The study aims were focused on profiling eight hydrolytic enzymes by fluorescence method using a multifunctional modular reader and studying the proportion of basic microorganism groups during composting and vermicomposting of sewage sludge mixed with straw pellets in several proportions (0, 25, 50, 75, and 100%). The greatest decrease in enzymatic activity occurred in the first half of composting and vermicomposting. After 4 months of these processes, the least enzymatic activity was observed in the sludge with 50% and also 25% straw addition, indicating that straw is an important means for the rapid production of mature compost from sewage sludge. Enzymatic activity was usually less in the presence of earthworms than in the control treatment because some processes took place in the digestive tract of the earthworm. For the same reason, we observed reduced enzyme activity during fresh feedstock vermicomposting than precomposted material. The final vermicompost from fresh feedstocks exhibited less microbial biomass, and few fungi and G- bacteria compared to precomposted feedstock. The enzymatic activity during composting and vermicomposting of sewage sludge and their mixtures stabilized at the following values: β-D-glucosidase-50 μmol MUFG/h/g dw, acid phosphatase-200 μmol MUFP/h/g dw, arylsulphatase-10 μmol MUFS/h/g dw, lipase-1,000 μmol MUFY/h/g dw, chitinase-50 μmol MUFN/h/g dw, cellobiohydrolase-20 μmol MUFC/h/g dw, alanine aminopeptidase-50 μmol AMCA/h/g dw, and leucine aminopeptidase-50 μmol AMCL/h/g dw. At these and lesser values, these final products can be considered mature and stable.

• Klíčová slova
• composting, earthworms, enzymatic activity, microorganisms, sewage sludge, straw pellets, vermicomposting,
• Publikační typ
• časopisecké články MeSH

It was originally shown [10] that urease retains its enzymatic activity when adsorbed at bare mercury and solid amalgam surfaces. However the opinion later prevailed that, when adsorbed at bare metal electrodes, proteins are irreversibly denatured. Here we confirm that urease is enzymatically active at a bare solid amalgam surface as found by Santhanam et al., and we show that this enzyme is equally active at a thiol-modified amalgam surface. We also show that it is the reduced form of urease, which is enzymatically active at Hg surfaces. Oxidation of the protein, resulting in formation of disulfide bonds, strongly decreases the enzyme activity. Using constant current chronopotentiometric stripping (CPS) we show that the exposure of surface-attached urease to negative potentials results in the protein unfolding. The extent of the unfolding depends upon the amount of time for which the protein is exposed to negative potentials, and at very short times this unfolding can be avoided. At thiol-modified Hg surfaces the protein is less vulnerable to the effects of the electric field. We conclude that the loss of enzymatic activity, resulting from a 10 min exposure of the protein to -0.58 V, is not due to reduction of the disulfide bonds as suggested by Santhanam et al. This loss is probably a result of protein reorientation, due to reduction of the Hg-S bonds (formed by accessible cysteines), followed by prolonged electric field effect on the surface-attached protein.

• Klíčová slova
• Constant-current chronopotentiometric stripping, Mercury containing electrodes, Protein denaturation at negatively charged surfaces, Protein structure at surfaces, Thiol-modified electrodes, Urease enzymatic activity,
• MeSH
• adsorpce MeSH
• cystein chemie   MeSH
• denaturace proteinů MeSH
• disulfidy chemie   MeSH
• dithiothreitol chemie   MeSH
• elektrochemické techniky MeSH
• elektrody MeSH
• katalýza MeSH
• oxidace-redukce MeSH
• povrchové vlastnosti MeSH
• rtuť chemie   MeSH
• sbalování proteinů MeSH
• sulfhydrylové sloučeniny chemie   MeSH
• teplota MeSH
• ureasa chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• disulfidy MeSH
• dithiothreitol MeSH
• rtuť MeSH
• sulfhydrylové sloučeniny MeSH
• ureasa MeSH

