Biomedical subjects

O N Okunev

Publications and source records attributed to O N Okunev.

At least 19 recordsLinked to original sources

PubMed2005

New effective method for analysis of the component composition of enzyme complexes from Trichoderma reesei.

A method for analysis of the component composition of multienzyme complexes secreted by the filamentous fungus Trichoderma reesei was developed. The method is based on chromatofocusing followed by further identification of protein fractions according to their substrate specificity and molecular characteristics of the proteins. The method allows identifying practically all known cellulases and hemicellulases of T. reesei: endoglucanase I (EG I), EG II, EG III, cellobiohydrolase I (CBH I), CBH II, xylanase I (XYL I), XYL II, beta-xylosidase, alpha-L-arabinofuranosidase, acetyl xylan esterase, mannanase, alpha-galactosidase, xyloglucanase, polygalacturonase, and exo-beta-1,3-glucosidase. The component composition of several laboratory and commercial T. reesei preparations was studied and the content of the individual enzymes in these preparations was quantified. The influence of fermentation conditions on the component composition of secreted enzyme complexes was revealed. The characteristic features of enzyme preparations obtained in "cellulase" and "xylanase" fermentation conditions are shown.

Binding Sites↗

PubMed2004

Cellulase complex of the fungus Chrysosporium lucknowense: isolation and characterization of endoglucanases and cellobiohydrolases.

Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44.

Adsorption↗

PubMed2003

Isolation and properties of pectinases from the fungus Aspergillus japonicus.

Using anion-exchange chromatography on different carriers and phenyl-Sepharose hydrophobic chromatography, five pectolytic enzymes were isolated from the culture liquid of a mutant strain of Aspergillus japonicus: two endo-polygalacturonases (I and II, 38 and 65 kD, pI 5.6 and 3.3), pectin lyase (50 kD, pI 3.8), and two pectinesterases (I and II) with similar molecular weights (46 and 47 kD) and the same pI (3.8). The pectinesterases apparently represent two isoforms of the same enzyme. All purified enzymes were homogenous according to SDS-PAGE and polyacrylamide gel-IEF, except for endo-polygalacturonase II that gave two bands on isoelectric focusing, but one band on electrophoresis. All enzymes had maximal activity in an acid medium (at pH 4.0-5.5). The pectin lyase and pectinesterase were stable at 40-50 degrees C. The thermal stability of both endo-polygalacturonases was much lower (after 3 h of incubation at 30 degrees C, endo-polygalacturonases I and II lost 40 and 10% of the activity, respectively). The activity of endo-polygalacturonases I and II towards polygalacturonic acid strongly depended on NaCl concentration (optimal concentration of the salt was 0.1-0.2 M); the enzymes were also capable of reducing the viscosity of pectin solution, but rather slowly. The pectin lyase had no activity towards polygalacturonic acid. The activity of the pectin lyase increased with increasing degree of methylation of pectins. Both endo-polygalacturonases demonstrated synergism with the pectinesterase during the hydrolysis of highly methylated pectin. On the contrary, in the mixture of pectin lyase and pectinesterase an antagonism between the two enzymes was observed.

Aspergillus↗

PubMed2003

Isolation and properties of major components of Penicillium canescens extracellular enzyme complex.

The composition of the enzyme complex secreted by Penicillium canescens was investigated. A scheme for purification of the main components of the complex by chromatofocusing on a Mono P column was developed. It was found that along with beta-galactosidase, the major components of the complex were endo-beta-1,4-xylanase (31 kD, pI 8.2-9.3), alpha-L-arabinofuranosidase (60 kD, pI 7.6), arabinoxylan-arabinofuranohydrolase (70 kD, pI 3.8-4.0), and endo-beta-1,3/1,4-glucanase (40 kD, pI 4.4). The substrate specificity, pH and temperature activity optima, adsorbability, thermal stability, and ability for synergic interaction of the isolated enzymes were studied.

Chromatography, Liquid↗

PubMed2003

Recombinant endo-beta-1,4-xylanase from Penicillium canescens.

Recombinant endo-beta-1,4-xylanase (Xyl-31rec, 31 kD, pI 8.2-9.3, the tenth family of glycosyl hydrolases) was isolated from the culture liquid of Penicillium canescens (strain with the amplified homologous xylanase gene) by chromatofocusing on Mono P and hydrophobic chromatography on phenyl-Superose. It is shown that the biochemical and kinetic parameters, substrate specificity, stability, and other properties of the recombinant and native enzymes are almost the same. It was found that Xyl-31rec can be used for biobleaching of cellulose, the recombinant P. canescens strains providing a high yield of extracellular Xyl-31rec (up to 800-900 U/ml of culture liquid) and not secreting cellulases.

Absorption↗

PubMed1999

Cellulase complex from Chaetomium cellulolyticum: isolation and properties of major components.

