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Biomedical subjects

A A Klesov

Publications and source records attributed to A A Klesov.

At least 19 recordsLinked to original sources
PubMed1990

Expression of the hAng gene in Escherichia coli; isolation and characterization of human recombinant Ser-(-1) angiogenin.

Recombinant human angiogenin has been synthesized in Escherichia coli with the aid of a human angiogenin gene (hAng) cloned by Neznanov et al (1990) from a human complementary DNA (cDNA) library. The gene has been expressed by use of a new type of expression vector called a 'TGATG vector' (plasmid pPR-TGATG-1; Mashko et al 1990a). The highest level of accumulation of the recombinant angiogenin (6%-8% of the total cell protein) was observed in E. coli strain BL21 carrying a temperature-amplifiable version of the plasmid. The synthesized polypeptide carries an additional serine residue at its N terminus in comparison with natural angiogenin. Furthermore, the initiator methionine residue of the recombinant protein is removed with high efficiency by E. coli terminal aminopeptidase. Simple procedures for purification of the recombinant angiogenin from the insoluble fraction of cell protein, and for refolding the protein allowed the isolation of almost 5 mg recombinant angiogenin g-1 wet bacterial biomass. The recombinant Ser-(-1) angiogenin displayed the same biological properties (specific RNAase activity and the ability to induce blood vessel growth on the sclera of experimental animals) as its natural counterpart isolated from human blood.

Amino Acid Sequence↗
PubMed1989

[A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E. coli. Some properties and identification in donor cells].

The properties of endoglucanase produced by the recombinant strain of E. coli carrying plasmid pCU 104 with a 2.9 kb insert of chromosomal DNA of C. thermocellum encoding the multiple forms of the 35.5 kD polypeptide (pI 4.3-4.7) were studied. The enzyme has a broad pH optimum of activity (6.0-7.5). The half-inactivation time for different forms of the enzyme at 65 degrees C is similar and is equal to 25-30 minutes. The enzyme is related to endoglucanases weakly adsorbed on cellulose (Kp = 0.065 1/g). Hydrolysis of microcrystalline cellulose is completed within 7 days (7-9%) and is accompanied by the formation of cellobiose and cellotriose. The enzyme splits dyed lichenan (mixed 1,3-1,4-beta-glucane) at a higher rate than the dyed CM-cellulose. A guinea pig antiserum to enzyme isoforms with a pI of 4.46-4.54 was obtained. Using direct solid phase immunoenzymatic analysis, it was demonstrated that all the enzyme isoforms under study (pI 4.3-4.7) are immunologically related (serum titers for different enzyme isoforms vary from 1:20,000 to 1:50,000). In the original culture fluid of C. thermocellum, the antigen related to the enzyme isolated from the recombinant strain was unobserved. However, SDS-PAAG electrophoresis of SDS- and mercaptoethanol-treated culture fluids revealed among 11 protein bands at least 4 antigens interacting with antibodies (Mr = 107, 76, 67 and 37 kD), although their antibody titers were far lower and did not exceed 1:300-1:500. The cumulative data suggest that the endoglucanase under study is not identical to the earlier described enzymes encoded by the cel A- and ceI B-genes of C. thermocellum.

Animals↗
PubMed1983

[Effects of the isoenzymes of endoglucanase from Trichoderma longibrachiatum differing in their ability to be adsorbed on cellulose and on soluble, amorphous and crystalline substrates: a different role of adsorption efficiency].

The mode of action of two purified endoglucanase (Mr 42 000) from Trichoderma longibrachiatum on soluble CM-cellulose as well as on amorphous and crystalline celluloses was studied. Both enzymes had the same Km values for CM cellulose (1.4 g/l), similar V values (51 and 47 mumole . min . mg of protein) and synthesized glucose and cellobiose from CM-cellulose in comparable amounts. However, the enzymes differed essentially in their ability to be adsorbed on soluble cellulose, i. e. their partition coefficients (cellulose surface/bulk solution) differed by one order of magnitude. When the enzyme concentrations on the cellulose surface were equal (in two independent experiments), the rate of degradation of amorphous (but not crystalline) cellulose was also almost identical. However, when crystalline cellulose was subjected to hydrolysis, the more tightly adsorbed enzyme revealed the initial solubilizing activity which was 20 times greater than that of the less tightly adsorbed enzyme (at the same endoglucanase activity on the surface). In the presence of high cellobiase concentrations sufficient to convert all intermediate cellobiose into glucose the effectively adsorbed endoglucanase was capable to convert at least 50% of crystalline cellulose to glucose.

