[Optimization of culture conditions of Aspergillus niger for the synthesis of alpha-N-acetylgalactosaminidase and alpha-galactosidase]. / Optimizatsiia uslovii kul'tivirovaniia Aspergillus niger, sinteziruiushchego al'fa-N-atsetilgalaktozaminidazu i alfa-galaktozidazu.

Different factors have been investigated for their effect on the process of biosynthesis of alpha-N-acetylgalactosaminidase and alpha-galactosidase of Aspergillus niger under deep-water cultivation. Optimal sources and concentrations of carbon (soy flour--25 g/l) and nitrogen (pepton--7.5 g/l) were estimated. It has been established that temperature of 25 degrees C, pH 6.0, growing in 50 ml medium at the swing velocity 220 rev/min. for 6-8 days are optimal cultivation parameters. It has been shown that dry borine blood (in concentration of 1%) and a number of guanidine derivatives (guanidine carbonate--0.25%, nitroaminoguanison dimethylaminobenzaldehyde--0.05%, nitroaminoguanison of salicylic aldehyde--0.1%) can play the part of inducers of the mentioned glycosidase synthesis. When growing fungal culture in selected conditions synthesis of enzymes increased almost 3 times. Activity of alpha-N-acetylgalactosaminidase was 0.46 E/ml, and alpha-galactosidase--1.9 E/ml.

[Study of functional groups in the active center of alpha-galactosidase of Penicillium sp. 23]. / Issledovanie funktsional'nykh grupp v aktivnom tsentre alpha-galaktozidazy Penicillium sp. 23.

Effect of metal ions and specific chemical reagents (EDTA, sodium aside, o-phenanthroline, n-chlormercurybenzoate, iodacetamide, N-ethylmaleinimide, L-cystein, dithiotreitol, beta-mercaptoethanol) on activity of alpha-galactosidase isolated from the culture liquid of micromycete Penicillium sp. 23 has been studied. It has been established that alpha-galactosidase is not metalloenzyme (lack of the inhibitor effects under treatment of enzyme by EDTA, sodium aside, o-phenanthroline). The heavy metal ions (Ag+ and Hg2+) inhibit the rate of alpha-galactosidase reaction; Ki for Ag+ and Hg2+ ions makes up 3.3 x 10(-5) and 3.0 x 10(-7) M, respectively. It has been established by the inhibitory and kinetic analysis that a group (groups) with ionization constant about 6.0, are in the enzyme active centre which apparently corresponds to histidine imidasole group. The sulfhydryl groups do not take part in catalysis but play the important role in maintaining active conformation of the protein molecule.

[Screening of alpha-N-acetylgalactosaminidase producers among microorganisms of different taxonomic groups]. / Skrining produtsentof al'fa-N-atsetilgalaktozaminidazy sredi mikroorganizmov razlichnykh taksonomicheskikh grupp.

Screening of producers of alpha-N-acetylgalactosaminidase among 854 strains of micromycetes, 171 yeast and 357 bacterial cultures has been carried out. A capacity to synthesize the enzyme was revealed in 11% of cultures. Representatives of Aspergillus genus (activity--0.11-0.142 un./ml) were most active in producing the enzyme. It has been established that glucosidases spectrum in the culture liquid of 18 most active strains was characterized by complete homogeneity and by the presence of a rather high level (0.5-0.9 un./ml) of alpha-galactosidase activity. Complex preparations of alpha-N-acetylgalactosaminidase and alpha-galactosidase have been obtained from the culture liquid of producers by fractionation by ammonium sulphate (30 and 90% saturation); their pH- and thermo-optimum, pH- and thermal stability have been studied. It was shown possibility to induce alpha-N-acetylgalactosaminidase synthesis by a number of carbohydrates (galactose, glucose, galactosamine, and glucosamine), complex-forming substances (guanidine HCl), nitroaminoguanidin and guanidine carbonate and bovine blood. As a result the strain of Aspergillus niger 185 III was chosen which activity level could be increased more than 3 times (activity--0.6 un./ml as compared to initial one).

[Functional groups in the alpha-galactosidase active site in Cladosporium cladosporioides]. / Issledovanie funktsional'nykh grupp v aktivnom tsentre alpha-galaktozidazy Cladosporium cladosporioides.

The activity of alpha-galactosidase isolated from culture fluid of micromycete Cladosporium cladosporioides (Fres.) de Vries 16,038 has been studied as affected by cations, anions and specific chemical reagents (p-chlormercurybenzoate (p-ChMB), iodacetamide, N-ethylmaleimide, L-cysteine, dithiotreitol, beta-mercaptoethanol, EDTA, o-phenanthroline, sodium azide). It has been established that Ag+ ions inhibited competitively alpha-galactosidase at pH 4.0 and 6.0, the inhibition constants (Ki) made 3.6 x 10(-5) M and 4.3 x 10(-6) M, respectively. Galactose in concentration of 1 mM to 5 mM preserved the enzyme from the negative effect of Ag+ ions, while L-cysteine did not manifest the protective effect. Ions of Hg2+ p-ChMB inhibited noncompetitively the activity of alpha-galactosidase, Ki for Hg2+ and p-ChMB made 5.7 x 10(-7) M and 4.7 x 10(-6) M, respectively. Preincubation with galactose does not preserve alpha-galactosidase from the inhibiting effect of Ag+ and p-ChMB, but th[not readable: see text] compounds (L-cysteine, dithiotreitol, beta-mercaptoethanol) restore the enzyme activity. Participation of histidine imidazole group in the catalytic action is supposed on the basis of the inhibitory and kinetic analysis. Sulphydryl groups do not take part in the catalysis but play an important role in supporting the active conformation of the protein molecule. The groups containing the atoms of metals are absent in the alpha-galactosidase molecule.