[Screening of the Producents of α-L-Rhamnosidases Amongst Representatives of Penicillium].

The aim of this work was to study α-L-rhamnosidase [КФ 3.2.1.40] - enzyme, which hydrolyse the terminal non-reduced α-1,2-, α-1,4- and α-1,6-linked L-rhamnose. As a result of screening conducted among 30 strains of micromycetes ability to synthesize α-L-rhamnosidase revealed in Penicillium tardum 60, 39, 2929, 2962, 2963, 2964, 2965, 2966, P. rugulosum 2778, 1652, 2766, P. restrictum 425, 2756, P. aculeatum 202, 217, 329, 2973, 2974, 2975, 2976, 2977, 2979 activity, which ranged from 0.07 to 0.53 OD/mg protein. The most active is brought out P. amleatum 202. From culture supernatant of this micromycete by fractionation with ammonium sulfate (90 % saturation) complex enzyme preparation was obtained and its physico-chemical properties were studied. It was shown that enzyme has pH optimum 3.0, thermooptimum - 60 °C and displayed stability in pH values from 2.0 to 4.0 during 90 min. At pH 5.0 the activity of complex enzyme preparation insignificantly decreased and appears to be up 40 % from initial one. At optimal pH value 3.0 and temperature 15 °C α-L-rhamnosidase tested was stable during 3 days. In addition to α-L-rhamnosidase enzyme preparation of P. arnleatum 202 exerted also ß-D-glucosidase activity.

[Kinetic characteristics of alpha-L-rhamnosidase of Eupenicillium erubescens].

It was established, as a result of investigations of the alpha-L-rhamnosidase of Eupenicillium erubescens kinetic properties, that K(m) and V(max) for the corresponding synthetic substrate were 1.0 mM and 120 micromol/min/mg of protein, respectively. alpha-L-Rhamnosidase was also competitively inhibited by the reaction product- L-rhamnose, the inhibition constant was 5.2 x 10(-2) M. One could also observe the inhibition of alpha-L-rhamnosidase reaction in the presence of 10(-3) M of glucose. It was shown that the rate of enzymatic hydrolysis of nitrophenyl substrate was directly proportional to the concentration of enzyme, and the increase of the substrate concentration leads to the increase of hydrolysis rate. The substrate concentration being increased above the optimal one (5.0 mg/ml), the reaction rate decreases due to the formation of inactive enzyme-substrate complex FS2.

[Purification and physico-chemical properties of Eupenicillium erubescens alpha-L- rhamnosidase].

A scheme of isolation and purification of the enzyme with alpha-L-rhamnosidase activity has been developed. It included fractionation by ammonium sulfate and chromatography on TSK-gels Toyopearl HW-60, Fractogel TSK DEAE-650-s and Sepharose 6B. The enzyme was purified 60 times with the yield of 0.5%. The enzyme preparation did not contain any glycosidase and proteolytic activity. Molecular mass of the alpha-L-rhamnosidase enzyme on the data of Sepharose 6B gel-filtration was 35 kDa. The enzyme preparation was stable during 48 hours at 20 degrees C, its pH- and thermal optimum were 5 and 60 degrees C, correspondingly.

[Influence of metal ions and specific chemical reagents on activity of alpha-L-rhamnosidase of Eupenicillium erubescens].

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide methiodide, EDTA, o-phenanthroline, dithiotreitol, L-cysteine, beta-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the activity of alpha-L-rhamnosidase of Eupenicillium erubescens has been investigated. The essential role of Ag+ and Hg2+ which inhibit the alpha-L-rhamnosidase activity by 47-73% has been shown. Whereas L-cysteine exhibits the protective effect, rhamnose in concentration of 1-5 mM does not protect the enzyme from negative effect of Ag+ and Hg2+. Basing on the inhibitory and kinetic analysis it was supposed that the carboxyl group of C-terminal aminoacid and imidazole group of histidine take part in the catalytic action of alpha-L-rhamnosidase. It was assumed that sulphydryl groups took part in catalysis, carried out by alpha-L-rhamnosidase of E. erubescens, since the activity of alpha-L-rhamnosidase inhibited by p-ChMB and thiol reagents such as dithiothreitol, L-cysteine, beta-mercaptoethanol did not remove its inhibitory action.

[Coordinative compounds of zinc with N-substituted thiocarbamoyl-N'-pentamethylensulfenamides--activity modifiers of enzymes of proteolytic and glycolytic action].

The influence of a number of coordinative compounds of zinc with N-substituted thiocarbamoil-N'-pentamethylensulfenamides on activity of elastase, alpha-L-rhamnosidase and alpha-galactosidases evidence for a possibility of their usage as stimulators or inhibitors of enzymes tested have been studied. It was shown that all the compounds in concentration of 0.1 and 0.01% inhibited by 90-100% Bacillus thuringiensis 27-88Els+ elastase activity. [Zn(L2)Br2], [Zn(L1)(NCS)2] and [Zn(L3)(NCS)2] at 20 h exposition activated Cryptococcus albidus 1001 alpha-L-rhamnosidase activity. The rest of compounds influenced it on the control level or inhibited it by 7-23%. The obtained results testify that essential role is not played by separate fragments (L-ligand and anions), but by molecules of zinc complexes as a whole. All the studied complexes, exept for [Zn(L3)(NCS)2], induced alpha-L-rhamnosidase activity of Eupenicillium erubescens 248 (7 to 60%). All zinc compounds (concentration 0.01%, exposition time - 60 min) influenced at the control level Aspergillus niger and Cladosporium cladosporioides alpha-galactosidases activity, however inhibited (up to 20%) activity of Penicillium canescens alpha-galactosidase. The increasing of exposition time of the compounds tested with enzymes up to 20 h testify to selective action of separate compounds on enzymes tested. The data obtained prove, that the character of interaction of zinc complexes is changed depending on the enzyme tested and its strain-producer.