1,721,042 research outputs found
Preparato detergente per superfici, del tipo pavimenti, pareti, vetrate, oggetti e simili, e procedimento per la pulizia di tali superfici.
No full textNuovo formulato per la detergenza di superfici
A continuous spectrophotometric enzyme-coupled assay for deoxynucleoside triphosphate triphosphohydrolases
No full textWe describe a continuous, spectrophotometric, enzyme-coupled assay useful to monitor reactions catalyzed by nucleoside triphosphohydrolases. In particular, using Escherichia coli deoxynucleoside triphosphohydrolase (Dgt), which hydrolyzes dGTP to deoxyguanosine and tripolyphosphate (PPPi) as the enzyme to be tested, we devised a procedure relying on purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) as the auxiliary enzymes. The deoxyguanosine released by Dgt can indeed be conveniently subjected to phosphorolysis by PNPase, yielding deoxyribose-1-phosphate and guanine, which in turn can be oxidized to 8-oxoguanine by XOD. By this means, it was possible to continuously detect Dgt activity at 297 nm, at which wavelength the difference between the molar extinction coefficients of 8-oxoguanine (8000 M-1 cm-1) and guanine (1090 M-1 cm-1) is maximal. The initial velocities of Dgt-catalyzed reactions were then determined in parallel with the enzyme-coupled assay and with a discontinuous high-performance liquid chromatography (HPLC) method able to selectively detect deoxyguanosine. Under appropriate conditions of excess auxiliary enzymes, the activities determined with our continuous enzyme-coupled assay were quantitatively comparable to those observed with the HPLC method. Moreover, the enzyme-coupled assay proved to be more sensitive than the chromatographic procedure, permitting reliable detection of Dgt activity at low dGTP substrate concentrations
HoLaMa: A Klenow sub-fragment lacking the 3′-5′ exonuclease domain
No full textThe design, construction, overexpression, and purification of a Klenow sub-fragment lacking the 30–50
exonuclease domain is presented here. In particular, a synthetic gene coding for the residues 515–928
of Escherichia coli DNA polymerase I was constructed. To improve the solubility and stability of the corresponding
protein, the synthetic gene was designed to contain 11 site-specific substitutions. The gene
was inserted into the pBADHis expression vector, generating 2 identical Klenow sub-fragments, bearing
or not a hexahistidine tag. Both these Klenow sub-fragments, denominated HoLaMa and HoLaMaHis,
were purified, and their catalytic properties were compared to those of Klenow enzyme. When DNA polymerase
activity was assayed under processive conditions, the Klenow enzyme performed much better
than HoLaMa and HoLaMaHis. However, when DNA polymerase activity was assayed under distributive
conditions, the initial velocity of the reaction catalyzed by HoLaMa was comparable to that observed in
the presence of Klenow enzyme. In particular, under distributive conditions HoLaMa was found to
strongly prefer dsDNAs bearing a short template overhang, to the length of which the Klenow enzyme
was relatively insensitive. Overall, our observations indicate that the exonuclease domain of the
Klenow enzyme, besides its proofreading activity, does significantly contribute to the catalytic efficiency
of DNA elongation
ENZYME COMPOSITION FOR REDUCING THE RELEASE OF PHARMACEUTICAL ACTIVE INGREDIENTS INTO THE ENVIRONMENT
No full textA composition comprising one or more enzymes selected from the group consisting of oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases for reducing the release into the environment of one or more pharmaceutical active ingredients, derivatives and metabolites thereof by a subject undergoing pharmacological therapy with the one or more pharmaceutical active ingredients, by means of an enzyme reaction of the one or more pharmaceutical active ingredients, derivatives and metabolites thereof with the one or more enzymes, the composition also comprising the one or more pharmaceutical active ingredients or being administered simultaneously, sequentially or separately with respect to the one or more pharmaceutical active ingredients
Substrate Activation of the Low-Molecular Weight Protein Tyrosine Phosphatase from Mycobacterium tuberculosis
No full textMycobacterium tuberculosis is known to express a low-molecular weight protein tyrosine phosphatase. This enzyme, denoted as MptpA (Mycobacterium protein tyrosine phosphatase A), is essential for the pathogen to escape the host immune system and therefore represents a target for the search of antituberculosis drugs. MptpA was shown to undergo a conformational transition during catalysis, leading to the closure of the active site, which is by this means poised to the chemical step of dephosphorylation. Here we show that MptpA is subjected to substrate activation, triggered by p-nitrophenyl phosphate or by phosphotyrosine. Moreover, we show that the enzyme is also activated by phosphoserine, with serine being ineffective in enhancing MptpA activity. In addition, we performed assays under pre-steady-state conditions, and we show here that substrate activation is kinetically coupled to the closure of the active site. Surprisingly, when phosphotyrosine was used as a substrate, MptpA did not obey Michealis-Menten kinetics, but we observed a sigmoidal dependence of the reaction velocity on substrate concentration, suggesting the presence of an allosteric activating site in MptpA. This site could represent a promising target for the screening of MptpA inhibitors exerting their action independently of the binding to the enzyme active site
