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Computer-assisted enzyme engineering by modification of tunnels, channels and gates
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Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification
PublikacjaRationally designed muteins of Candida albicans glucosamine-6-phosphate synthase, an enzyme known as a promising target for antifungal chemotherapy, were constructed, overexpressed in Escherichia coli and purified to near homogeneity. To facilitate and to optimize the purification of the enzyme, three recombinant versionscontaining internal oligoHis fragments were constructed: (i) by substituting residues 343 - 348...
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Enzyme Conjugation - A Promising Tool for Bio-catalytic and Biotransformation Applications – A Review
PublikacjaEnzymes have revolutionized conventional industrial catalysts as more efficient, eco-friendly, and sustainable substitutes that can be used in different biotechnological processes, food, and pharmaceutical industries. Yet, the enzymes from nature are engineered to make them adapt and enhance their durability in the industrial environment. One promising approach involves the combined use of multiple enzymes that catalyze highly...
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Enzyme-conjugated MXene nanocomposites for biosensing and biocatalysis acuities
PublikacjaEngineered two-dimensional (2-D) MXenes-based materials with tunable characteristics and multi-functionalities have brought up new paradigms in the biosensing and catalysis of chemical compounds. The profusion of electroactive functional moieties on the surface of few/multi-layer MXenes facilitates their ability to retain biomolecules such as enzymes resulting in unique dimensions for bioanalytical and biosensing applications....
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Glucosamine-6-phosphate synthase with an oligoHis insert
PublikacjaGlucosamine-6-phosphate (GlcN-6-P) synthase known also as L-Glutamine: D-fructose-6-phosphate aminotransferase (EC 2.6.1.16), catalyzes the first committed step in the amino sugar biosynthetic pathway in prokaryotic and eukaryotic organisms. The final product of this pathway is an activated precursor of numerous macromolecules containing amino sugars, including chitin and mannproteins in fungi, peptydoglican and lipopolysaccharides...
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Polysaccharides-Based Hybrid Materials for Bio- and Non-Bio Sectors. Edytorial
PublikacjaThis special edition spotlights the recent research in the design, development, and emerging applications of polysaccharides-based hybrid materials for biotechnological and biomedical purposes. All the articles published in this issue underscore the significance of materials derived from cellulose, alginate, chitosan, starch, and carrageenan for various applications, including enzyme production, encapsulation, targeted drug delivery,...
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The Development of Toxoplasma gondii Recombinant Trivalent Chimeric Proteins as an Alternative to Toxoplasma Lysate Antigen (TLA) in Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Immunoglobulin G (IgG) in Small Ruminants
PublikacjaThis study presents an evaluation of seventeen newly produced recombinant trivalent chimeric proteins (containing the same immunodominant fragment of SAG1 and SAG2 of Toxoplasma gondii antigens, and an additional immunodominant fragment of one of the parasite antigens, such as AMA1, GRA1, GRA2, GRA5, GRA6, GRA7, GRA9, LDH2, MAG1, MIC1, MIC3, P35, and ROP1) as a potential alternative to the whole-cell tachyzoite lysate (TLA) used...
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Synthesis of Trehalose by the Erythritol-Producing Yeast Yarrowia lipolytica Co-Displaying Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase
PublikacjaIndustrial trehalose production faces economic challenges with costly enzyme preparations, prompting the exploration of eco-friendly alternatives. Here, we established a coupled functional sugar production line leveraging erythritolproducing cells as an innovative enzyme preparation for trehalose synthesis. The erythritol-producing Yarrowia lipolytica was modified to express a fusion protein consisting of maltooligosyltrehalose synthase...
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Mapping the Transglycosylation Relevant Sites of Cold-Adapted β-D-Galactosidase from Arthrobacter sp. 32cB
PublikacjaB-Galactosidase from Arthrobacter sp. 32cB (ArthbetaDG) is a cold-adapted enzyme able to catalyze hydrolysis of beta-D-galactosides and transglycosylation reaction, where galactosyl moiety is being transferred onto an acceptor larger than a water molecule. Mutants of ArthbetaDG D207A and E517Q were designed to determine the significance of specific residues and to enable formation of complexes with lactulose and sucrose and to...
