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M L Rabinovich

Publications and source records attributed to M L Rabinovich.

9 recordsLinked to original sources
PubMed1991

[Cellobiohydrolase from Clostridium thermocellum, synthesized by a recombinant E. coli strain].

Clostridium thermocellum cellobiohydrolase was isolated in preparative amounts from the recombinant strain of E. coli K12 C600 carrying plasmid pCU 304 with a C. thermocellum chromosomal DNA insertion. The isolation procedure included chromatography on Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose CL-6B, rechromatography on Ultrogel and FPLC on Mono Q resulting in a 17.6% yield and 1530-fold purification. According to data from sodium dodecylsulfate polyacrylamide gel electrophoresis performed under nondenaturing conditions and analytical gel isoelectrofocusing, the enzyme preparation contains only one active protein band with Mr 56.2 +/- 1.0 kDa and pI 4.15. The enzyme does not reduce the viscosity of the CM-cellulose solution but forms reducing sugars from this soluble substrate. Cellobiose (93-97%) is the major component produced by the enzyme from crystalline and amorphous cellulose (specific activity 2.3 x 10(-3) and 2.8 x 10(-2) U/mg, respectively). The activity optimum of the enzyme is at pH 5.6, 60 degrees C. The half-inactivation time at 60 degrees C and 65 degrees C is 450 and 15.5 min, respectively. The action pattern of the enzyme on the low molecular fluorogenic cellooligosaccharides suggests that the enzyme pertains to typical cellobiohydrolases.

Binding Sites↗
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

[Ability of cellulases to degrade crystalline cellulose as a result of their effective adsorption on the substrate: experimental confirmation and theoretical interpretation].

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.

Adsorption↗
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

[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↗
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

[Rapid method for determining cellobiase activity].

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%.

Aspergillus↗
PubMedDate not supplied

[Properties of Fusarium graminearum galactose oxidase].

The kinetics and action mechanism of the galactose oxidase from Fusarium graminearum were studied. pH-optimum of the enzyme activity and stability was 7.0, the activity and stability of the galactose oxidase being decreased at any other values of pH. The enzyme is destabilized at acidic pH that is connected with protonization of its ionogenic group with pK 4.7. The temperature optimum of the galactose oxidase is 35 degrees C. When studying the enzyme thermoinactivation, it was found that at temperatures below 30 degrees C the energy of activation of denaturation was about 40 kcal/mole and at temperatures ranging from 30 to 70 degrees C - 13 kcal/mole. On the basis of the data obtained it was concluded that a low-temperature form of the galactose oxidase, possessing a higher energy of activation of denaturation, is more active than a high-temperature form. The value of Km for the enzyme in respect to galactose was 0.19 M, and the value of Vmax = 360 mumole/min per g of the preparation.

Drug Stability↗