• Here, enzymatic activity of five hydrolases was measured fluorometrically in the fluid collected from traps of four aquatic Utricularia species and in the water in which the plants were cultured. • In empty traps, the highest activity was always exhibited by phosphatases (6.1-29.8 µmol l-1 h-1 ) and β-glucosidases (1.35-2.95 µmol l-1 h-1 ), while the activities of α-glucosidases, β-hexosaminidases and aminopeptidases were usually lower by one or two orders of magnitude. Two days after addition of prey (Chydorus sp.), all enzymatic activities in the traps noticeably decreased in Utricularia foliosa and U. australis but markedly increased in Utricularia vulgaris. • Phosphatase activity in the empty traps was 2-18 times higher than that in the culture water at the same pH of 4.7, but activities of the other trap enzymes were usually higher in the water. Correlative analyses did not show any clear relationship between these activities. • Trap comensals (Euglena) could be partly responsible for production of some trap enzymes. The traps can produce phosphatases independently of catching prey. Taking into account the enzymatic activities in traps, phosphorus uptake from prey might be more important than that of nitrogen for the plants.

• Klíčová slova
• aminopeptidase, aquatic carnivorous plants, chitinase, extracellular enzymatic activity, glucosidase, phosphatase, trap fluid pH, β-hexosaminidase,
• Publikační typ
• časopisecké články MeSH

Determination of free cyanide (fCN) is required for various industrial, environmental, food, and clinical samples. Enzymatic methods are not widely used in this field despite their selectivity and mild conditions. Therefore, we present here a proof of concept for new spectrophotometric enzymatic assays of fCN. These are based on the hydrolysis of fCN affording the readily detectable NADH. fCN is hydrolyzed either in one step by cyanide dihydratase (CynD) or in two steps by cyanide hydratase (CynH) and formamidase (AmiF). An advantage of the latter route is the higher activity of CynH and AmiF compared to CynD. In both cases, the resulting formate is then transformed by an NAD-dependent formate dehydrogenase (FDH). The NADH thus formed is quantified colorimetrically using a known method based on a reduction of a tetrazolium salt (WST-8) with NADH. The developed assays of fCN are selective except for formic acid interference, proceed under mild conditions, and, moreover, fCN is detoxified during the reactions. The assays proceeded in a microtiter plate format. The limit of detection (LOD) and the limit of quantification (LOQ) were lower for the three-enzyme (CynH-AmiF-FDH) method (7.00 and 21.2 µmol/L, respectively) than for the two-enzyme (CynD-FDH) method (10.7 and 32.4 µmol/L, respectively). In conclusion, the new fCN assays presented in this work are selective, high-throughput, do not require harsh conditions, and use only small amounts of chemicals and enzymes.

• Klíčová slova
• Cyanide dihydratase, Cyanide hydratase, Enzymatic assays, Formamidase, Formate dehydrogenase, Free cyanide,
• MeSH
• dehydratasy chemie   metabolismus   MeSH
• enzymatické testy * metody   MeSH
• hydrolýza MeSH
• kolorimetrie metody   MeSH
• kyanidy * analýza   metabolismus   MeSH
• limita detekce MeSH
• NAD chemie   MeSH
• spektrofotometrie metody   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• dehydratasy MeSH
• kyanidy * MeSH
• NAD MeSH

The effect of low-dose, commercially available wastewater sludge compost (WSC; 15 t ha-1) treatment was examined with or without arbuscular mycorrhizal fungal (AMF) inoculation on the nutritional status, heavy metal (HM) concentration and the rhizosphere activity of giant reed (Arundo donax L. var. BL clone (Blossom)) plants. Funneliformis mosseae (BEG12; AMF1), F. geosporum (BEG11; AMF2) or their combination (AMFmix) were applied as AMF treatments in a short-term pot experiment. The physiological and growth parameters of the host plants, the AMF root colonization and the microbiological enzyme activity of the mycorrhizosphere were examined. We assumed that the combined treatment (WSC + AMF) enhances the fertility of low-fertility acidic sandy soil. Neither the WSC treatment nor the AMF inoculations changed the extent of root colonization. Based on the results of root electrical capacitance and the phosphorous uptake, plant nutritional status was improved by WSC addition, without any negative impacts among the measured parameters. AMF treatments increased the enzyme activity in the soil and decreased the concentrations of the potentially toxic HMs (Cu, Mn, Pb, Zn) in roots, but that mitigation of Cu and Zn was compensated in shoots. According to the results of MicroResp™ measurements, the catabolic activity profile of the soil microbial community was changed in case of the AMF2 treatment. The efficient regulatory mechanism of giant reed might be able to adjust optimal/maximal colonization rate, and to select the preferential AMF partners, this supposed mechanism might be responsible for its invasiveness and tolerance to a wide range of environmental conditions.

