Magdalena Jaszek

Activity of free and immobilized extracellular Cerrena unicolor laccase in water miscible organic solvents

The extracellular laccase of white rot fungus Cerrena unicolor was purified from culture by ion-exchange chromatography on DEAE-Toyopearl column and immobilized on silanized porous glass heads. During bonding procedure 94.35% protein and 100 % laccase activity were coupled to the support. Optimum pH for immobilized laccase compared to free enzyme was shifted from 5.5 to 5.7. The immobilized laccase was more resistant for thermal denaturation: at 70°C, the activity of immobilized enzyme was around 20 % higher than that of the free enzyme. It was also more stable during storage at 4°C. After about seven months the immobilized laccase retained 94.81 % of its initial activity, whereas free enzyme only 39.59 %. Nine water-miscible organic solvents tested in an anhydrous form caused an inhibitory effect on both laccase forms, but, in the mixture with water, the enzyme in all cases shows activity. It decreased, however, when the concentration of solvents increased. Among all solvents, the best results were obtained with ethylene glycol and methoxyethanol. When the reaction mixture contained 50 % of ethylene glycol, free and immobilized laccase retained 24 and 31 % respectively of their activity in the buffer. In the case of 50 % methoxyethanol these data were 6 and 36 %. Organic solvents shifted pH optima of both laccase forms to pH 6.0. The calculation from Lineweaver-Burk plot oxidizing capacity of laccase towards syringaldazine was slightly higher for the free enzyme than for immobilized one, 18.5 and 20.0 M/l respectively. These results show that Cerrena unicolor laccase both in free and immobilized form is able to catalyze the oxidation of syringaldazine in organic solvents. It may be usable in transformation of substrates insoluble or sparingly soluble in water. The immobilized laccase, as more stable and temperature resistant than free enzyme, seems to be more useful.

New Bioactive Fungal Molecules with High Antioxidant and Antimicrobial Capacity Isolated from Cerrena unicolor Idiophasic Cultures

Three bioactive fractions, extracellular laccase (ex-LAC), crude endopolysaccharides (c-EPL), and a low molecular subfraction of secondary metabolites (ex-LMS), were isolated from the idiophasic cultures of the white rot fungus Cerrena unicolor. For the first time, we determined the antioxidant properties of these samples by chemiluminometric measurement (a) and assessment of the scavenging effect on ABTS (b) and the DPPH reduction rate (c). The highest reducing capability was found for the ex-LMS fraction: 39–90% for (a), 20–90% for (b), and 10–59% for (c) at the concentration of 6.25–800 µg/mL. The scavenging abilities of the C. unicolor c-EPL were between 36 and 70% for (a), 2 and 60% for (b), and 28 and 32% for (c) at the concentration of 6.25–800 µg/mL. A very high prooxidative potential was observed for the ex-LAC probes. The preliminary toxicity tests were done using the Microtox system and revealed the following percentage of the toxic effect against Vibrio fischeri: 85.37% for c-EPL, 50.67% for ex-LAC, and 99.8% for ex-LMS, respectively. The ex-LAC sample showed the antibacterial activity against Escherichia coli, c-EPL against Staphylococcus aureus, and ex-LMS against both bacterial strains, respectively, but the stronger inhibitory effect was exerted on S. aureus.

The response of the Rhizobium leguminosarum bv. trifolii wild-type and exopolysaccharide-deficient mutants to oxidative stress

AimsThe aim of this study was investigation of the response of R. leguminosarum bv. trifolii wild-type and its two rosR and pssA mutant strains impaired in exopolysaccharide (EPS) synthesis to oxidative stress conditions caused by two prooxidants: a superoxide anion generator- menadione (MQ) and hydrogen peroxide (H2O2).MethodsThe levels of enzymatic (catalase, superoxide dismutase, pectinase and β-glucosidase) and non-enzymatic (superoxide anion generator, formaldehyde, phenolic compounds) biomarkers were monitored using biochemical methods in both the supernatants and rhizobial cells after treatment with 0.3 mM MQ and 1.5 mM H2O2. The viability of bacterial cells was estimated using fluorescent dyes and confocal laser scanning microscopy. In addition, the effect of prooxidants on symbiosis of the R. leguminosarum bv. trifolii strains with clover was established.ResultsThe tested stress factors significantly changed enzymatic patterns of the rhizobial strains, and the wild-type strain proved to be more resistant to these prooxidants than both pssA and rosR mutants. Significantly higher activities of both catalase and superoxide dismutase have been detected in these mutants in comparison to the wild-type strain. H2O2 and MQ also increased the levels of pectinase and β-glucosidase activities in the tested strains. Moreover, pre-incubation of R. leguminosarum bv. trifolii strains with the prooxidants negatively affected the viability of bacterial cells and the number of nodules elicited on clover plants.ConclusionsEPS produced in large amounts by R. leguminosarum bv. trifolii plays a significant protective role as a barrier against oxidative stress factors and during symbiotic interactions with clover plants.

