Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization.

Kinetics of refolding of bovine carbonic anhydrase B have been studied by the "double-jump" technique (i.e. the dependence of protein refolding on delay time in the unfolded state after fast unfolding). It is shown that two stages (the slow with a relaxation time of t1/2 approximately equal to 120 s and the superslow with t1/2 approximately equal to 600 s) observed during refolding of bovine carbonic anhydrase B are due to trans-cis isomerization of proline residues. The dependences of rate constants of these processes on temperature and on the final denaturant concentration were measured. Activation energies of both processes are the same, Ea = 18(+/- 2) kcal/mol. The rate constants of protein refolding do not depend on the final concentration of urea under native conditions. In addition, the rate of isomerization of essential proline residues in the "molten globule" intermediate state of bovine carbonic anhydrase was measured and found to be equal to that for unstructural polypeptides. The effect of several proline residues on carbonic anhydrase refolding is discussed.

Sequential mechanism of refolding of carbonic anhydrase B.

The kinetics of refolding of bovine carbonic anhydrase B was studied by a variety of methods over a wide range of times (from milliseconds to hours). It has been shown that protein refolding proceeds through three stages. At the first stage (t1/2 approximately equal to 0.03 s) hydrophobic clusters and a compact state of the chain are formed. At the second stage (t1/2 approximately equal to 140 s) hydrophobic clusters are desolvated and the rigid native-like hydrophobic core is formed. At the third stage (t1/2 approximately equal to 600 s) the native active protein is formed.

[Staged equilibrium of carbonic anhydrase unfolding in strong denaturants]. / Stadiinost' ravnovesnogo razvorachivaniia karboangidrazy v sil'nymi denaturantami.

It has been shown by 1H-NMR, circular dichroism, fluorescence and viscometry techniques that equilibrium unfolding of carbonic anhydrase B (a one-domain globular protein) in urea guanidine hydrochloride consists of two sequential stages. The first stage is connected with a decrease of intramolecular interactions, stabilizing the rigid tertiary structure and with the increase of mobility of aliphatic side chain groups. At the second stage the decrease of protein secondary structure and hydrophobic interactions take place as well as the increase of mobility of massive aromatic side chain groups.

[Denatured transitions of the molecular chaperone GroEL from Escherichia coli]. / Denaturatsionnye perekhody molekulianogo shaperona GroEL iz Escherichiacoli.

Conformational changes of oligomeric particle of GroEL chaperone from E. coli in solution were studied, which proceed during its denaturation upon the action of elevated urea concentration, temperature, and extremal pH values by the methods of CD, light scattering, scanning microcalorimetry, hydrophobic probe binding, and ATPase activity measurements. The ranges of changing the external conditions; within which GroEL retains its structure and functions, were determined. Denaturation transitions were found to be cooperative, pronounced, and irreversible. In the pH range from 6.0 to 9.6, the three-step change of the ATPase activity of GroEL was shown to occur with half-transition pH1/2 of 6.3, 8.5, and 9.3. It does not result in any essential structural changes and is probably associated with a protonation/deprotonation of amino acid residues important for the GroEL ATPase activity.

[Localization of catalysis enzyme systems that degrade higher plants' cell wall polysaccharides. Pectinases (review)]. / O lokalizatsii sistem fermentov, kataliziruiushchikh rasshcheplenie polisakharidov rastitel'nykh kletochnykh stenok u vysshikh rastenii. Pektinazy (obzor).

This paper reviews recent data on the locations and compositions of enzyme systems catalyzing the cleavage of cellulose, hemicelluloses, and pectin polysaccharides in higher plants. The physiological functions and physicochemical properties of certain enzymes degrading pectin polymers in higher plants, fungi, and bacteria are described.

[Properties of the exo-1,4-beta-xylosidase of Aspergillus niger 15]. / Svoistva ékzo-1,4-beta-ksilozidazy Aspergillus niger 15.

Properties of exo-1,4-beta-xylosidase from the fungus Aspergillus niger 15 were investigated. The enzyme was homogeneous during gel filtration, electrophoresis in polyacrylamide gel in the presence and absence of Na dodecyl sulfate, ultracentrifugation and isoelectric focusing. The enzyme had a temperature optimum at 70 degrees, pH optimum 3.8-4.0 for p-nitrophenyl-beta-D-xylopyranoside (p-NPX), was stable at pH 3-8, retained its 100% activity for 1 hour at 50 degrees and 42% activity at 60 degrees. Km was 0.23 mM for p-NPX and 0.67 mM for xylobiose. Xylose was a competitive inhibitor of exo-1,4-beta-xylodidase with Ki = 2.9 mM. The enzyme showed a transglycosilase activity. The aminoacid analysis of exo-1,4-beta-xylosidase showed that the enzyme molecule contained predominantly dicarboxylic and hydrophobic amino acids as well as serine. The enzyme contained no carbohydrates. Its activity was inhibited by p-chloromercury benzoate.

