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.

A method for detection of cellulases in polyacrylamide gels using 5-bromoindoxyl-beta-D-cellobioside: high sensitivity and resolution.

The assay of endo-1,4-beta-glucanases (cellulases) from Trichoderma reesei, T. longibrachiatum, and Sporotrichum pulverulentum by 5-bromoindoxyl-beta-D-cellobioside is described. The substrate is enzymatically cleaved to afford 5-bromoindoxyl and latter undergoes immediate azo coupling with Fast Red or oxidation by nitroblue monotetrazolium chloride, various forms of endoglucanases which can thus be assayed in polyacrylamide gel.

Continuous photometric determination of endo-1,4-beta-D-glucanase (cellulase) activity using 4-methylumbelliferyl-beta-D-cellobioside as a substrate.

A very simple and sensitive procedure for the determination of the activity of highly purified endo-1,4-beta-glucanase from the microscopic fungus Trichoderma reesei using 4-methylumbelliferyl-beta-D-cellobioside has been developed. The HPLC study has shown that this substrate is cleaved by endo-1,4-beta-glucanase to form predominantly free 4-methylumbelliferone, Km and kcat being 1.25 mM and 7.9 s-1, respectively (30 degrees C, pH 5.0). The possibility of continuous photometric determination of the enzyme using the difference absorptivity coefficient of 1600 M-1 cm-1 at 350 nm has been demonstrated.

[A new approach to titrating active enzyme centers upon the use of surface-active micellar substances in organic solvents]. / Novyi podkhod k titrovaniiu aktivnykh tsentrov fermentov pri ispol'zovanii mitsell poverkhnostno-aktivnykh veshchestv v organicheskikh rastvoriteliakh.

Micellar solutions of surfactant in organic solvents with rubber additions are proposed for determination of active enzyme concentration. A kinetic theory of enzymatic reactions in reversed micellar systems is developed, suggesting the intermicellar transport of the substrate to be the limiting step in viscous medium. Under these conditions, it is shown that fraction of the product formed after quick transformation of the substrate located in the enzyme-containing micelles depends upon active enzyme concentration and aggregation number of surfactant molecules. The proposed approach is used for the active-site titration of trypsin and cellobiase and for the determination of the aggregation number of Aerosol OT (AOT) molecules in the ternary system AOT/water/cyclohexane.

[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.

[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.