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Aryl and N-arylamide carbon nanotubes for electrical coupling of laccase to electrodes in biofuel cells and biobatteries
PublikacjaSingle walled carbon nanotubes (SWCNTs) were equipped with aryl residues by chemical reactions. These insoluble materials were used to substitute classical soluble mediators, which help to transfer electrical charge between the conducting electrode and the redox active center of enzyme molecules. The effect of different aryl residues on the efficiency of the catalytic reduction of dioxygen in the presence of laccase was systematically...
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Increased concentration of Taq DNA polymerase as a solution for GC-rich templates from clinical and environmental samples
PublikacjaDNA polymerase is an enzyme which plays crucial role in replication and DNA repair. It found application in PCR (polymerase chain reaction) where catalyses process of in vitro DNA synthesis. To meet the demands posed by mod- ern diagnostic, molecular biology or genetic engineering it is necessary to improve DNA polymerases to obtain new or better features useful in these fields. So far implemented modifications in majority are...
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The first study on the usefulness of recombinant tetravalent chimeric proteins containing fragments of SAG2, GRA1, ROP1 and AMA1 antigens in the detection of specific anti-Toxoplasma gondii antibodies in mouse and human sera
PublikacjaThis study presents an evaluation of four tetravalent recombinant chimeric proteins containing fragments of the Toxoplasma gondii antigens, SAG2, GRA1, ROP1 and AMA1, as potential replacements of a the soluble, whole-cell tachyzoite lysate (TLA) used in serological assays. Recombinant chimeric proteins (SAG2-GRA1-ROP1-AMA1N, AMA1N-SAG2-GRA1-ROP1, AMA1C-SAG2-GRA1-ROP1, and AMA1-SAG2-GRA1-ROP1) obtained by genetic engineering were...
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Możliwości zastosowań, potencjalne źródła oraz ewolucja technologiczna sposobu otrzymywania trehalozy ze szczególnym uwzględnieniem enzymu syntazy trehalozy [EC 5.4.99.16]
PublikacjaJedna z wiodących gałęzi współczesnej biotechnologii opiera się na produkcji białek – przede wszystkim enzymów. Są one powszechnie wykorzystywane: od przemysłu chemicznego poprzez przemysł spożywczy aż po medycynę i życie codzienne (czy ktoś wyobraża sobie w dzisiejszych czasach proszek do prania „bez enzymu”). Przed rozwojem nowoczesnych metod biologii molekularnej i inżynierii genetycznej pozyskiwano interesujące substancje z...
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Electrokinetic properties of n-tetradecane/ethanol emulsions with DPPC and enzyme lipase or phospholipase A2
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A comprehensive approach to SBR modelling for monitoring and control system design
PublikacjaThe aim of this research is to provide a comprehensive description of the Sequencing Batch Reactor (SBR) modelling for monitoring, control, and plant operational optimisation validation. The paper provides a detailed modelling of the SBR, its components and constituent processes. For this purpose, the mass balance principle and continuity equations were provided with a reactive term implemented using Activated Sludge Model (ASM)....
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Enzymatic cross-linking of β-lactoglobulin in solution and at air–water interface: Structural constraints
PublikacjaEffective and controlled use of cross-linking enzymes in structure engineering of food systems depends on characterization of the favorable conditions for enzyme-substrate complex and the limiting factors for the desired modification. In this respect, we analyzed the susceptibility of bovine β-lactoglobulin (BLG) to enzymatic cross-linking by Trichoderma reesei tyrosinase (TrTyr) and transglutaminase (TG). Changes in BLG molecular...
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THE GENE CLONING, OVEREXPRESSION, PURIFICATION AND BIOCHEMICAL CHARACTERISATION OF A NEW COLD-ADAPTED β–GALACTOSIDASE FROM ARTHROBACTER SP. VII-4
PublikacjaINTRODUCTION. β-Galactosidase [EC 3.2.1.23] is an enzyme that catalyzes the hydrolysis of O-glycosidic linkages in galactosides. It is commercially used in dairy industry for the production of milk with reduced lactose content. Potentially, the best method for lactose removal under cooling conditions should be carried out with a cold-adapted enzyme. AIM. The aim of this study was to determine the taxonomic affiliation of the isolate...