• Klíčová slova
• Arundo donax L., AMF inoculation, Soil enzymatic activity, Substrate-induced respiration, Wastewater sludge compost,
• Publikační typ
• časopisecké články MeSH

Enzymes are biological catalysts that play an important role in biochemical reactions necessary for normal growth, maturation and reproduction through whole live world. Their accurate quantitation in biological samples is important in many fields of biochemistry, not only in routine biochemistry and in fundamental research, but also in clinical and pharmacological research and diagnosis. Since the direct measurement of enzymes by masses is impossible, they must be quantified by their catalytic activities. Many different methods have been applied for this purpose so far. Although photometric methods are undoubtedly the most frequently used, separation methods will further gain their position in this field. The article reviews different possibilities for the assay of enzymatic activity by means of capillary electrophoresis (CE). Both the off-line and on-line enzyme assays based on CE are discussed.

• MeSH
• beta-galaktosidasa analýza   metabolismus   MeSH
• elektroforéza kapilární přístrojové vybavení   metody   MeSH
• enzymy analýza   metabolismus   MeSH
• karboxypeptidasy analýza   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• beta-galaktosidasa MeSH
• enzymy MeSH
• karboxypeptidasy MeSH

Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.

• Klíčová slova
• cloning, comparative modeling, leishmaniasis, molecular dynamics, proteolytic activity,
• MeSH
• fylogeneze MeSH
• Leishmania * genetika   metabolismus   MeSH
• leishmanióza * parazitologie   MeSH
• metaloendopeptidasy metabolismus   MeSH
• paraziti * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glycoprotein gp63, Leishmania MeSH Prohlížeč
• metaloendopeptidasy MeSH

BACKGROUND: S1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn2+-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non‑zinc-dependent S1-like nucleases and what is the molecular basis of their activities. METHODS: S1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay. RESULTS: We discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues. CONCLUSIONS: Our findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases. GENERAL SIGNIFICANCE: Our findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non‑zinc-dependent members of this family.

• Klíčová slova
• Acanthamoeba castellanii, Active-site, Non‑zinc-dependent activity, Physcomitrella patens, S1-like nuclease,
• MeSH
• endonukleasy * chemie   MeSH
• eukaryotické buňky MeSH
• katalytická doména MeSH
• katalýza MeSH
• rostliny * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• endonukleasy * MeSH

• Klíčová slova
• ENZYMES/chemistry *, LENS, CRYSTALLINE/chemistry *,
• MeSH
• čočky * MeSH
• enzymy chemie   MeSH
• Fabaceae * MeSH
• oční čočka chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• enzymy MeSH

The time course of structural and enzymatic changes in cardiac myosin was studied in the right and left ventricle of rats exposed to intermittent high altitude (IHA) hypoxia. In the controls, ATPase activity and myosin structure in both ventricles was the same. After the third exposure to simulated high altitude (2 600 m), myosin enzymatic activity rose significantly in the left ventricle and a significant right-left difference appeared. In the next phase of adaptation (11 exposures, 6 000 m), myosin ATPase activity fell in both ventricles and the right-left difference disappeared. After the 16th exposure (7 000 m), enzymatic activity increased again in both ventricles and attained control values. IHA also produced significant structural changes in cardiac myosin, particularly in the rigaht ventricle. The changes were characterized by the formation of myosin aggregates with significantly lower ATPase activity that the myosin monomer. The time course and localization of structural and enzymatic changes in cardiac myosin corresponded to the morphological damage to the heart fibres.

• MeSH
• adenosintrifosfatasy metabolismus   MeSH
• atmosférický tlak * MeSH
• chromatografie DEAE-celulózová MeSH
• chromatografie iontoměničová MeSH
• fyziologická adaptace MeSH
• hypoxie enzymologie   patofyziologie   MeSH
• krysa rodu Rattus MeSH
• myokard enzymologie   metabolismus   MeSH
• myosiny analýza   metabolismus   MeSH
• nadmořská výška * MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenosintrifosfatasy MeSH
• myosiny MeSH