Four major components of the cellulase complex of Chaetomium cellulolyticum have been isolated by gel-filtration, ion-exchange chromatography on DEAE-Toyopearl and Macro Prep Q, and chromatofocusing on Mono P. These components include three endoglucanases (19, 35, and 40 kD) and a cellobiohydrolase (45 kD). The isoelectric points of the enzymes vary from 3.8 to 4.2. The optimal pH values for catalytic activity are in the range 4.5-6.0, and the optimal temperatures are in the range 60-70 degrees C. Of these enzymes the 19 kD endoglucanase is the most stable; it retained high activity within a broad pH range (from 5.0 to 9.6) at 50 degrees C for 3 h. This enzyme also had the highest topolytic activity determined by the efficiency of removal of indigo from the surface of cotton.

Cellulase↗

PubMed1991

Description of two anaerobic fungal strains from the bovine rumen and influence of diet on the fungal population in vivo.

Neocallimastix sp. NC71 and Piromyces sp. PC12 isolated from the calf remen grew optimally at 39 degrees C and pH 6.5-6.7, utilized a wide range of mono-, oligo- and polysaccharides and exhibited CMCase, Avicelase, cellobiase, amylase and xylanase activities. The end-products of wheat straw fermentation by both strains were acetate, formate, ethanol and lactate. The number of Neocallimastix sp. zoospores in the rumen of cows in the first 3 h after feeding with hay-silage-concentrate diets varied from 7 x 10(3) to 5.4 x 10(5) ml-1; the number of uniflagellate zoospores varied from 10(4) to 10(5) ml-1. Fungal zoosporgenesis and colonization of plant substrates in the rumen were induced by feed intake and were favoured by increased levels of crude fibre in the diet.

Animals↗

PubMedDate not supplied

[Change in composition of structural components of leaf cellulose sulfate during fermentation bleaching by xylanases].

The possibility of the use of xylanase preparations for hydrolysing hemicelluloses in a non-bleached kraft pulp in order to facilitate its bleaching was studied. The effects of enzymatic preparations of the fungal and bacterial origins were examined, and the optimal conditions for xylanase activity were determined. UV spectroscopy demonstrated that the treatment of kraft pulp with the enzymatic preparations containing xylanase facilitated the subsequent removal of lignin and increased the brightness by 5%. The effect of enzymatic treatment was retained in the case of peroxide bleaching. The enzymatic preparations studied are promising for the development of chlorine-free pulp bleaching technologies.

Cellulose↗

PubMedDate not supplied

[Change in composition of hydrolyzed lignin during composting].

Microbial assemblages were composed for composting hydrolysed lignin. Data on bioconversion of aromatic compounds with various types of substitution in the ring were used for this purpose. Composting of hydrolysed lignin reduced the contents of lignin, low-hydrolyzable polysaccharides, resins, and low-molecular-weight phenols and resulted in accumulation of humic acids. The resulting compost showed no phytotoxicity.

Hydrolysis↗

PubMedDate not supplied

[Isolation and properties of cellobiase from Penicillium verruculosum].

Cellobiase (beta-D-glucosidase) with a molecular weight of 100 kDa and pI 5.2 was isolated from the cellulolytic system of Penicillium verruculosum. Kinetic parameters of enzymatic hydrolysis of cellobiose, gentiobiose, sophorose, and synthetic substrates, i.e. methylumbelliferyl and p-nitrophenyl sugar derivatives were determined. Glucose and D-glucose-delta-lactone competitively inhibited cellobiase (Ki = 0.19 mM and 17 microM, respectively). Glucosyl transfer reactions were studied with cellobiose as a single substrate and in the mixture of cellobiose and methylumbelliferyl cellobioside. The product composition was determined in these systems. The ratio of hydrolysis and transfer reaction rates for cellobiose conversion was calculated.

Chromatography, Gel↗

PubMedDate not supplied

[Studies of hydrolytic activity of enzyme preparations of Penicillium and Trychoderma fungi].

Enzyme preparations were isolated from the culture liquid of five mutant strains of the cellulase producer Penicillium verruculosum. The hydrolytic activities of these preparations against unbleached eucalypt cellulose was compared to that of commercial preparations of Trichoderma reesei (T. longibrachiatum). In the majority of cases, P. verruculosum enzymes provided higher yields of reducing sugars (RSs) and glucose. A correlation was found between the yield of RSs and the avicelase activity of the preparations in the reaction mixture.

Culture Media↗

PubMedDate not supplied

[The selection and properties of Penicillium verruculosum mutants with enhanced production of cellulases and xylanases].

The paper describes three Penicillium verruculosum 28K mutants with about threefold enhanced production of five industrially important carbohydrases. The two-stage fermentation process that we developed provided a further two- to threefold increase in the production of carbohydrases. Physiological and biochemical studies showed that the synthesis of all five carbohydrases is inducible. Carboxymethylcellulase, xylanase, and beta-glucanase are synthesized under a common regulatory control, as is evident from the concurrent increase in the synthesis of these enzymes in the presence of microcrystalline cellulose. The synthesis of avicelase and beta-glucosidase is evidently induced by other cellulose- and hemicellulose-containing compounds present in the fermentation medium and, hence, is regulated independently of the three aforementioned enzymes.

Cellulase↗

PubMedDate not supplied

[Properties of hemicellulases of the enzyme complex from Trichoderma longibrachiatum].

