Tag Archives: purification

Purification and physico-chemical properties of Bacillus thuringiensis ІМВ В-7324 peptidase with elastolytic and fibrinolytic activity

18.05.2016   Uncategorized   No comments

О. V. Matselyukh, N. A. Nidialkova, L. D. Varbanets

Institute of Microbiology and Virology, National Academy of Sciences of Ukraine, Kyiv;
e-mail: oivanko@yahoo.com

The scheme of isolation and purification of Bacillus thuringiensis ІMV B-7324 peptidase has been developed. This scheme includes ammonium sulfate precipitation and chromatography on neutral and charged TSK-gels. It was found that the enzyme hydrolyzes elastin and fibrin. The molecular weight is 26 kDa. It was shown that the enzyme is an alkaline serine peptidase. The optimal pH of hydrolysis of elastin and fibrin were 9.0 and 10.0, respectively. The optimal temperature of elastin and fibrin hydrolysis are 40 and 50 °C, respectively.  The high stability of the purified preparation in the studied range of pH and temperature was shown. The stabilizing effect of zinc at a concentration of 1 mM on the elastase activity, and the inhibitory effect of other divalent cations under study have been established. The investigated chloride and acetate anions reduced activity by 20%, while phosphate anions increased activity by 15–30%.

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A new mannose-specific lectin from daylily (Hemerocallis fulva L.) rhizome: purification and properties

06.10.2015   Uncategorized   No comments

V. O. Antonyuk1,2, L. V. Panchak1,2, M. O. Starykovych1, R. S. Stoika1

1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine;
2Danylo Halytsky Lviv National Medical University, Ukraine;
e-mail: antonyuk@meduniv.lviv.ua

A new lectin was purified from the daylily (Hemerocallis fulva L.) with the yield of approximately 10 mg per kg of fresh plant rhizome. The purification procedure was based on application of the affinity chromathography on the column with yeast mannan and the ion-exchange chromatography on the column with DEAE-Toyopearl. The lectin possessed low affinity for α-methyl-D-mannopyranoside, D-fructose, D-turanose and 2-acetamido-D-galactopyranose and hight affinity for the yeast mannan. The lectin bound with greatly less affinity for the mannose-containig glycoproteins, such as ovoalbumin, ovomucoid and horseradish peroxidase. According to the results of electrophoresis in 20% DSNa-PAGE, the lectin consists of subunits of 12 kDa molecular weight. According to the results of gel-chromatography on the Toyopearl HW-55, the lectin’s molecular weight is 48 kDa. It agglutinated rabbit erythrocytes very well, while rat and guinea-pig erythrocytes were agglutinated worse, and human erythrocytes were not agglutinated at all.  Lectin’s dialysis against 1% EDTA or heating to 60 ºC for 60 min did not stop its hemagglutinating activity.

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Isolation and properties of polyphenol oxidase from basidiocarps of Lactarius pergamenus Fr. (Fr.) fungi

26.05.2015   Uncategorized   No comments

M. V. Tsivinska1,2, V. O. Antonyuk3,4, R. S. Stoika1,3

1Ivan Franko Lviv National University, Ukraine;
е-mail: antonyukvo@gmail.com;
2Scientific-Research Center of Criminalistic Expertise in Lviv Region
at the Ministry of Internal Affairs of Ukraine;
3Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv;
4Danylo Halytskyi Lviv National Medical University, Ukraine

Fresh juice of basidiocarps of Lactarius pergamenus Fr. (Fr.) fungi was subjected to ion exchange chromatography with used DEAE-toyopearl and CM-cellulose columns, as well as preparative electrophoresis in 7.5% polyacrylamide gels (pH 8.6). Three isoforms of polyphenol oxidase (PPO) were discovered and two isoforms (1-1 and 1-2) were purified with a release of protein 0.42 mg/kg and 0.15 mg/kg of basidiocarps, respectively. These isoforms differ in the mobility at disc-electrophoresis in 7.5% PAGE in alkaline buffer system (pH 8.6). Specific activity of isoform 1-2 is 4.8 times higher than that of the isoforms 1-1. The molecular weight determination by gel chromatography on the Toyopearl HW-55 demonstrated that both isoforms 1-1 and 1-2 have the same 64 ± 2 kDa molecular mass. Electrophoresis in 15% PAGE in the presence of sodium dodecylsulphate and β-mercaptoethanol revealed one band with molecular mass of 64 ± 1 kDa which suggests the presence of one polypeptide chain in the molecule of the enzyme. The enzyme has demonstrated the highest activity at pH 6.0 and temperature +10 ºC, and at +70 ºC the enzyme was inactivated. The PPO activity was the highest in young mushrooms and it decreased with their age and positively correlated with the content of the milky juice. Ortho-aminophenol was most effective among all the tested substrates to determine the activity of PPO (o-, m– and p-aminophenol, catechol, tyrosine, resorcinol, phloroglucinol) and its relative activity was 129% of the activity of catechol. Ascorbic acid was the most effective inhibitor of the polyphenol oxidase activity which was completely blocked at 1 mM concentration, whereas the same concentration of thiourea and sodium sulphite decreased the enzymatic activity by 40-45%. The PPO in L. pergamenus fungi basidiocarps was mainly localized in the mushroom milky juice where its high activity may be associated with protection of basidiocarps against various pathogens.

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