[Properties of beta-glucosidase from the fungus Geotrichum candidum 3C]. / Svoistva beta-gliukozidazy griba Geotrichum candidum 3C.

The ability of beta-glucosidase from the fungus Geotrichum candidum 3C to degrade cell oligosaccharides, disaccharides and glycosides was examined. It was demonstrated that the rate of enzymic hydrolysis of cell oligosaccharides depended on the length of the substrate chain. The major product was glucose. The beta-glucosidase purified showed high specificity towards beta-glucopyranosyl and degraded only the glucosides which incorporated beta-glucopyranosyl. The enzyme had a relatively low specificity towards aglycon, and hydrolyzed both disaccharides and aryl-glucosides. The enzyme also showed traansglycosylase activity.

[Transglycosylation reactions catalyzed by Aspergillus niger 15 beta-xylosidase]. / Izuchenie reaktsii transglikozilirovaniia, kataliziruemykh beta-ksilozidazoi Aspergillus niger 15.

The products of transglycosylation formed as a result of action of beta-xylosidase from Aspergillus niger 15 on p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside were studied by means of chromatography on sephadexes. They are formed at the substrate concentration of 10--100 mM with the amount 7--10 times less than that of hydrolysis products. Peculiarities of chromatography of substrates, products of transglycosylation and p-nitrophenol on Sephadexes G-15 and G-25 were analysed.

[Preparation of cellulolytic enzymes from Geotrichum candidum]. / Poluchenie tselliuloliticheskikh fermentov iz Geotrichum candidum.

The ability of different fungi cultivated on cellulose to synthesize extracellular cellulase was examined. The fungus Geotrichum candidum str. 3c was the most active among the fungi tested, including Trichoderma fungi. The formation of cellulolytic enzymes in the culture filtrate of G. candidum occurred, when the entire glucose and the major portion of reducing substances were utilized. The formation of cellulases depended on the content of ammonium ions. The enzyme accumulation reached the highest level at the stationary phase of the culture growth and depended on the inoculum amount. When the inoculum was used as a submerged culture at a concentration of 3%, accumulation of C1-enzyme, endo-(1 leads to 4)beta-glucanases, and beta-glucosidases reached maximum by 44, 52 and 56 hours of cultivation. The fats that are frequently used as a foam catcher inhibited the cellulase formation by the abouve strain. From the filtrate cellulolytic enzymes were isolated via spray drying and precipitation with 3 ethanol volumes to yield 6.5 and 5.0 g/l, respectively. The cellulases when stored at 5 degrees C did not lose their activity for a year and became inactivated by 40-50% in 5 years.

[Purification of exo-1,4-beta-xylosidase from Aspergillus niger 15]. / Ochistka ékzo-1,4-beta-ksilozidazy Aspergillus niger 15.

From the preparation of xylanase of the fungus Aspergillus niger 15 exo-1,4-beta-xylosidase was isolated by means of ethanol fractionation and chromatography on columns of Sephadex G-50, cellulose DE-52, Sephadex SP C-50, Sephadex G-200. The isolated enzyme (with the purification degree as calculated per protein 199, and yield with respect to activity 42.5%) was homogeneous during gel filtration, polyacrylamide gel electrophoresis in the presence and absence of Na dodecyl sulfate, ultracentrifugation and isoelectric focusing. Exo-1,4-beta-xylosidase had a molecular mass of 253,000 as shown by gel filtration and 122,000 as shown by Na dodecyl sulfate polyacrylamide gel electrophoresis; its sedimentation coefficient was 10.6 S and isoelectric point was pI 4.9. The specific activity of the homogeneous enzyme was 35.2 u./mg protein with respect to p-nitrophenyl-beta-D-xylopyranoside and 30.2 u./mg protein with respect to xylobiose.

[Isolation of endo-1,4-beta-xylanase from Geotrichum candidum 3C with various ability of sorption on an insoluble substrate]. / Razdelenie éndo-1,4-beta-ksilanaz Geotrichum candidum 3C s razlichnoi sposobnost'iu k sorbtsii na nerastvorimom substrate.

Culture liquid from Geotrichum candidum 3C was shown to contain three endoxylanase types: endoxylanase I that binds to cellulose, endoxylanase II that sorbs to insoluble xylan, and endoxylanase III that cannot sorb to dissoluble substrate. The catalytic and substrate-binding domains of endoxylanase II were isolated.