Cooperation of fungal laccase and glucose 1-oxidase in transformation of Björkman lignin and some phenolic compounds

Summary A screening of wood-rotting basidiomycete fungi was conducted for glucose 1-oxidase (GOD) and laccase (LAC) production as well as for ligninolytic activity measured by a Rhemazol reaction. The results showed that genera rich in GOD are lignin degraders as well as effective producers of extracellular LAC. The fungi poor in GOD neither showed LAC, nor ligninolytic activity. The Björkman lignin and 3 phenolic compounds, hydroquinone and syringic and vanillic acids, were tested on the sequential activity of LAC and GOD. In the presence of LAC, quinoid intermediates formed from Björkman lignin and phenolic compounds were observed. The addition of GOD caused a diminution of the quinone level. During incubation of Björkman lignin with LAC and GOD depolymerization occurred, and in the experiments omitting GOD the quantities of low molecular products were markedly lower. Consequently, the consecutive ping-pong activity of LAC and GOD reduced the polymerization and improved the efficiency of depolymerization processes.

Profiles of the body-surface proteolytic system of honey bee queens, workers and drones: Ontogenetic and seasonal changes in proteases and their natural inhibitors*

We describe how protease and protease inhibitor activity patterns vary during ontogenesis, with season, and in relation to caste and sex in the honey bee (Apis mellifera). Extraction of body surfaces with water and detergent was followed by the in vitro analysis of proteolytic activity and protease inhibitor level, as well as the electrophoretic separation of extracts in polyacrylamide gels. In in vitro experiments, we compared two groups of detectable proteolytic activities: neutral and alkaline water-soluble versus acidic detergent-soluble. The most active proteases appeared to be acidic ones and were detected on drone pupae in spring. The most distinct and most active protease bands in electrophoretic separations were those obtained for neutral and alkaline activities on queens in all seasons. The highest levels of protease inhibitor activities in vitro were obtained from worker samples in all seasons. The enzymatic properties suggest that all catalytic types of proteases were present in the extracts, but at different activity levels, depending on pH: asparagine and cysteine proteases at pH 2.5, cysteine proteases and metalloproteases at pH 7.0, and serine proteases at pH 11.5, respectively.ZusammenfassungBei verschiedenen Organismen, einschliesslich Schaben, Amphibien und Menschen, wurde bereits eine beschränkte Anzahl an Proteasen und/oder Proteaseinhibitoren auf der Körperoberfläche identifiziert. Dabei sind die Unterschiede innerhalb der Invertebraten und im Vergleich mit Vertebraten nur relativ gering. Untersuchungen basierend auf dem Genom der Honigbienen weisen bereits auf eine bestimmte Anzahl neuer Proteine, einschliesslich Kutikulaproteinen, hin, die auch Proteasen umfassen könnten. Ziel dieser Studie war die Beschreibung der Muster, der in vitro Aktivität und der Zymogramme der Proteasen der Körperoberfläche und ihrer natürlichen Inhibitoren im Verlauf der Ontogenese der Honigbiene (Apis mellifera). Dabei wurden auch Kasten- und Saisonunterschiede in Betracht gezogen.Adulte Honigbienen wurden entweder am Stockeingang abgefangen (Arbeiterinnen) oder aus dem Volk entommen (Königinnen und Drohnen). Puppen, Larven und Eier wurden aus Brutwaben gewonnen. Larven der ersten drei Larvenstadien wurden als „kleine Larven“ und die des vierten und fünften Larvenstadiums als „grosse Larven“ gepoolt. Alle Proben wurden mit destilliertem Wasser abgewaschen, um Verunreinigungen und nicht festhaftende Mikroorganismen zu entfernen. Anschliessend wurde mittels feuchter Baumwollstäbchen die Körperoberfläche abgewaschen. Aus den Baumwollstäbchen wurden dann die biologischen Proben ausgepresst. Für eine zweite Waschung wurde destilliertes Wasser mit 1 % Detergenz verwendet. Allgemeine Proteasenaktivitäten wurden am optimalen pH mittels Hämoglobin als Substrat bestimmt (Tab. I) In den Extrakten waren alle katalytischen Typen an Proteasen vertreten. Bei pH 2,5 hatten die Asparaginproteasen (inhibiert durch Pepstatin und DAN) und Cysteinproteasen (inhibiert durch Iodoacetamid und pCMB) ihre höchsten Werte; bei pH 7,0 erreichten die Cysteinproteasen und Metalloproteasen (inhibiert durch o-Phenantrolin) und bei pH 11,5 die Serinproteasen (inhibiert durch PMSF und STI) die höchsten Aktivitätswerte.Die Bestimmung der allgemeinen Niveaus der Aktivitäten der Proteaseninhibitoren wurde mit Pepsin als Proteasemarker bei saurem pH und mit Trypsin als Markerprotease für neutrale und basische pH-Werte durchgeführt. Elektrophoresen in 10 % Gelen erfolgten bei nichtdenaturierenden Bedingungen, mit 1 % Gelatine als vorgegebenem Proteinsubstrat. In in vitro-Versuchen verglichen wir unter identischen Bedingungen zwei Gruppen proteolytischer Aktivitäten: die neutralen und basischen wasserlöslichen und die sauren, detergenzlöslichen. Die aktivsten Proteasen waren saure Proteasen auf Drohnenpuppen, die im Frühling gesammelt worden waren (Abb. 2), neutrale und basische Proteasen auf adulten Drohnen im Sommer (Abb. 3), neutrale auf adulten Königinnen im Herbst, sowie alle Proteasentypen auf Königinnenpuppen im Frühjahr (Abb. 4 und Abb. 2). Die geringsten Proteaseaktivitäten fanden wir auf Arbeiterinnen im Frühjar (Abb. 2) und auf Drohnen im Herbst (Abb. 4). In allen Fällen waren die Aktivitäten auf Puppen und Imagines höher als auf den anderen Stadien. Im zymographischen Vergleich in Gelen mit Gelatine zeigten sich einige Unterschiede. Die schärfsten Banden mit der höchsten Aktivität fanden wir für neutrale und basische Werte auf Königinnen in allen Jahreszeiten. Die verschwommensten Banden waren für neutrale und basische Werte auf Drohnen in allen Jahreszeiten zu verzeichnen. Bei Arbeiterinnenproben erhielten wir für alle Jahreszeiten sowohl klare als auch verschwommen Banden. Letzteres steht in Übereinstimmung mit den niedrigen Proteaseaktivitäten, die wir für Arbeiterinnen in den in vitro-Versuchen gefunden hatten. Vergleichbar hohe Werte für Proteaseinhibitoren fanden wir bei Drohnen im Sommer (Abb. 6) und bei adulten Königinnen im Herbst (Abb. 7).

