35, and 65 days, respectively.

[Acid-stable and acid-unstable alpha-amylases of the mold fungi Aspergillus]. / Kislotostabil'naia i kislotolabil'naia alf'a-amilazy plesnevykh gribov Aspergillus.

Acid-sable alpha-amylase of Asp. niger and acid-unstable, alpha-amylase of Asp. oryzae were studied. It was demonstrated, that beside being a more acid-stable properties, alpha-amylase Asp. niger has increased thermal stability as compared to alpha-amylase Asp. oryzae. The molecular weights of acid-stable alpha-amylase and acid-unstable alpha-amylase are 58 000 and 51 000, respectively. The amino acid composition, and the C- and N-terminal amino acids of both forms of alpha-amylases were determined. It was demonstrated, that the enzymes under study contain one sylfhydryl group per mole of enzyme, which in the Ca2+-bound form plays an important role in the maintenance of the catalytically active enzyme conformation.

[Immobilization of alpha-amylase on porous glass and silochrome]. / Immobilizatsiia al'fa-amilazy na poristom stekle i silokhrome.

Immobilized forms of alpha-amylase from Aspergillus oryzae were prepared on the porous glass and silochrome by the glutaraldehyde method. An addition of calcium ions at a concentration of 0.05 M to the reaction mixture during immobilization stabilized the enzyme activity. pH optimum of the insoluble form of alpha-amylase was 5.8 and that of the soluble form was 4.7. Storage of the insoluble enzyme as water suspension in 0.015 M CaCl2 at 4 degrees C for six months and twenty times repeated specific reaction did not affect significantly the activity of insoluble alpha-amylase.

[Effect of the composition of the nutrient medium on the synthesis of acid-fast alpha-amylase by different strains of Aspergillus]. / Vliianie sostava pitatel'noi sredy na biosintez kislotoustoichnivoi Al'fa-amilazy razlichnymi vidami gribov Aspergillus.

The capacity of 86 strains of the Aspergillus fungus to synthesize acid stable alpha-amylase was examined. The strains of Asp. niger showing a high capacity of synthesizing the enzyme were isolated. Repeated cultivation of the selected cultures on the Minoda agar medium led to a 200% increase in the enzyme activity in the submerged culture. Addition of sodium nitrate to the Minoda medium during submerged cultivation allowed a 3-fold increase of the synthesis of acid stable alpha-amylase.