[Formation of an extracellular system of enzymes during growth of Geotrichum candidum 3C on cell walls isolated from cereal grain capsules]. / Obrazovanie vnekletochnykkh sistem fermentov pri vyrashchivanii Geotrichum candiudum 3C na izolirovannykh kletchnykh stenkakh obolochek zerna zlakov

The activities of extracellular systems of hemicellulases, pectinases, and cellulases was studied during a 72-h cultivation of Geotrichum candidum 3C. The culture was grown on a medium containing 3% cell walls isolated from wheat grain capsules, which served as the sole carbon source. Enzymes catalyzing the degradation of pectin substances (beet pectin, alpha-L-arabinan, and 1,4-beta-D-galactan), as well as beta-D-galactosidase and alpha-L-arabinofuranosidase involved in their hydrolysis, were formed first (4 h after the beginning of cultivation). Enzymes hydrolyzing 4-O-methyl-alpha-D-glucurono-beta-D-xylan and sodium carboxymethyl xylan were also found in the culture liquid after 4 h of fungal growth. The contents of pectin-degrading and xylanolytic enzymes reached their maximum levels after 52-56 and 72 h of growth, respectively. Cellulolytic enzymes were detected after 8-28 h of cultivation. Enzymes degrading alpha-D-galacto-beta-D-mannan were found 24 h after the beginning of growth; their content was maximum after 72 h of cultivation.

[Formation of xylitol in Candida guilliermondii 2581 culture]. / Obrazovanie ksilita v kul'ture Candida guilliermondii 2581.

The yeast strain Candida guilliermondii 2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.

[Ethylene-induced activation of xylanase in adventitious roots of maize as a response to the stress effect of root submersion]. / Indutsirovannia étilenom aktivatsiia ksilanazy v pridatochnykh korniakh kukuruzy kak otvet na stressovoe vliianie kornevogo zatopleniia.

Submersion of roots of ten-day-old maize (Zea mays L.) seedlings was accompanied by a decrease in pO2 and an increase in pCO2 of the medium adjacent to roots. These changes stimulated ethylene evolution in intact plants. Enhanced biosynthesis of ethylene was accompanied by xylanase activation in adventitious roots. As a result, an enhanced formation of aerenchyma was observed in the cortex of adventitious roots. Therefore, these processes resulted in the development of a ventilation system by which O2 can reach the root system exposed to hypoxia. The volume of aerenchyma was assessed by the volume of gas cavities (porosity). In contrast to the main root, the growth of adventitious roots was not inhibited under these conditions. Enlargement of the stem base and increase in the number of aerenchymatous adventitious roots facilitated the oxygen supply to submerged organs of plants.

[Purification and characteristic of endo-(1--4)-beta-xylanase from Geotrichum candidum 3C]. / Ochistka i kharakteristika éndo-(1--4)-beta-ksilinazy iz Geotrichum candium 3C.

A method of purification of endo-(1-->4)-beta-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid of Geotrichum candidum 3C, grown for three days, is described. The enzyme purified 23-fold had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30-45 degrees C for 1 h. With xylan from beach wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50 degrees C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10, 12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.

[Use of the enzyme preparation cellocandin from Geotrichum candidum 3C-106 for difibering of waste paper and dessication of cellulose suspension]. / Primenenie fermentnogo preparata tsellokandin iz Geotrichum candidum 3C-106 dlia rospuska makulatury i obezvozhivaniia suspenzii tselliulozy.

The effect of cellocandin, an enzymatic preparation from Getrichum candidum 3C-106 with cellulolytic and xylanolytic activities, on defibring of waste paper and acceleration of desiccation of paper pulp was studied. It was shown that cellocandin accelerated defibring of waste paper to cellulose fibers and desiccation of cellulose suspension.

[An immunoregulatory effect of endoglucanase preparations on plant resistance to fungal infections]. / Immunoreguliruiushchee deistvie fermentnykh preparatov éndoglikanaz na ustoichivost' rastenii k gribnoi infektsii.

Effects of enzymatic preparations--pectomacerin, hemicellulase, and Trichoderma viride 13/10 cellulase--on the plant immunological status were studied by examples of the pathosystems carrot root-white rot agent (Sclerotinia libertiana) and carrot root-black rot agent (Rhizopus nigricans). It was demonstrated that these preparations reduced the damage of infections, namely, decreased the permeability of cell membranes in infected tissue and stimulated its defense responses, which manifested itself in a stable elevation in the content of phenolic compounds and formation of tissue protective barriers.