Exopolysaccharide from Ganoderma applanatum as a Promising Bioactive Compound with Cytostatic and Antibacterial Properties

A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS) was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory effect. The results indicate that the tested GpEPS (at concentrations above 22.85 µg/mL and 228.5 µg/mL) may exhibit selective activity against tumor cells (cell lines SiHa) and stimulate production of TNF-α THP-1-derived macrophages at the level of 752.17 pg/mL. The GpEPS showed antibacterial properties against Staphyloccoccus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage). High cholesterol-binding capacity and triglycerides-binding capacity (57.9% and 41.6% after 24 h of incubation with the tested substances, resp.) were also detected for the investigated samples of GpEPS.

Effect of coniferyl alcohol addition on removal of chlorophenols from water effluent by fungal laccase

The effect of coniferyl alcohol on removal of chlorinated phenols from a water environment byRhizoctonia praticola andCerrena unicolor laccases was studied. At optimal conditions in which 7 mM coniferyl alcohol and laccase were added to chlorinated phenols over 20h, about 34% of the radioactivity of 4-chlorophenol, 57% of 2,4-dichlorophenol, 66% of 2,4,5-trichlorophenol, and 85% of pentachlorophenol were removed from the supernatants, compared to the level without laccase activity. After 12-h incubation periods at the optimal concentrations of coniferyl alcohol and laccase (added simultaneously), the fast first phase of chlorophenol removal was complete in 1 h, and eventually coniferyl alcohol enhanced the removal of 4-chlorophenol by 40%, 2,4-dichlorophenol by 54%, 2,4,5-trichlorophenol by 60%, and pentachlorophenol by 76%.