Method for preventing and controlling biofouling on marine objects
No full textA method for preventing and controlling the formation of biofouling on an object that is immersed or partially immersed in a water environment in which there is one or more organic or inorganic compounds capable of releasing gas, comprising the step of applying to the surface of the object a composition comprising a polymeric resin and one or more enzymes or a paint or a coating and one or more enzymes, wherein the one or more enzymes are adapted to catalyze a reaction of the one or more compounds that leads to the formation of gas
METHOD FOR PREVENTING AND CONTROLLING ORGANISMS THAT INFEST AQUEOUS SYSTEMS
No full textMethod for preventing and controlling organisms that infest aqueous systems in which one or more organic or inorganic compounds capable of releasing gas are present, comprising the step of dispersing on and/or in the aqueous mass a preparation comprising enzymes adapted to catalyze a reaction of the one or more compounds that leads to the formation of gas
Method for preventing and controlling biofouling on marine objects
No full textA method for preventing and controlling the formation of biofouling on an object that is immersed or partially immersed in a water environment in which there is one or more organic or inorganic compounds capable of releasing gas, comprising the step of applying to the surface of the object a composition comprising a polymeric resin and one or more enzymes or a paint or a coating and one or more enzymes, wherein the one or more enzymes are adapted to catalyze a reaction of the one or more compounds that leads to the formation of gas
Production in Escherichia coli of recombinant COVID-19 spike protein fragments fused to CRM197
No full textDuring 2020, the COVID-19 pandemic affected almost 108 individuals. Quite a number of vaccines against COVID-19 were therefore developed, and a few recently received authorization for emergency use. Overall, these vaccines target specific viral proteins by antibodies whose synthesis is directly elicited or indirectly triggered by nucleic acids coding for the desired targets. Among these targets, the receptor binding domain (RBD) of COVID-19 spike protein (SP) does frequently occur in the repertoire of candidate vaccines. However, the immunogenicity of RBD per se is limited by its low molecular mass, and by a structural rearrangement of full-length SP accompanied by the detachment of RBD. Here we show that the RBD of COVID-19 SP can be conveniently produced in Escherichia coli when fused to a fragment of CRM197, a variant of diphtheria toxin currently used for a number of conjugated vaccines. In particular, we show that the CRM197-RBD chimera solubilized from inclusion bodies can be refolded and purified to a state featuring the 5 native disulphide bonds of the parental proteins, the competence in binding angiotensin-converting enzyme 2, and a satisfactory stability at room temperature. Accordingly, our observations provide compulsory information for the development of a candidate vaccine directed against COVID-19
A single-molecule FRET sensor for monitoring DNA synthesis in real time
Full text linkWe developed a versatile DNA assay and framework for monitoring polymerization of DNA in real time and at the single-molecule level. The assay consists of an acceptor labelled DNA primer annealed to a DNA template that is labelled on its single stranded, downstream overhang with a donor fluorophore. Upon extension of the primer using a DNA polymerase, the overhang of the template alters its conformation from a random coil to the canonical structure of double stranded DNA. This conformational change increases the distance between the donor and the acceptor fluorophore and can be detected as a decrease in the Förster resonance energy transfer (FRET) efficiency between both fluorophores. Remarkably, the DNA assay does not require any modification of the DNA polymerase and albeit the simple and robust spectroscopic readout facilitates measurements even with conventional fluorimeters or stopped-flow equipment, single-molecule FRET provides additional access to parameters such as the processivity of DNA synthesis and, for one of the three DNA polymerases tested, the detection of binding and dissociation of the DNA polymerase to DNA. We furthermore demonstrate that primer extensions by a single base can be resolved
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