As a result of the screening of glycosidase activities among 1,330 strains of micromycetes, yeasts, and bacteria isolated from soils of temperate latitudes, Antarctica, the Chornobyl Exclusion Zone, the water of rivers and seas, bottom sediments of the Black Sea, plants, invertebrates, and industrial waste new data were obtained on distribution of α-galactosidases and α-L-rhamnosidases among microorganisms of different taxonomic and ecological groups. The dominant groups of α-galactosidase and α-L-rhamnosidases producers were both collection and freshly isolated soil micromycetes of the genera Aspergillus, Fusarium, and Penicillium. For the first time, β-mannanase and α-galactosidase activities were detected for the species Rhizomucor oryzae, Penicillium cyclopium, and P. expansum, and α-L-rhamnosidase activities were described for the species P. restrictum and P. roseopurpureum.
Glycosidases were isolated from the culture fluid of Eupenicillium erubescens, P. commune, P. tardum, P. restrictum, P. canescens, Cryptococcus albidus, Aspergillus niger, Cladosporium cladosporioides and purified to a homogeneous state. A comparative study of the component composition, physicochemical, catalytic, and kinetic properties made it possible to show a wide variability of characteristics of glycosidases in microorganisms of different species. All glycosidases, except α-L-rhamnosidases of P. commune, had high activity and stability in the pH range of 4.0-6.0 and thermal optimum at 60 and 65 oС. It was shown for the first time that α-L-rhamnosidases of C. albidus and E. erubescens, obtained when grown on different carbon sources (rhamnose or naringin), display different stability under thermal denaturation conditions. In the composition of P. commune, P. tardum, C. albidus, and E. erubescens α-L-rhamnosidases the carbohydrates were found, the content of which was 15, 12, 5, and 1 %, respectively. The mixed pattern of glycosylation of α-galactosidases from A. niger, P. canescens, and C. cladosporioides was established. The process of thermal inactivation of microbial oligomeric α-galactosidases and monomeric α-L-rhamnosidases was studied. It was established that the thermal inactivation of A. niger and C. cladosporioides α-galactosidases at 55 and 60 oC corresponds to the kinetic mechanism of two-stage dissociative thermal inactivation of the oligomer. The imidazole group of histidine (electrophile) and the carboxyl group of dicarboxylic acids (nucleophile) are assumed to be functionally active groups of all glycosidases, as well as the conformational role of the SH-groups of cysteine. It was established that complex compounds of germanium and divalent metals are effective activators of E. erubescens, P. tardum, P. restrictum, and C. albidus α-L-rhamnosidases. Complex zinc compounds with N-substituted thiocarbamoyl-N′-pentamethylene sulfenamides had an inhibitory effect on A. niger, C. cladosporioides, and P. canescens α-galactosidases. The most effective stabilization methods of α-galactosidases were immobilization on celluloses, dextrans, precipitation on polyethylene glycols, hydrophobic modification with alcohols and succinic anhydride, and encapsulating in lecithin capsules. The stabilization strategy of α-L-rhamnosidases consisted of inhibiting primary reversible stages of thermal inactivation, hydrophobic modification, and aggregation. Therefore, the proposed methods made it possible obtaining of α-galactosidases and α-L-rhamnosidases preparations for long-term storage and repeated use. Advantageous functional properties and substrate specificity of studied enzymes for modifying rhamno- and galactoglycosides suggest their broad-range applicability for food and animal feed processing, as well as the pharmaceutical industry.