Six xylan-hydrolyzing enzymes have been isolated from the preparations Celloviridin G20x and Xybeten-Xyl, obtained previously based on the strain Trichoderma longibrachiatum (Trichoderma reesei) TW-1. The enzymes isolated were represented by three xylanases (XYLs), XYL I (20 kDa, pi 5.5), XYL II (21 kDa, pI 9.5), XYL III (30 kDa, pI 9.1); endoglucanase I (EG I), an enzyme exhibiting xylanase activity (57 kDa, pI 4.6); and two exodepolymerases, beta-xylosidase (beta-XYL; 80 kDa, pI 4.5) and alpha-L-arabinofuranosidase I (alpha-L-AF I; 55 kDa, pI 7.4). The substrate specificity of the enzymes isolated was determined. XYL II exhibited maximum specific xylanase activity (190 U/mg). The content of the enzymes in the preparation was assessed. Maximum contributions to the total xylanase activities of the preparations Celloviridin G20x and Xy-beten-Xyl were made by EG I and XYL II, respectively. Effects of temperature and pH on the enzyme activities, their stabilities under various conditions, and the kinetics of exhaustive hydrolysis of glucuronoxylan and arabinoxylan were studied. Combinations of endodepolymerases (XYL I, XYL II, XYL III, or EG I) and exodepolymerases (alpha-L-AF I or beta-XYL) produced synergistic effects on arabinoxylan cleavage. The reverse was the case when endodepolymerases, such as XYL I or EG I, were combined with alpha-L-AF I.

Enzyme Stability↗

PubMedDate not supplied

[Synthesis of xylanolytic enzymes and other carbohydrases by the fungus Penicillium canescens: transformants with an altered copy number of the gene xlnR and an encoding transcriptional activator].

A fragment of Penicillium canescens genomic DNA carrying the xlnR gene coding for a translational activator of xylanolytic genes was isolated. It was demonstrated that a loss of this function in genetically modified transformants resulted in a drastic decrease in the production of P. canescens major xylanases and had a negative effect on the syntheses of several other extracellular xylanases. An increase in the dose of the xlnR gene elevated the xylanolytic activity as well as the activities of a number of other auxiliary enzymes involved in xylan degradation. The activities of two P. canescens major secreted enzymes--beta-galactosidase and alpha-L-arabinofuranosidase-appeared weakly dependent on the translational activator xlnR.

Amino Acid Sequence↗

PubMedDate not supplied

[New cellulases efficiently hydrolyzing lignocellulose pulp].

Commercial and pilot enzyme preparations from fungi of the genera Penicillium and Trichoderma have been compared with regard to their action on conifer wood pretreated with acidified aqueous ethanol (organosolve). In most experiments, enzymes from the genus Penicillium allowed higher yields of reducing sugars and glucose than those from Trichoderma. High beta-glucosidase activity is essential for deep pulp hydrolysis.

Cellulases↗

PubMedDate not supplied

[Use of a preparation from fungal pectin lyase in the food industry].

A new enzyme preparation of fungal pectin lyase (EC 4.2.2.10) was shown to be useful for the production of cranberry juice and clarification of apple juice in the food industry. A comparative study showed that the preparation of pectin lyase is competitive with commercial pectinase products. The molecular weight of homogeneous pectin lyase was 38 kDa. Properties of the homogeneous enzyme were studied. This enzyme was most efficient in removing highly esterified pectin.

Beverages↗

PubMedDate not supplied

[Application of neutral-alkaline pectate lyases to cotton fabric boil off].

Commercial and pilot pectate lyase preparations (EC 4.2.2.2) have been compared. They differ in their effect on pectins with different esterification degrees (ED). The activity of the pilot preparation with respect to a substrate with ED = 70% is tenfold lower than with respect to unesterified polygalacturonic acid. For commercial preparations, this activity ratio ranged within 1.5-2. At equal pectate lyase activities, the commercial preparations better remove pectin from crude cotton fabric during its boil off. The laboratory preparation is more efficient for improving the capillarity (wettability) of the fabric owing to the cooperative effect of the pectate lyase, cellulase, and hemicellulase present in the preparation.

Bacillus↗

PubMedDate not supplied

[Anaerobic cellulolytic fungi from cattle rumen].

Two anaerobic fungal strains, Neocallimastix sp. 71 and Piromonas sp. 12, were isolated from cattle rumen. Their motile zoospores have either one (strain 12) or several (strain 71) flagella. The spores get attached to a substrate, germinate and form a branched rhizoid with one sporangium within which a new generation of zoospores appears after 24 h, on the average. Both fungi have a temperature optimum at 39 degrees C and a pH optimum at 6.5-6.7. They can utilize a wide spectrum of mono-, oligo- and polysaccharides. Strain 71 produces acetate, formate, ethanol and lactate while strain 12 forms acetate, formate, lactate and traces of ethanol as main products accumulated upon the fermentation of wheat straw, filter paper and cellobiose in a liquid semidefined medium. Both strains exert the activities of CMCase, Avicelase, cellobiase, amylase and xylanase.

Anaerobiosis↗