Fungus Cerrena unicolor as an effective source of new antiviral, immunomodulatory, and anticancer compounds

In the report, three bioactive fractions from Cerrena unicolor: laccase (LAC), endopolysaccharides (c-EPL), and low molecular weight (ex-LMS) were tested for the first time towards their antiviral, immunostimulatory, cytotoxic and antiproliferative effect. The immunomodulatory activity was studied by means of THP-1-derived macrophages able to synthesize and secrete IL-6 and TNF-α. We used cervical carcinoma cell lines SiHa (ATCC, HTB-35) and CaSki (ATCC, CRL 1550) to determine antitumor activity and human skin fibroblasts (HSF) as a control. SiHa and L929 cell lines were used in the antiviral activity assay to propagate HHV-1 and EMCV, respectively. LAC was the most active against HSV at an early stage of viral replication, whereas the activity of laccase against EMCV was evident after incubation of the virus with LAC before and after the adsorption step. Moreover, the investigations showed that the fungal c-EPL fraction stimulated the production and secretion of TNF-α and IL-6 by THP-1-derived macrophages up to a level of 2000 pg/ml and 400 pg/ml, respectively. It was indicated for the first time that the LAC and ex-LMS fractions exhibited anticancer activity. This resulted from their cytotoxic or antiproliferative action against the investigated tumor cells at concentrations above 250 μg/ml and 10 μg/ml, respectively.

Laccase purified from Cerrena unicolor exerts antitumor activity against leukemic cells.

Chronic lymphocytic leukemia (CLL) is the most commonly observed adult hematological malignancy in Western countries. Despite the fact that recent improvements in CLL treatment have led to an increased percentage of complete remissions, CLL remains an incurable disease. Cerrena unicolor is a novel fungal source of highly active extracellular laccase (ex-LAC) that is currently used in industry. However, to the best of our knowledge, no reports regarding its anti-leukemic activity have been published thus far. In the present study, it was hypothesized that C. unicolor ex-LAC may possess cytotoxic activity against leukemic cell lines and CLL primary cells. C. unicolor ex-LAC was separated using anion exchange chromatography on diethylaminoethyl cellulose-Sepharose and Sephadex G-50 columns. The cytotoxic effects of ex-LAC upon 24- and 48-h treatment on HL-60, Jurkat, RPMI 8226 and K562 cell lines, as well as CLL primary cells of nine patients with CLL, were evaluated using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Annexin V/propidium iodide staining of Jurkat cells treated with ex-LAC was used to investigate apoptosis via flow cytometry. Ex-LAC induced changes in Jurkat and RPMI 8226 cells, as visualized by fluorescence and scanning electron microscopy (SEM). The XTT assay revealed high cytotoxic rates following treatment with various concentrations of ex-LAC on all the cell lines and CLL primary cells analyzed, with a half maximal inhibitory concentration ranging from 0.4 to 1.1 µg/ml. Fluorescence microscopy and SEM observations additionally revealed apoptotic changes in Jurkat and RPMI 8226 cells treated with ex-LAC, compared with control cells. These results were in agreement with the apoptosis analysis of Jurkat cells on flow cytometry. In conclusion, C. unicolor ex-LAC was able to significantly induce cell apoptosis, and may represent a novel therapeutic agent for the treatment of various hematological neoplasms.

New alkaline lipase from Rhizomucor variabilis: Biochemical properties and stability in the presence of microbial EPS

A new strain of Rhizomucor variabilis producing an active extracellular lipase was identified and characterized in the present studies. The culture conditions were optimized and the highest lipase production amounting to 136 U/mL was achieved after 4 days of cultivation. The optimum pH (5.5) and temperature (28 °C) were determined as the best conditions for R. variabilis lipase production. The isolated enzyme preparation exhibited maximum activity at 40 °C and pH 8.0. Lipase from R. variabilis was stable up to 50 °C during 2 H retaining 80% of its initial activity. The enzyme was highly stable in the pH range of 7.0–9.0. Moreover, the addition of naturally obtained exopolysaccharides (EPS) significantly enhanced lipase activity. The presence of EPS derived from Ganoderma applanatum and Rhizobium leguminosarum enhanced the lipase activity, which was 22% and 31%, respectively, higher than that in the control experiments. Simultaneously, the pH activity profiles remained unchanged. The Michaelis–Menten constant and the turnover number of the enzyme for p‐nitrophenyl palmitate in the standard assay conditions were estimated at a level of 0.631 mM and 0.674 Sec−1. In conclusion, the results obtained in this work present a newly isolated lipase preparation stabilized with EPS or without modification as a very effective tool for industrial application.