Enzymes, Immobilized↗
PubMed1982

[Enzymatic hydrolysis of cellulose. Regulatory effect of the non-soluble substrate on the effectiveness of the enzymatic reaction].

It was shown that one of the cellulase components, i.e. cellobiase, can be adsorbed on cellulose surface with the concomitant decrease of activity (by 10 times and more). The specific activity of the adsorbed cellobiase depends on the enzyme concentration in the adsorption layer and is increased with the increase in the surface concentration of cellobiase. It was found that variations in the amount of non-soluble cellulose and the corresponding changes in cellobiase activity in the system (as a result of the adsorption) can lead to a certain alteration in the shape of the kinetic curves for formation of intermediate cellobiose, which in its turn controls the rate of formation of the end product, i.e. glucose. Thus, the substrate surface causes a regulatory effect on the rate and kinetic mechanism of the enzymatic conversion of cellulose to glucose due to the adsorption effects.

Cellulase↗
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↗
PubMed1982

[Non-steady state kinetics for action of a multienzyme cellulase system toward insoluble cellulose].

A kinetic theory for multienzyme cellulase systems in non-steady state conditions (in relation to intermediate metabolites) of hydrolysis of insoluble cellulose has been developed. To verify the kinetic regularities obtained the action of 12 different cellulases from the fungi Trichoderma, Geotrichum and Aspergillus as well as from bacterial cells Thermomonospora sp. and Rapidase preparation has been studied with respect to ball milled cotton linters and microcrystalline cellulose. The experimental data were discussed in the framework of mechanism of enzymatic hydrolysis of cellulose suggest by us recently and the main routes for glucose and cellobiose formation from insoluble cellulose have been elucidated. The effects of concentrations of cellulase preparations and the initial substrate on (i) time lags for glucose formation, (ii) stationary rate of glucose formation, and (iii) kinetics of cellulose conversion to glucose at low and high conversion degrees, were studied. A theoretical analysis of maxima and inflection points on the kinetic curves for intermediates (oligosaccharides and cellobiose) was performed. The effects and intensity of stirring on the time lag values for glucose formation were studied and the relative role of kinetic and diffusional factors in this process was evaluated. The data obtained are unequivocally indicative of the common mechanism of conversion of cellulose to glucose by cellulase complexes irrespective of their source and composition.

Actinomycetales↗
PubMed1982

[Adsorption of cellulolytic enzymes on cellulose and the kinetics of the adsorbed enzymes. Two modes for interaction of the enzymes with the insoluble substrate].

The affinity of cellulolytic enzymes of various origin for microcrystalline cellulose (MCC) in a column type reactor has been studied. It was shown that there exist two types of the enzymes differing in their ability to bind to cellulose and to degrade MCC. One group of cellulases is characterized by weak affinity for MCC and by a rather low degree of conversion of this substrate into soluble products, but shows a burst of soluble products during the initial period of the hydrolysis. On the contrary, the other group of the enzymes irreversibly binds to cellulose and shows a noticeable ability to solubilize MCC. The results obtained suggest that the weak binding reflects a productive enzyme--cellulose interaction by the enzyme active center, like its interaction with soluble polymeric substrates. The tight binding, on the other hand, is less productive and reflects the interaction of peripheral parts of the enzymes with the cellulose surface. The hydrolytic action of the tightly adsorbed cellulases proceeds on the substrate surface consecutively, without leaving the insoluble substrate between the catalytic acts and the enzymes might steadily penetrate into the cellulose matrix. This in turn might induce mechanical fragmentation of the substrate. The rate of action of adsorbed cellulases can be limited by their diffusion along the surface or into the cellulose matrix. A decrease of the ionic strength weakens the cellulase affinity for cellulose, which in its turn leads to an increase in the initial burst of the soluble products of cellulose hydrolysis and a respective decrease of efficiency of cellulose conversion into glucose. It is concluded that the principal factor which determines the ability of cellulases to degrade crystalline cellulose is the affinity (i. e. degree of adsorption) of the enzymes for the insoluble substrate.

Cellulase↗
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↗
PubMed1981

[Enzymatic conversion of polymers. Nature of apparent product inhibition in the course of enzymatic degradation of polymer substrates].

