Tag Archives: Cryptococcus albidus

Substrate specificity of Cryptococcus albidus and Eupenicillium erubescens α-L-rhamnosidases

Е. V. Gudzenko, L. D. Varbanets

Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
е-mail: varbanets@serv.imv.kiev.ua

The substrate specificity of Cryptococcus albidus and Eupenicillium erubescens α-L-rhamnosidases has been investigated. It is shown that the enzymes are able to act on synthetic and natural substrates, such as naringin, neohesperidin. α-L-Rhamnosidases hydrolysed the latter ones very efficiently, in this case E. erubescens enzyme was characterized by higher values of Vmax in comparison with the enzyme of C. albidus. However the C. albidus α-L-rhamnosidase showed greater affinity for naringin and neohesperidin than the enzyme of E. erubescens (Km 0.77 and 3.3 mM and 5.0 and 3.0 mM, respectively). As regards the synthetic derivatives of monosaccharides, both enzymes exhibited narrow specificity for glycon: E. erubescens α-L-rhamnosidase – only to the p-nitrophenyl-α-L-rhamnopiranoside (Km 1.0 mM, Vmax 120 µmol/min/mg protein), and C. albidus – to p-nitrophenyl-α-D-glucopyranoside (Km 10 mM, Vmax 5 µmol/min/mg protein). Thus, it was found that the enzyme preparations of E. erubescens and C. albidus are differed by their substrate specifici­ty. The ability of E. erubescens and C. albidus α-L-rhamnosidases to hydrolyse natural substrates: naringin and neohesperidin, evidences for their specificity for α-1,2-linked L-rhamnose. Based on these data, we can predict the use of E. erubescens and C. albidus α-L-rhamnosidases in various industries, food industry in particular. This is also confirmed by the fact that the investigated α-L-rhamnosidases were stable at 20% concentration of ethanol and 500 mM glucose in the reaction mixture.

Thermal stability of Cryptococcus albidus α-L-rhamnosidase

O. V. Gudzenko, N. V. Borzova, L. D. Varbanets

Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv, Ukraine;
e-mail: nv_borzova@bigmir.net

Yeast as well as micromycetes α-L-rhamnosidases, currently, are the most promising group of enzymes. Improving of the thermal stability of the enzyme preparation are especially important studies. Increase in stability and efficiency of substrate hydrolysis by α-L-rhamnosidase will improve the production technology of juices and wines. The aim of our study was to investigate the rate of naringin hydrolysis by α-L-rhamnosidase from Cryptococcus albidus, and also some aspects of the thermal denaturation and stabilization of this enzyme. We investigated two forms of α-L-rhamnosidase from C. albidus, which were obtained by cultivation of the producer on two carbon sources – naringin and rhamnose. A comparative study of properties and the process of thermal inactivation of α-L-rhamnosidases showed that the inducer of synthesis had no effect on the efficiency of naringin hydrolysis by the enzyme, but modified thermal stability of the protein molecule. Hydrophobic interactions and the cysteine residues are involved in maintaining of active conformation of the α-L-rhamnosidase molecule. Yeast α-L-rhamnosidase is also stabilized by 0.5% bovine serum albumin and 0.25% glutaraldehyde.

Complexes of cobalt (II, III) with derivatives of dithiocarbamic acid – effectors of peptidases of Bacillus thuringiensis and α-L-Rhamnozidase of Eupenicillium erubescens and Cryptococcus albidus

L. D. Varbanets1, E. V. Matseliukh1, I. I. Seifullina2,
N. V. Khitrich2, N. A. Nidialkova1, E. V. Gudzenko1

1D. K. Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
2I. I. Mechnikov Оdеssa National University, Ukraine;
e-mail: varbanets@serv.imv.kiev.ua

The influence of  cobalt (II, III) coordinative compounds with derivatives of dithiocarbamic acid on Bacillus thuringiensis IMV B-7324 peptidases with elastase and fibrinolytic activi­ty and Eupenicillium erubescens and Cryptococcus albidus α-L-rhamnosidases have been studied. Tested coordinative compounds of cobalt (II, III) on the basis of their composition and structure are presented by 6 groups: 1) tetrachlorocobaltates (II) of 3,6-di(R,R′)-iminio-1,2,4,5-tetratiane – (RR′)2Ditt[CoCl4]; 2) tetrabromocobaltates (II) of 3,6-di(R,R′)-iminio-1,2,4,5-tetratiane – (RR′)2Ditt[CoBr4]; 3) isothiocyanates of tetra((R,R′)-dithiocarbamatoisothiocyanate)cobalt (II) – [Co(RR′Ditc)4](NCS)2]; 4) dithiocarbamates of cobalt (II) – [Co(S2CNRR′)2]; 5) dithiocarbamates of cobalt (III) – [Co(S2CNRR′)3]; 6) molecular complexes of dithiocarbamates of cobalt (III) with iodine­ – [Co(S2CNRR′)3]∙2I2. These groups (1-6) are combined by the presence of the same complexing agent (cobalt) and a fragment S2CNRR′ in their mole­cules. Investigated complexes differ by a charge of intrinsic coordination sphere: anionic (1-2), cationic (3) and neutral (4-6). The nature of substituents at nitrogen atoms varies in each group of complexes. It is stated that the studied coordination compounds render both activating and inhibiting effect on enzyme activity, depending on composition, structure, charge of complex, coordination number of complex former and also on the enzyme and strain producer. Maximum effect is achieved by activating of peptidases B. thuringiensis IMV B-7324 with elastase and fibrinolytic activity. So, in order to improve the catalytic properties of peptidase 1, depending on the type of exhibited activity, it is possible to recommend the following compounds: for elastase – coordinately nonsaturated complexes of cobalt (II) (1-4) containing­ short aliphatic or alicyclic substituents at atoms of nitrogen and increasing activity by 17-100% at an average; for fibrinolytic – neutral dithio­carbamates of cobalt (II, III) (4-5) (by 29-199%). For increasing the fibrinolytic activity of peptidase it is better to use dibenzyl- or ethylphenyldithiocarbamates of cobalt (III), which increase activity by 15-40% at an average. The same complexes, and also compound {(CH2)6}2Ditt[CoCl4] make an activating impact on α-L-rhamnosidase C. albidus (by 10-20%).