[New conjugates of antitumor antibiotic doxorubicin with water-soluble galactomannan: synthesis and biological activity].

New water-soluble conjugates in the form of Schiff bases (DGM-1 and DGM-2) were prepared by the interaction of water-soluble periodate-oxidized galactomannan with doxorubicin or N-(L-lysyl)doxorubicin, respectively. The water-soluble galactomannan (DAVANAT a commercial product of Pro-Pharmaceuticals company) was obtained by partial acidic hydrolysis of high-molecular-mass galactomannan from Cyamopsis tetragonoloba (guar gum) seeds. The conjugate stability was studied in aqueous solutions. The DGM-1 antiproliferative activity was comparable with that of doxorubicin on three models: cell lines of murine melanoma B 16-F1, human breast cancer MCF-7 (HTB-22), and human colon cancer HT-29 (HTB-38). DGM-2 was poorly active in all the three tests. DGM- 1 can thus be regarded as a high-molecular-mass depot form of doxorubicin.

[The quantitative assessment of the biological activity of angiogenin]. / Kolichestvennaia otsenka biologicheskoi aktivnosti angiogenina.

Methods have been developed for the in vivo quantitative estimation of biological activity of angiogenin, a protein growth factor of blood vessels. The quantitative analysis of angiogenin with the methods of angiogenetic disks in isotopic modification requires nanogram amounts of the angiogenetic. The cutireaction test is less sensitive though technically simple. The methods are developed on animal (mouse and rat) tissues.

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.

[A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E. coli. Some properties and identification in donor cells]. / Novyi tip éndogliukanazy Clostridium thermocellum, obrazuemoi rekombinantnym shtammom E. coli. Svoistva i identifikatsiia v organizme donora.

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.

[Mathematical modeling of flow-tube extracorporeal shunts with immobilized enzymes]. / Matematicheskoe modelirovanie protochnykh trubchatykh ékstrakorporal'nykh shuntov s immobilizovannymi fermentami.

A complex of computer programs "IF-reactor" for model calculations and processing of experimental data related to flow tube extracorporeal shunts with immobilized enzymes has been developed. Qualitative and quantitative comparison of theory with experiment has been made.

[Rapid method for determining cellobiase activity]. / Ekspressnyi metod opredeleniia aktivnosti tsellobiaz.

A simple and rapid method for determining the cellobiase activity in purified enzyme preparations was developed. It is based on a series of consecutive enzymatic reactions, i. e. hydrolysis of cellobiose by cellobiase, oxidation of the forming glucose by glucose oxidase, and formation of a dyed product under peroxidase action in the same reaction system. The dyed product is recorded spectrophotometrically at 460 nm. One measurement takes from 2-3 to 7-10 min depending on a particular method of the activity determining. The reagent which is used for the activity determining can be obtained in the yophylized form and used repeatedly. The relative deviation of the method is 5-7%.

[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]. / Deistvie izofermentov éndogliukanazy Trichoderma longibrachiatum, otlichaiushchikhsia po prochnosti adsorbtsii na tselliuloze, na rastvorimy, amorfnyi i kristallicheskii substraty: razlichnaia rol' éffektivnosti adsorbtsii.

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.

[Ability of cellulases to degrade crystalline cellulose as a result of their effective adsorption on the substrate: experimental confirmation and theoretical interpretation]. / Sposobnost' tselliulaz k degradatsii kristallicheskoi tselliulozy kak rezul'tat ikh éffektivnoi adsorbtsii na substrate: éksperimental'noe podtverzhdenie i teoreticheskaia interpretatsiia.

It was shown experimentally that the increase in the adsorption ability of cellulases leads to a sharp increase of the rate of the enzymatic degradation of crystalline cellulose. At the same time the rate of degradation of amorphous cellulose does not depend on the adsorption ability of cellulases. These effects have been explained using a model which takes into consideration the influence of the structure of the solid substrate on the surface mobility of the adsorbed molecules of the enzyme. The equations were derived which inter-relate the catalytic and the adsorptive properties of cellulases.

[Enzymatic hydrolysis of cellulose. Inactivation and stabilization of the enzymes of the cellulose complex]. / Fermentativnyi gidroliz tselliulozy. Inaktivatsiia i stabilizatsiia fermentov tselliulaznogo kompleksa.