The general regularities for the kinetics of enzymatic conversion of polymers were studied. It was proposed that the formal kinetic analysis of progress kinetic curves for enzymatic degradation of polymers inevitably results in the apparent effects of product inhibition, even in the case when the products are not virtually bound to the enzyme and there is no real product inhibition at all. An experimental verification of this hypothesis was performed, using maltodextrins hydrolysis by Aspergillus niger glucoamylase as an example. It was shown that a progressive decrease of the enzyme reactivity with respect to a polymer substrate in the course of its degradation is kinetically equivalent to a progressive decrease of the velocity of an enzymatic reaction due to the product inhibition. These two cases cannot practically be distinguished by conventional methods of analysis of progress kinetic curves.

Aspergillus niger↗
PubMed1981

[Effect of composition of multienzyme cellulase complexes on rate-limiting steps for non-soluble (native) cellulose hydrolysis].

It was shown that the kinetics of multienzyme cellulase complexes are generally characterized by the absence of any single rate-limiting step of hydrolysis, even when the rates of individual steps of the process are substantially different. This peculiarity of the kinetic behaviour of cellulases can be explained in terms of certain "shunt ways" in a series of individual steps of enzymatic degradation of cellulose into glucose. A basic kinetic scheme for the degradation of cellulose was developed and proved experimentally for hydrolysis of milled cotton linters under the action of ten various cellulase complexes from the fungi Trichoderma, Geotrichum and Aspergillus. It was found that the value of the stationary rate of glucose formation in all cases is determined by the effect of two or three cellulolytic components of the complexes. It was concluded that the mechanism of native cellulose hydrolysis is the same for all cellulase complexes under study irrespective of their composition and origin.

Aspergillus niger↗
PubMed1980

[Effect of progressive chemical modification on the activity and thermal stability of soluble and immobilized glucoamylase].

The chemical modification of glucoamylase from Aspergillus niger with acryloylchloride has been studied. It was shown that the first 60--70% of the total amount of available amino groups of the enzyme readily interact with the reagent: the activity and thermal stability of the resulting soluble acryloylglucoamylase are only slightly less than those of the native enzyme. The modification of the remaining 30% of the total amount of amino groups is less intensive and results in a sufficient loss of activity (6 times at 92% modification) and thermal stability (3,5 times at the same degree of modification) by the enzyme. The multipoint immobilization of glucoamylase by mutual covalent attachment of its acryloyl derivative to polyacrylamide gel also causes destabilization of the enzyme. The resulting increase of the number of the enzyme--support binding points leads to a progressive decrease of the enzyme thermal stability as compared to native glucoamylase despite a slight increase in stability (up to 1,5 times) as compared to soluble acryloylglucoamylase. It was shown that destabilization of glucoamylase at 65 degrees due to chemical modification is more pronounced than its negligible stabilization due to the multipoint fixation of glucoamylase on a high polymer support.

Acrylates↗
PubMed1980

[Purification and properties of low molecular weight endoglucanase of the cellulase complex from Trichoderma koningii].

A homogenous low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) has been isolated from a crude commercial preparation of cellokoningine P10X of T. koningii origin. The molecular weight of the enzyme as determined by polyacrylamide gel electrophoresis is 13 000. The endoglucanase was obtained as a lyophylized preparation free of the cellobiase activity. It was shown that cellobiose or methylcellobioside activate the effect of the low molecular weight endoglucanase (measured by the viscometric technique with respect to CMC hydrolysis) and at the same time almost completely suppress the activity of high molecular weight endoglucanases from the sane source. A detailed kinetic study of the effects showed that the low molecular weight enzyme is activated by a transglycosylation mechanism, where cellobiose acts as an added nucleophile. The activation is 6-fold at saturating concentrations of cellobiose (Ks = 15 mM). It was shown that diverse kinetic behaviour of cellobiose which can act both as activatory and inhibitor for endoglucanases from different sources can be explained, firstly, by different ratios of low to high molecular weight endoglucanases in the cellulase complexes, and, secondly, by their ability to catalyze transglycosylation reactions, which, in turn, results in a transfer of reducing end groups of the reaction products onto cellobiose as an added nucleophile.

Cellobiose↗
PubMed1980

[Relationship between the kinetics of hydrolysis of soluble and native celluloses under the action of cellulase multi-enzyme complexes].

