Biomedical subjects

A P Sinitsyn

Publications and source records attributed to A P Sinitsyn.

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↗

PubMed2002

Viscometric method for assaying of total endodepolymerase activity of pectinases.

An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, polymethylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5 min at 40 degrees C and the appropriate pH. Depending on the pH-optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH 5.0, and 2) for neutral (mildly alkaline) pectinases at pH 8.0. The modifications differed in the control and in the calculation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was linear in the range of at least 10-fold change in the enzyme concentration). The relative error of the method did not exceed 10%.

Aspergillus↗

PubMed2001

Application of microassays for investigation of cellulase abrasive activity and backstaining.

Model microassays were used for testing the denim-washing performance and indigo backstaining for Trichoderma reesei and Chrysosporium lucknowense commercial cellulase preparations on a 'test-tube scale'. C. lucknowense preparation demonstrated a higher potential in the denim biostoning process. The performance of four purified cellulases (two endoglucanases and two cellobiohydrolases) from C. lucknowense on cotton textiles was assayed, and the key enzyme (endoglucanase with a molecular weight of 25 kDa) responsible for high abrasion effects on denim fabrics was found.

Cellulase↗

PubMed2001

Study of protein adsorption on indigo particles confirms the existence of enzyme--indigo interaction sites in cellulase molecules.

Adsorption of several crude and purified cellulases (from Trichoderma reesei, Penicillium verruculosum and Chrysosporium lucknowense) on indigo particles and Avicel cellulose was studied. Much higher amounts of protein were bound to indigo than to cellulose under similar conditions. For different purified enzymes, the quantity of bound protein per mg of adsorbent (indigo or cellulose) varied in the range of 57-111 and 0-62 microg x mg(-1), respectively. However, in general, the enzyme adsorption on indigo was less specific than the adsorption on cellulose. Three endoglucanases, having the highest indigo-binding ability, demonstrated the best washing performance in the process of enzymatic denim treatment. These data confirmed our previous findings that certain cellulases, which have indigo-binding sites (clusters of closely located aromatic and other non-polar residues) on the surface of their molecules, may remove indigo from the denim fabric better than cellulases with lower content of hydrophobic residues exposed to solvent.

Adsorption↗

PubMed2000

Electrochemical assay of endo-depolymerase activity of cellulases.

A method for determination of endo-1,4-beta-D-glucanase activity of cellulase samples based on the indirect measurement of decrease in viscosity of a carboxymethylcellulose solution in an electrochemical cell in the presence of an electron carrier was developed. A rotating disk electrode is used as the working electrode. When two reactions (enzymatic and electrochemical) proceeded in the cell simultaneously, the limiting diffusion current at a constant applied potential increases as the viscosity of the solution decreases. Conditions where the initial rate of change of diffusion current (dI/dt) is proportional to the enzyme concentration were found. A good correlation between the new method and a previously known viscometric method for determination of endoglucanase activity was observed.

Carboxymethylcellulose Sodium↗

PubMed1993

[The inhibiting effect of dimethylaminomethylferrocene on amylase].

The effects of dimethylaminomethylferrocene (DMAMF) on amylose and maltodextrins destruction by gluco-, alpha- and beta-amylases have been studied. The nature of DMAMF effects depends on the action mechanism of amylases and structure of their active sites. The effect observed is interpreted in terms of a hypothesis on a subsite structure of the amylase active centers.

Amylases↗

PubMed1991

Transglycosylation activity of cellobiohydrolase I from Trichoderma longibrachiatum on synthetic and natural substrates.

Using 4-methylumbelliferyl (MUF) beta-D-cellobioside as a substrate, the ability of cellobiohydrolase I from Trichoderma longibrachiatum to catalyze transglycosylation has been demonstrated. At substrate concentrations greater than 2 mM, the formation of MUF-tetrasaccharide was detected using HPLC. In the course of enzymatic reaction, a concentration of the transglycosylation product passed through a maximum, since at later stages of the reaction the product was further hydrolyzed. At MUF-beta-D-cellobioside concentrations of 2-10 mM, the maximum weight content of MUF-tetrasaccharide amounted to 1-4% of the total content of saccharides. In the reaction system, containing 2.5 mM MUF-beta-D-cellobioside and 10 mM MUF-beta-D-glucoside, MUF-trisaccharide was formed as the main transglycosylation product. In hydrolysis of natural substrates (cellulose and cellotriose) in the presence of MUF-beta-D-glucoside a formation of MUF-trisaccharide was also observed.

