Tag Archives: α-L-rhamnosidase

The influence of coordinative tartrate and malatogermanate compounds on the activity of α-L-rhamnosidase preparations from Penicillium tardum, Eupenicillium erubescens and Cryptococcus albidus

О. V. Gudzenko1, L. D. Varbanets1*, І. I. Seifullina2,
E. А. Chebanenko2, E. Е. Martsinko2, Е. V. Аfanasenko2

1Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
2Mechnikov Odessa National State University, Ukraine;
*e-mail: varbanets_imv@ukr.net

Received: 26 November 2019; Accepted: 15 May 2020

Recently enzyme preparations of microbial origin become increasingly important in different industries. Preparations of α-L-rhamnosidase are used in the pharmaceutical industry as well as in scientific work as a tool for analytical research. We have obtained purified α-L-rhamnosidase preparations from Penicillium tardum, Eupenicillium erubescens and Cryptococcus albidus microorganism strains which are effective enzyme producers. The aim of the study was to estimate the ability of germanium coordination compounds to enhance enzyme catalytic activity. The effects of 11 heterometal mixed ligand tartrate (malate-)germanate compounds at 0.01 and 0.1% concentration on the activity of α-L-rhamnosidase preparations from Penicillium tardum IMV F-100074, Eupenicillium erubescens and Cryptococcus albidus 1001 were studied at 0.5 and 24 h exposition. The inhibitory effect of [Ni(bipy)3]4[{Ge2(OH)2(Tart)2}3Cl2]·15H2 on P. tardum α-L-rhamnosidase was revealed. All studied compounds except [CuCl(phen)2][Ge(OH)(HMal)2] were shown to increase activity of P. tardum α-L-rhamnosidase at a longer term of exposition. Activity of E. erubescens α-L-rhamnosidase was shown to be stimulated by d-metal cation-free compounds. C. albidus α-L-rhamnosidase occurred to be insensitive to all compounds studied.

Influence of metal ions and specific chemical reagents on activity of α-L-rhamnosidase of Eupenicillium erubescens

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

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

The effect of cations, anions and specific chemical reagents: 1-[3-(dimethylamino)propyl]-3-ethylcarbodiіmide methiodide, ЕDТА, о-phenanthroline, dithiotreitol, L-cysteine, β-mercaptoethanol, p-chlormercurybenzoate (p-ChMB), N-ethylmaleimide on the activity of α-L-rhamnosidase of Eupenicillium erubescens has been investigated. The essential role of Ag+ and Hg2+ which inhibit the α-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 α-L-rhamnosidase. It was assumed that  sulphydryl groups took part in catalysis, carried out by α-L-rhamnosidase of E. erubescens, since the activity of α-L-rhamnosidase inhibited by p-ChMB and thiol reagents such as dithiothreitol, L-cysteine, β-mercaptoethanol did not remove its inhibitory action.

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%).