Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn2+ and Mg2+ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg2+. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.

• Klíčová slova
• Characterization, Curvularia lunata, Laccase, Purification,
• MeSH
• antiinfekční látky * MeSH
• azosloučeniny * MeSH
• benzothiazoly * MeSH
• Curvularia * MeSH
• koncentrace vodíkových iontů MeSH
• kyseliny sulfonové * MeSH
• lakasa * metabolismus   MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid MeSH Prohlížeč
• antiinfekční látky * MeSH
• azosloučeniny * MeSH
• benzothiazoly * MeSH
• kyseliny sulfonové * MeSH
• lakasa * MeSH
• mordant yellow 3 MeSH Prohlížeč

Two isoforms of NAD(P)(+)-dependent malic enzyme (EC 1.1.1.39) were isolated from hydrogenosomes of Trichomonas vaginalis. A positively charged isoform at pH 7 was obtained in a single purification step using cation-exchange chromatography. The second isoform, negatively charged at pH 7.5, was partially purified using a combination of anion-exchange and affinity chromatography. Both isoforms displayed similar physical and kinetic properties. Molecular weight determination of the native enzyme suggested a homotetrameric arrangement of the 60 kDa subunits. The enzyme utilized NAD+ (Km, 6-6.3 microM) preferentially to NADP+ (Km, 125-145 microM). The NAD(+)-dependent activity showed a broad pH optimum with maximum under alkaline conditions (pH 9) likely to be present inside hydrogenosomes. Immunocytochemical studies using a polyclonal rabbit antibody raised against purified T. vaginalis malic enzyme proved hydrogenosomal localization of the enzyme. Subfractionation of hydrogenosomes suggested an association of the malic enzyme with the hydrogenosomal membranes. The 60 kDa malic enzyme subunit was highly sensitive to non-enzymatic cleavage by an iron-ascorbate system resulting in two enzymatically inactive fragments of about 31 kDa. Microsequencing of the fragments revealed that the 60 kDa subunit was cleaved at the metal-binding site between Asp279-Ile280. The enzyme inactivation was inhibited by an excess of manganese. Iron-dependent posttranslational modification might contribute to the regulation of malic enzyme activity in vivo.

• MeSH
• chloridy farmakologie   MeSH
• frakcionace buněk MeSH
• intracelulární membrány enzymologie   MeSH
• izoenzymy chemie   izolace a purifikace   metabolismus   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• kyselina askorbová farmakologie   MeSH
• malátdehydrogenasa analýza   chemie   izolace a purifikace   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• NAD metabolismus   MeSH
• organely enzymologie   MeSH
• sekvence aminokyselin MeSH
• sekvenční analýza MeSH
• sloučeniny manganu farmakologie   MeSH
• Trichomonas vaginalis enzymologie   MeSH
• železnaté sloučeniny farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chloridy MeSH
• ferrous sulfate MeSH Prohlížeč
• izoenzymy MeSH
• kyselina askorbová MeSH
• malátdehydrogenasa MeSH
• malate dehydrogenase (decarboxylating) MeSH Prohlížeč
• manganese chloride MeSH Prohlížeč
• NAD MeSH
• sloučeniny manganu MeSH
• železnaté sloučeniny MeSH

Fungi are producers of lignolytic extracellular enzymes which are used in industries like textile, detergents, biorefineries, and paper pulping. This study assessed for the production, purification, and characterization of novel p-diphenol oxidase (PDO; laccase) enzyme from lignolytic white-rot fungal isolate. Fungi samples collected from different areas of Pakistan were initially screened using guaiacol plate method. The maximum PDO producing fungal isolate was identified on the basis of ITS (internal transcribed spacer sequence of DNA of ribosomal RNA) sequencing. To get optimum enzyme yield, various growth and fermentation conditions were optimized. Later PDO was purified using ammonium sulfate precipitation, size exclusion, and anion exchange chromatography and characterized. It was observed that the maximum PDO producing fungal isolate was Schizophyllum commune (MF-O5). Characterization results showed that the purified PDO was a monomeric protein with a molecular mass of 68 kDa and showed stability at lower temperature (30 °C) for 1 h. The Km and Vmax values of the purified PDO recorded were 2.48 mM and 6.20 U/min. Thermal stability results showed that at 30 °C PDO had 119.17 kJ/K/mol Ea value and 33.64 min half-life. The PDO activity was stimulated by Cu2+ ion at 1.0 mM showing enhanced activity up to 111.04%. Strong inhibition effect was noted for Fe2+ ions at 1 mM showing 12.04% activity. The enzyme showed stability against 10 mM concentration oxidizing reducing agents like DMSO, EDTA, H2O2, NaOCl, and urea and retained more than 75% of relative activity. The characterization of purified PDO enzyme confirmed its tolerance against salt, metal ions, organic solvents, and surfactants indicating its ability to be used in the versatile commercial applications.

