Laccase is predominantly found in lignin degrading filamentous white rot fungi, where it is involved in the oxidative degradation of this recalcitrant heteropolymer. In brown rot fungi it is much less prevalent: laccases from only a few brown rots have been detected and only two have been characterized. This study tries to understand the role of this ligninolytic enzyme in brown rots by investigating the catalytic properties of laccases secreted by Fomitopsis pinicola FP58527 SS1. When grown on either poplar or spruce wood blocks, several laccases were detected in the secretome. Two of them (FpLcc1 and FpLcc2) were heterologously produced using Trichoderma reesei QM9414 Δxyr1 as expression host and purified to homogeneity by consecutive steps of hydrophobic interaction, anion exchange and size exclusion chromatography. With the substrates 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol both laccases showed similar, low pH-optima below 3 for ABTS and 2,6-DMP and at pH 3.5 for guaiacol which is at the acidic end of laccases isolated from white rot fungi. The determined KM values were low while kcat values measured at acidic conditions were comparable to those reported for other laccases from white rot fungi. While both enzymes showed a moderate decrease in activity in the presence of oxalic and citric acid FpLcc2 was activated by acetic acid up to 3.7 times. This activation effect is much more pronounced at pH 5.0 compared to pH 3.0 and could already be observed at a concentration of 1 mM acetic acid.
- MeSH
- Coriolaceae * genetika MeSH
- Hypocreales MeSH
- lakasa * genetika MeSH
- lignin MeSH
- Publikační typ
- časopisecké články MeSH
Detection tubes are small devices for the colorimetric enzymatic detection of cholinesterase inhibitors such as sarin, soman, VX nerve agents and substances denoted as Novichok. These detectors contain carriers in the form of pellets with immobilized cholinesterase, substrate and detection reagent. Their advantages are portability, sensitivity and simplicity, enabling fast detection of such compounds from air and water in case of a terrorist attack or war. In general, maintaining the stability of an enzyme for a longer time is very problematic; therefore, its further enhancement is required for safety and financial reasons. In this study, the stability of our patented carriers in the form of pellets with immobilized butyrylcholinesterase containing an increasing amount of the unique sorbent Neusilin® US2 was evaluated. The samples containing Neusilin maintained the stability of the immobilized enzyme for a longer time even at higher temperature and humidity than the currently commercially used carrier without Neusilin, allowing improved detection of nerve agents.
- MeSH
- biosenzitivní techniky metody MeSH
- butyrylcholinesterasa chemie metabolismus MeSH
- cholinesterasové inhibitory analýza MeSH
- enzymy imobilizované chemie metabolismus MeSH
- kolorimetrie metody MeSH
- nosiče léků chemie MeSH
- silikáty metabolismus MeSH
- sloučeniny hliníku metabolismus MeSH
- sloučeniny hořčíku metabolismus MeSH
- stabilita enzymů účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
Human lysosomal β-N-acetylhexosaminidases from the family 20 of glycoside hydrolases are dimeric enzymes catalysing the cleavage of terminal β-N-acetylglucosamine and β-N-acetylgalactosamine residues from a broad spectrum of glycoconjugates. Here, we present a facile, robust, and cost-effective extracellular expression of human β-N-acetylhexosaminidase B in Pichia pastoris KM71H strain. The prepared Hex B was purified in a single step with 33% yield obtaining 10mg of the pure enzyme per 1L of the culture media. The enzyme was used in the inhibition assays with the known mechanism-based inhibitor NAG-thiazoline and a wide variety of its derivatives in the search for specific inhibitors of the human GH20 β-N-acetylhexosaminidases over the human GH84 β-N-acetylglucosaminidase, which was expressed, purified and used in the inhibition experiments as well. Moreover, enzyme-inhibitor complexes were analysed employing computational tools in order to reveal the structural basis of the results of the inhibition assays, showing the importance of water-mediated interactions between the enzyme and respective ligands. The presented method for the heterologous expression of human Hex B is robust, it significantly reduces the costs and equipment demands in comparison to the expression in mammalian cell lines. This will enhance accessibility of this human enzyme to the broad scientific community and may speed up the research of specific inhibitors of this physiologically important glycosidase family.
- MeSH
- acetylglukosamin analogy a deriváty farmakologie MeSH
- beta-hexosaminidasa, beta řetězec antagonisté a inhibitory genetika izolace a purifikace MeSH
- exprese genu MeSH
- inhibitory enzymů farmakologie MeSH
- katalytická doména MeSH
- kinetika MeSH
- lidé MeSH
- molekulární modely MeSH
- Pichia enzymologie genetika MeSH
- rekombinantní proteiny účinky léků genetika izolace a purifikace MeSH
- thiazoly farmakologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
In recent years, there has been an increase in efforts to improve wastewater treatment as the concentration of dangerous pollutants, such as endocrine disrupting chemicals, in wastewater increases. These compounds, which mimic the effect of hormones, have a negative impact on human health and are not easily removed from water. One way to effectively eliminate these pollutants is to use enzymatically activated materials. In this study, we report on the use of laccase from the white rot fungus Trametes versicolor immobilized onto polyamide 6/chitosan (PA6/CHIT) nanofibers modified using two different spacers (bovine serum albumin and hexamethylenediamine). We then tested the ability of the PA6/CHIT-laccase biocatalysts to eliminate a mixture containing 50μM of two endocrine disrupting chemicals: bisphenol A and 17α-ethinylestradiol. The PA6/CHIT nanofiber matrix used in this study not only proved to be a suitable carrier for immobilized and modified laccase but was also efficient in the removal of a mixture of endocrine disrupting chemicals in three treatment cycles.
- MeSH
- biodegradace MeSH
- chitosan chemie MeSH
- endokrinní disruptory metabolismus MeSH
- enzymy imobilizované metabolismus MeSH
- fungální proteiny metabolismus MeSH
- kaprolaktam analogy a deriváty chemie MeSH
- lakasa metabolismus MeSH
- lidé MeSH
- nanovlákna chemie ultrastruktura MeSH
- polymery chemie MeSH
- Trametes enzymologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
