Tag Archives: α-galactosidase
Influence of chemical reagents and UV irradiation on the activity of Penicillium canescens α-galactosidase
N. V. Borzova, L. D. Varbanets
Danylo Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
е-mail: nv_borzova@bigmir.net
Investigations of the influence of chemical and physical factors on the conformational and functional properties of enzymes make a significant contribution to the study of the mechanism of action of industrially important proteins. The aim of the work was to evaluate the effect of chemical reagents and UV irradiation on the catalytic properties of Penicillium canescens α-galactosidase. Enzyme activity was assessed with p-nitrophenyl-α-D-galactopyranoside. Studies of the functionally active glycosidase groups were carried out on the basis of inhibitory and kinetic analysis using Dixon and Luinuiver-Burke methods with help of specific chemical reagents. A significant decrease in the activity of α-galactosidase in the presence of carbodiimides, diethylpyrocarbonate, the reagents on sulfhydryl groups was shown. A UV-induced decrease in enzyme activity in the dose range of 900-7200 J/m2 was noted. Based on the data obtained, the imidazole group of histidine, carboxyl groups of C-terminal amino acids and the SH-groups of cysteine are assumed to play an important role in the manifestation of the activity of P. canescens α-galactosidase.
Stability of native and modified α-galactosidase of Cladosporium cladosporioides
N. V. Borzova, L. D. Varbanets
Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
e-mail: nv_borzova@bigmir.net
By modifying carbohydrate component of glycoproteins it is possible to elucidate its role in manifestation of structural and functional properties of the enzyme. The comparison of activity and stability of the native and modified by oxidation with sodium periodate α-galactosidase of Cladosporium cladosporioides was carried out. To determine α-galactosidase activity the authors used n-nitrophenyl synthetic substrate, as well as melibiose, raffinose and stachyose. Modification of the carbohydrate component had a significant effect on catalytic properties of the enzyme. Both the reduction of Vmax and enzyme affinity for natural and synthetic substrates were observed. The native enzyme retained more than 50% of the maximum activity in the range of 20-60 °C, while for the modified enzyme under the same conditions that temperature range was 30-50 °C. The modified α-galactosidase demonstrated a higher thermal stability under neutral pH conditions. The residual activity of the modified α-galactosidase was about 30% when treated with 70% (v/v) methanol, ethanol and propanol. About 50% of initial activity was observed when 40% ethanol and propanol, and 50% methanol were used. It was shown that the modification of C. cladosporioides α-galactosidase by sodium periodate is accompanied by a significant decrease in enzyme activity and stability, probably caused by topological changes in the tertiary and quaternary structure of the protein molecule.
Role of glycosylation in secretion and stability of micromycetes α-galactosidase
N. V. Borzova, O. V. Gudzenko, L. D. Varbanets
Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
e-mail: nv_borzova@bigmir.net
The effect of the glycosylation inhibitors (tunicamycin and 2-deoxy-D-glucose) on the activity, stability and production of fungal glycosidases has been studied. It was shown that inhibition of N-glycosylation sites did not affect the secretion of Aspergillus niger α-galactosidase, however reduced yield of Cladosporium cladosporioides and Penicillium canescens α-galactosidases. Changes in the level of O-glycosylation resulted in a significant reduction in the activity and stability of α-galactosidases of all three producers tested. Activity of the modified enzymes was significantly lower than that of the native ones, and was 2.6 and 0.33 U/mg for A. niger α-galactosidase, 3.3 and 32.5 U/mg for C. cladosporioides α-galactosidase, 11.66 and 31.1 U/mg for P. canescens α-galactosidase, respectively. A. niger α-galactosidase completely lost activity during purification and storage. The decrease of thermal stability at 55 °C by 20% was shown for C. cladosporioides and P. canescens α-galactosidases. It was also noted that O-deglycosylation led to a decrease in resistance of these enzymes to the action of proteases.