• Klíčová slova
• 2′-azino-di(3-ethylbenzthiazoline-6-sulfonate), ABTS 2, Characterization, Fungi, Guaiacol, PDO (p-diphenol oxidase/laccase), Process optimization, Schizophyllum commune,
• MeSH
• koncentrace vodíkových iontů MeSH
• lakasa * metabolismus   MeSH
• peroxid vodíku MeSH
• Schizophyllum * genetika   metabolismus   MeSH
• stabilita enzymů MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• lakasa * MeSH
• peroxid vodíku MeSH

The white-rot fungus Daedalea quercina produced the ligninolytic enzymes laccase and Mn-dependent peroxidase. Laccase was purified using anionexchange and size-exclusion chromatographies. SDS-PAGE showed the purified laccase to be a monomeric protein of 69 kDa (71 kDa using gel filtration) with an isoelectric point near 3.0. The optimum pH for activity was below 2.0 for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (K(m)=38 microM), 4.0 for 2,6-dimethoxyphenol (K(m)=48 microM), 4.5 for guaiacol (K(m)=93 microM) and 7.0 for syringaldazine (K(m)=131 microM). The temperature optimum was between 60 and 70 degrees C depending on the pH and buffer used. The enzyme was stable up to 45 degrees C, and stability was higher at alkaline pH. Enzyme activity was increased by the addition of Cu(2+) and inhibited by Mn(2+), sodium azide, dithiothreitol, and cysteine. Laccase from Daedalea quercina was able to decolorize the synthetic dyes Chicago sky blue, poly B-411, remazol brilliant blue R, trypan blue and reactive blue 2.

• MeSH
• aktivátory enzymů farmakologie   MeSH
• anthrachinony metabolismus   MeSH
• azosloučeniny metabolismus   MeSH
• barva MeSH
• barvicí látky metabolismus   MeSH
• benzothiazoly MeSH
• chromatografie iontoměničová MeSH
• gelová chromatografie MeSH
• guajakol metabolismus   MeSH
• hydrazony metabolismus   MeSH
• inhibitory enzymů farmakologie   MeSH
• izoelektrický bod MeSH
• koncentrace vodíkových iontů MeSH
• kovy farmakologie   MeSH
• kyseliny sulfonové metabolismus   MeSH
• lakasa biosyntéza   chemie   izolace a purifikace   metabolismus   MeSH
• lignin metabolismus   MeSH
• molekulová hmotnost MeSH
• peroxidasy biosyntéza   MeSH
• Polyporales enzymologie   MeSH
• pyrogalol analogy a deriváty   metabolismus   MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• teplota MeSH
• triaziny metabolismus   MeSH
• trypanová modř metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid MeSH Prohlížeč
• aktivátory enzymů MeSH
• anthrachinony MeSH
• azosloučeniny MeSH
• barvicí látky MeSH
• benzothiazoly MeSH
• Cibacron Blue F 3GA MeSH Prohlížeč
• guajakol MeSH
• hydrazony MeSH
• inhibitory enzymů MeSH
• kovy MeSH
• kyseliny sulfonové MeSH
• lakasa MeSH
• lignin MeSH
• manganese peroxidase MeSH Prohlížeč
• peroxidasy MeSH
• pontamine sky blue MeSH Prohlížeč
• pyrogallol 1,3-dimethyl ether MeSH Prohlížeč
• pyrogalol MeSH
• Remazol Brilliant Blue R MeSH Prohlížeč
• syringaldazine MeSH Prohlížeč
• triaziny MeSH
• trypanová modř MeSH