Cellobiose↗

PubMed1982

[Enzymatic hydrolysis of cellulose. Inactivation and stabilization of the enzymes of the cellulose complex].

The thermal inactivation of the individual cellulolytic components (endoglucanase, EC 3.2.1.4; exoglucosidase, EC 3.2.1.74; cellobiase, EC 3.2.1.21) from the fungi Trichoderma reesei, T. viride, T. lignorum an Aspergillus foetidus has been studied without resolution of the cellulase complexes. The kinetics of the thermal inactivation follow the first order for cellobiase of Asp. foetidus alone and show a more complex picture which is typical for a number of isoenzymes of different thermal stabilities for other cellulolytic components of all the cellulases under study. It was shown that selective elimination of acid proteinase from the cellulase preparations by affinity chromatography did not affect the time course of the thermal inactivation. Covalent attachment of the cellulases to porous glass and to some soluble high polymer supports only resulted in slight stabilizing effects (200-250%). Some polymer effectors (e. g. polyethylene glycols with Mr of 4000 and 40,000 and maltodextrins), as well as the end products of enzymatic hydrolysis of cellulose (i. e. glucose) did not affect the thermal stability of the cellulases under study. In some cases cellulose itself (cotton, CM-cellulose) produced a thermostabilizing effect (3-4-fold) on cellulolytic enzymes.

Aspergillus↗

PubMed1981

[Comparative role of exo-1,4-beta-glucosidase and cellobiase in the enzymatic hydrolysis of cellulose].

An inhibitory action of glucono-delta-lactone on individual components of cellulase complexes from Trichoderma reesei, T. longibrachiatum, T. lignorum and Aspergillus foetidus has been studied. It was shown that gluconolactone exerts an inhibiting effect on cellobiases only (the inhibition constants varied within the range of 0.03-0.1 mM) and does not influence the activities of endoglucanases, cellobiohydrolases and exoglucosidases of the complexes. This formed a basis for a new method for determination of the exoglucosidase activity in a mixture with other components of the cellulase complexes. The complete and selective inhibition of cellobiases by gluconolactone with exoglucosidases activity being intact allowed to evaluate the relative contribution of these enzymes in glucose formation in the course of enzymatic hydrolysis of cellulose (CM-cellulose, filter paper and Avicel). It was found that for most of the cellulase complexes studied the crucial role in glucose formation both from soluble and insoluble cellulose at early steps of hydrolysis belongs to exoglucosidase. On the other hand, the role of exoglucosidase (comparatively with cellobiase) progressively decreases in the course of cellulose hydrolysis. The latter effect does not presumably reflect the changes in the mechanism of cellulose conversion in the course of hydrolysis, but is due to a specific kinetic behaviour of the multienzyme cellulase system.

Aspergillus↗

PubMed1978

[Study of E. coli penicillin amidase. The pH-dependence of the enzymatic inactivation kinetics].

The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.

Amidohydrolases↗

PubMed1977

[Applicability of quantitative kinetic spectrophotometric method for glucose determination].

A simple kinetic method for quantitative determination of glucose has been developed. The method is based on two enzyme reactions, catalyzed by glucose oxidase (E. C. 1.1.3.4) and peroxidase (E. C. 1.11.1.7). The advantages of the method are high sensitivity (up to 2 X 10(-5) M glucose in a sample) and rapidity (3-4 min per analysis). The method gives reliable results for determination of small glucose concentrations in the presence of cellobiose. The conditions have been found when the reproducibility of the method does not depend on the glucose oxidase activity.

Disaccharides↗

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↗