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.

[Regulation of the formation and conversion of intermediate cellooligosaccharides and cellobiose during ezymatic hydrolysis of insoluble cellulose]. / Zakonomernosti obrazovaniia i raskhodovaniia promezhutochnykh tsellooligosakharidov i tsellobiozy pri fermentativnom gidrolize nerastvorimoi tselliulozy.

A kinetic analysis of the main regularities for formation and consumption of the intermediate metabolites (i.e. cellooligosaccharides and cellobiose) in the course of enzymatic hydrolysis of cellulose has been performed. The boundary relationships between the velocities of the individual steps of hydrolysis controlling the transition of the multienzyme cellulase system to be steady-state with respect to the intermediate metabolites were revealed. Using the kinetic approaches developed here, the values of the steady-state concentrations of insoluble intermediate oligosaccharides were determined experimentally for a number of cellulases from the fungi Trichoderma and Geotrichum. It was shown experimentally that when studying the kinetics of enzymatic hydrolysis of cellulose it is necessary to take into account the structural irregularities of the insoluble substrate even when a practically negligible fraction of it (with respect to its weight) is converted to soluble products. In other cases the kinetic parameters obtained could not be corresponding to the actual reactivity of the cellulose/cellulase system, reflecting rather a difference in reactivity between the labile (amorphous) and stable (crystalline) fractions of a cellulose substrate.

[Adsorption of cellulolytic enzymes on cellulose and the kinetics of the adsorbed enzymes. Two modes for interaction of the enzymes with the insoluble substrate]. / Adsorbtsiia tselliuloliticheskikh fermentov na tselliuloze i kinetika deistviia adsorbirovannykh fermentov. Dba tipa vzaimodeistviia fermentov s nerastvorimym substratom.

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.

[Enzymatic hydrolysis of cellulose. Regulatory effect of the non-soluble substrate on the effectiveness of the enzymatic reaction]. / Fermentativnyi gidroliz tselliulozy. Reguliatornoe vliianie nerastvorimogo substrata na éffektivnost' fermentativnoi reaktsii.

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.

[Non-steady state kinetics for action of a multienzyme cellulase system toward insoluble cellulose]. / Kineticheski zakonomernosti gidroliza nerastvorimoi tselliulozy pod deistviem polifermentnykh tselliulaznykh sistem v nestatsionarnom rezhime reaktsii.

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.

[Enzymatic conversion of polymers. Nature of apparent product inhibition in the course of enzymatic degradation of polymer substrates]. / Fermentativnoe prevrashchenie polimerov. O prichinakh kazhushchegosia ingibirovaniia produktami v reaktsiiakh fermentativnoi degradatsii polimernykh.

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.

[Comparative role of exo-1,4-beta-glucosidase and cellobiase in the enzymatic hydrolysis of cellulose]. / Sravintel'naia rol' ekzo-1,4-beta-gliukozidazy i tsellobiazy pri fermentativnom gidrolize tselliulozy.

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.

[Effect of composition of multienzyme cellulase complexes on rate-limiting steps for non-soluble (native) cellulose hydrolysis]. / Vliianie sostava polifermentnykh tselliulaznykh kompleksov na kharakter stadii, limitiruiushchikh skorost' gidroliza nerastvorimoi (prirodnoi) tselliulozy.

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.

[Hydrolysis of microcrystalline cellulose by multienzyme cellulase complexes of various origins]. / Gidroliz mirokristallicheskoi tselliulozy pod deistviem polifermentnykh tselliulaznykh kompleksov razlichnogo proiskhozhdeniia.

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.

[Effect of progressive chemical modification on the activity and thermal stability of soluble and immobilized glucoamylase]. / Vliianie posledovatel'noi khimicheskoi modifikatsii na aktivnost' i termostabil'nost' rastvorimoi i immobilizovannoi gliukoamilazy.

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.

[Purification and properties of low molecular weight endoglucanase of the cellulase complex from Trichoderma koningii]. / Ochistka i svoistva nizkomolekuliarnoi endogliukanazy tselliulaznogo kompleksa iz 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.