Acidic chitinase (Chia) has been implicated in asthma, allergic inflammations, and food processing. We have purified Chia enzymes with striking acid stability and protease resistance from chicken and pig stomach tissues using a chitin column and 8 M urea (urea-Chia). Here, we report that acetic acid is a suitable agent for native Chia purification from the stomach tissues using a chitin column (acetic acid-Chia). Chia protein can be eluted from a chitin column using 0.1 M acetic acid (pH 2.8), but not by using Gly-HCl (pH 2.5) or sodium acetate (pH 4.0 or 5.5). The melting temperatures of Chia are not affected substantially in the elution buffers, as assessed by differential scanning fluorimetry. Interestingly, acetic acid appears to be more effective for Chia-chitin dissociation than do other organic acids with similar structures. We propose a novel concept of this dissociation based on competitive interaction between chitin and acetic acid rather than on acid denaturation. Acetic acid-Chia also showed similar chitinolytic activity to urea-Chia, indicating that Chia is extremely stable against acid, proteases, and denaturing agents. Both acetic acid- and urea-Chia seem to have good potential for supplementation or compensatory purposes in agriculture or even biomedicine.

• Klíčová slova
• GlcNAc, acetic acid, acidic chitinase, chitin, chitin column, competitive manner, natural enzyme, supplementation purposes, therapeutic agents, urea,
• MeSH
• chitin chemie   metabolismus   MeSH
• chitinasy chemie   metabolismus   MeSH
• kur domácí MeSH
• kyselina octová chemie   MeSH
• prasata MeSH
• vazba proteinů MeSH
• žaludek enzymologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chitin MeSH
• chitinasy MeSH
• kyselina octová MeSH

Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overproducing arginase I (rhARG1) from human liver under the control of the efficient copper-inducible promoter CUP1, was constructed. The (His)(6)-tagged rhARG1 was purified in one step from the cell-free extract of the recombinant strain by metal-affinity chromatography with Ni-NTA agarose. The maximal specific activity of the 40-fold purified enzyme was 1600 μmol min(-1) mg(-1) protein.

• MeSH
• arginasa chemie   genetika   izolace a purifikace   metabolismus   MeSH
• chromatografie afinitní metody   MeSH
• histidin chemie   genetika   metabolismus   MeSH
• játra enzymologie   MeSH
• klonování DNA MeSH
• kyselina nitrilotrioctová analogy a deriváty   MeSH
• lidé MeSH
• oligopeptidy chemie   genetika   metabolismus   MeSH
• organokovové sloučeniny MeSH
• rekombinantní fúzní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arginasa MeSH
• His-His-His-His-His-His MeSH Prohlížeč
• histidin MeSH
• kyselina nitrilotrioctová MeSH
• nickel nitrilotriacetic acid MeSH Prohlížeč
• oligopeptidy MeSH
• organokovové sloučeniny MeSH
• rekombinantní fúzní proteiny MeSH

Tannase-producing filamentous fungi residing alongside tannin-rich ambient in the Northwest Himalayas were isolated at laboratory conditions and further identified by 18S ribosomal RNA gene sequencing. Five most potent tannase producing strains (EI ≥ 2.0), designated Aspergillus fumigatus AN1, Fusarium redolens AN2, Penicillium crustosum AN3, Penicillium restrictum AN4, and Penicillium commune AN5, were characterized. The strain Penicillium crustosum AN3 exhibited a maximum zone dia (25.66 mm ± 0.38). During solid-state fermentation, a maximal amount of tannase was attained with Penicillium crustosum AN3 using pine needles (substrate) by adopting response surface methodology for culture parameter optimization. Gel filtration chromatography yielded 46.48% of the partially purified enzyme with 3.94-fold of tannase purification. We found two subunits in enzyme-117.76 KDa and 88.51 KDa, respectively, in the SDS-PAGE. Furthermore, the characterization of partially purified tannase revealed a maximum enzyme activity of 8.36 U/mL at 30 °C using a substrate concentration (methyl gallate) of 10 mM. To broaden the knowledge of crude enzyme application, dye degradation studies were subjected to extracellular crude tannase from Penicillium crustosum AN3 where the maximum degradation achieved at a low enzyme concentration (5 ppm).