The kinetics of cellulase multi-enzyme complex under steady- and non-steady-state conditions have been studied. To verify the kinetic regularities obtained the action of eight different cellulases from Trichoderma, Geotrichum and Aspergillus has been studied with respect to ball milled cotton linters. On the other hand, the activities of the individual components of cellulase complexes, i.e. endoglucanase, exoglucosidase and cellobiase have been determined with respect to soluble cellulose derivatives. Theoretical and experimental evidence for the existence of a linear correlation between the rate of hydrolysis of native cellulose under certain experimental conditons, on the one hand, and that of its soluble polymeric compounds (particularly, carboxymethyl cellulose) on the other, have been obtained. The kinetics of the cellulase multi-enzyme system suggest that this correlation is a substantiation of the fact that endoglucanse is a cellulolytic component which is the first to attack native cellulose. The data obtained contradict the hypothesis being discussed in literature for the last 30 years about the presence in cellulase complexes of some "pre-hydrolytic C1-enzyme", which presumably attacks native cellulose. Evidently, a hypothetical "C1-enzyme" is the endoglucanase of random action.

Aspergillus↗
PubMed1980

[Hydrolysis of microcrystalline cellulose by multienzyme cellulase complexes of various origins].

The kinetic regularities of glucose and cellobiose formation from microcrystalline cellulose (MCC) under the action of cellulase complexes from eight different sources were studied. By means of successive addition of selected components of the cellulase complexes (endoglucanase and cellobiase) to the reaction system the rate-limiting steps for multienzymatic hydrolysis of MCC were determined. It was shown that in most cases the rate-limiting step of glucose formation (via hydrolysis of the intermediate cellobiose) is the cellobiase action. In a single case only (with a cellulase complex from Aspergillus foetidus enriched with cellobiase) the rate of glucose formation from MCC was limited by the endoglucanase action. In accordance with the kinetic theory developed it was shown that the addition of cellobiase excess to the reaction system resulted in changes of the rate-limiting step over to endoglucanase attack on the non-soluble cellulose for all cellulase complexes under study. Under the given experimental conditions a linear correlation between the steady-state ready of glucose formation from MCC under the action of all cellulase complexes on the on hand, and the endoglucanase activity of these complexes, on the other, was established. It was shown that the action of all cellulase (arbitrarily selected ones) is described by principally the same kinetic regularities, which, in turn, is indicative of identical mechanisms for hydrolysis of the insoluble cellulose under effects of cellulase complexes of various origin.

Actinomycetales↗
PubMed1979

[Substrate thermostabilization of soluble and immobilized glucoamylase].

A new kinetic approach to the study of enzyme thermal inactivation in the presence of a substrate, which influences the rate of inactivation has been developed. The method was applied to investigation of inactivation kinetics of soluble and porous silica-immobilized glucoamylase. It was found that the binding of a substrate (maltose or maltodextrines Star-Dri 24-R) increases the thermal stability of glucoamylase, the stabilizing effect being more pronounced in the case of the soluble enzyme (40-fold stabilization) as compared to the immobilized one (15-fold stabilization). The stabilizing effect does not depend on the length of the substrate (maltose, d. p. 2 or dextrines, d. p. 7). Glucose, a product of the enzymatic hydrolysis, has a much lower stabilizing effect. It was concluded that the main role in the glucoamylase thermostabilization is played by the substrate stabilization rather than by the immobilization itself (3-fold stabilization). However, a combined effect of thermostabilization of glucoamylase due to both immobilization and/or substrate stabilization is restricted by the same limit of value for immobilized and soluble enzymes, which is equal to 40--50-fold in comparison with the soluble enzyme in the absence of the substrate.

Aspergillus niger↗
PubMed1979

[Cellobiose as a regulator of endoglucanase activity of cellulase complexes. Mechanism of the regulation].

Cellobiose may exert different effects on the activities of various endoglucanases. The endoglucanases of T. reesei and Rapidase are noticeably suppressed by cellobiose at concentrations above 3 mM. On the other hand, a low molecular weight endoglucanase from T. koningii is activated by cellobiose, whereas high molecular weight endoglucanases from the same source are inhibited by cellobiose. A detailed kinetic analysis of the effects showed that the low molecular weight endoglucanase is activated by a transglycosylation mechanism, in which cellobiose acts as an additional nucleophile. At saturating concentrations of cellobiose (Ks = 15 mM) the enzyme activity is increased 6-fold. Such a specific mechanism of activation manifests itself in an acceleration of random cleavage of CM-cellulose by the low molecular weight endoglucanase, which can be recorded by a viscosimetric technique. However, its action does not accelerate the production of soluble reducing sugars.

Cellobiose↗
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↗