• Klíčová slova
• Application, Fungus, Pine needles, Purification, Solid-state fermentation, Tannase,
• MeSH
• barvicí látky metabolismus   chemie   MeSH
• fermentace MeSH
• fungální proteiny genetika   metabolismus   izolace a purifikace   chemie   MeSH
• Fusarium enzymologie   genetika   MeSH
• fylogeneze MeSH
• houby enzymologie   genetika   MeSH
• karboxylesterhydrolasy * metabolismus   genetika   izolace a purifikace   chemie   MeSH
• kultivační média chemie   MeSH
• molekulová hmotnost MeSH
• Penicillium * enzymologie   genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• stabilita enzymů MeSH
• substrátová specifita MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• barvicí látky MeSH
• fungální proteiny MeSH
• karboxylesterhydrolasy * MeSH
• kultivační média MeSH
• RNA ribozomální 18S MeSH
• tannase MeSH Prohlížeč

The high-molar mass form of beta-glucosidase from Aspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6-5.3 and 70 degrees C, respectively. The K(m) and kcat for 4-nitrophenyl beta-D-glucopyranoside at 40 degrees C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0-9 mol/L transverse urea-gradient-PAGE for 105 min at 12 degrees C, the nonpurified beta-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.

• MeSH
• Aspergillus niger enzymologie   MeSH
• beta-glukosidasa antagonisté a inhibitory   izolace a purifikace   metabolismus   MeSH
• chemická precipitace MeSH
• chromatografie iontoměničová MeSH
• fungální proteiny antagonisté a inhibitory   izolace a purifikace   metabolismus   MeSH
• gelová chromatografie MeSH
• inhibitory enzymů farmakologie   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• molekulová hmotnost MeSH
• síran amonný farmakologie   MeSH
• thermolysin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• beta-glukosidasa MeSH
• fungální proteiny MeSH
• inhibitory enzymů MeSH
• síran amonný MeSH
• thermolysin MeSH

The intra- and extra-cellular monophenolase and diphenolase activities of the tyrosinase produced by Amylomyces rouxii were determined in submerged culture using Melin-Norkrans medium supplemented with 12.5 mg/L pentachlorophenol (PCP) and 0.1 g/L tyrosine. Maximal intracellular monophenolase activity was 180 U/mL while maximal extracellular monophenolase activity was 80 U/mL, both using p-cresol as substrate. For diphenolase, the highest intracellular activity was 2233 U/mL using 4-tert-butylcatechol (TBC) as substrate and extracellular diphenolase activity was 975 U/mL with catechol as substrate. The peak tyrosinase activity (mono- and diphenolase) was observed at 48 h of culture. The transformant A412-3 exhibited the highest extracellular activities, with a 2.14-fold increase in monophenolase and a 3.02-fold increase in diphenolase activity compared to the parental strain of A. rouxii. Additionally, it was confirmed that the enzyme secreted was in its active form. Extracellular tyrosinase from the transformant A412-3 was partially purified, achieving a purification factor of 10.6. SDS-PAGE analysis of partially purified tyrosinase revealed three bands of 40, 53, and 130 kDa. These bands were sequenced by LC-MS/MS, revealing eight peptides that showed similarity to tyrosinases from different fungi. It was determined that purified tyrosinase exhibited higher diphenolase activity than monophenolase activity, in line with previous studies on fungal tyrosinases.

• Klíčová slova
• Amylomyces rouxii, Silencing, Tyrosinase partial purification, Zygomycetes,
• Publikační typ
• časopisecké články MeSH

Dehydrogenase/reductase SDR family member 7 (DHRS7, SDR34C1, retSDR4) is one of the many endoplasmic reticulum bound members of the SDR superfamily. Preliminary results indicate its potential significance in human metabolism. DHRS7 containing TEV-cleavable His10 and FLAG-tag expressed in the Sf9 cell line was solubilised, purified, and reconstituted into liposomes to enable the improved characterisation of this enzyme in the future. Igepal CA-630 was determined to be the best detergent for the solubilisation process. The solubilised DHRS7 was purified using affinity chromatography, and the purified enzyme was subjected to TEV cleavage of the affinity tags and then repurified using subtractive Ni-IMAC. The cleaved and uncleaved versions of DHRS7 were successfully reconstituted into liposomes. In addition, using tobacco specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as the substrate, the cleaved liposomal DHRS7 was found to be inactive, whereas the pure and uncleaved liposomal DHRS7 were confirmed as enzymes, which reduce carbonyl group of the substrates.

• Klíčová slova
• Carbonyl reductase activity, DHRS7, Purification, Reconstitution, Short-chain dehydrogenases/reductases, Solubilisation,
• MeSH
• buněčná membrána MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• oxidoreduktasy chemie   genetika   izolace a purifikace   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Sf9 buňky MeSH
• Spodoptera MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• oxidoreduktasy MeSH
• rekombinantní proteiny MeSH