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Isolation of the GFA1 gene encoding glucosamine-6-phosphate synthase of Sporothrix schenckii and its expression in Saccharomyces cerevisiae
PublikacjaGlucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases...
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Homoisocitrate dehydrogenase from Candida albicans: properties, inhibition, and targeting by an antifungal pro-drug
PublikacjaThe LYS12 gene from Candida albicans, coding for homoisocitrate dehydrogenase was cloned and expressed as a His-tagged protein in Escherichia coli. The purified gene product catalyzes the Mg2+- and K+-dependent oxidative decarboxylation of homoisocitrate to alpha-ketoadipate. The recombinant enzyme demonstrates strict specificity for homoisocitrate. SDS-PAGE of CaHIcDH revealed its molecular mass of 42.6±1 kDa, while in size-exclusion...
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Nanostructured biocatalysis for biotechnological applications
PublikacjaThe purpose immobilization process is to enhance the performance of an enzyme for commercial processes. A large number of structures have been reported in the literature to boost the effectiveness of immobilized enzymes. The nanomaterials have the optimal properties for equilibrating key parameters that govern the performance of biocatalysts, such as high enzyme loading ability, specific surface area, and mass transfer resistance....
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Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization
PublikacjaDNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the...
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Enzyme-linked carbon nanotubes as biocatalytic tools to degrade and mitigate environmental pollutants
PublikacjaA wide array of organic compounds have been recognized as pollutants of high concern due to their controlled or uncontrolled presence in environmental matrices. The persistent prevalence of diverse organic pollutants, including pharmaceutical compounds, phenolic compounds, synthetic dyes, and other hazardous substances, necessitates robust measures for their practical and sustainable removal from water bodies. Several bioremediation and...
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Efficient production of Staphylococcus simulans lysostaphin in a benchtop bioreactor by recombinant Escherichia coli.
PublikacjaLysostaphin is an enzyme with bactericidal activity against Staphylococcus aureus and other staphylococcal species. In spite of many advantages and promising results of preliminary research, the enzyme is still not widely used in medicine, veterinary or as a food preservative. One of the most important factors limiting application of the enzyme in clinical or technological practice is the high costs of its production. In the present...
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Construction of glucosamine-6-phosphate synthase with an internal hexahistydyl fragment
PublikacjaL-Glutamine: D-fructose-6-phosphate aminotransferase (EC 2.6.1.16), known also as glucosamine-6-phosphate (GlcN-6-P) syntase, catalyzes the first committed step in the amino sugar biosynthetic pathway in prokaryotic and eukaryotic organisms. The enzyme carries out a complex reaction involving ammonia transfer and sugar phosphate isomerization. GlcN-6-P syntase is an important point of metabolic control of amino sugar biosynthesis...
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Structural features responsible for control of the eukaryotic GlcN-6-P synthase activity
PublikacjaGlucosamine-6-phosphate (GlcN-6-P) synthase is responsible for catalysis of the first and practically irreversible step in hexosamine metabolism. As UDP-GlcNAc, the final product of the hexosamine path, constitutes one of the essential substrates for assembly of bacterial and fungal cell walls, the enzyme is an interesting target for antimicrobial therapy.The structure of E. coli enzyme, known since 2001, forms a dimer of two identical...
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The psychrotrophic yeast Sporobolomyces roseus LOCK 1119 as a source of a highly active aspartic protease for the in vitro production of antioxidant peptides
PublikacjaA psychrotrophic yeast strain producing a cold-adapted protease at low temperature was classified as Sporobolomyces roseus. In standard YPG medium, S. roseus LOCK 1119 synthesized an extracellular protease with an activity of approximately 560 U/L. Optimization of medium composition and process temperature considerably enhanced enzyme biosynthesis; an approximate 70% increase in activity (2060 U/L). The native enzyme was purified...
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A new cold-adapted beta-D-galactosidase from the Antarctic Arthrobacter sp. 32c - gene cloning, overexpression, purification and properties
PublikacjaThe development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production. In this article, we present a new β-D-galactosidase as a candidate to be applied in the above mentioned biotechnological...
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Heterogeneity of quaternary structure of glucosamine-6-phosphate deaminase from Giardia lamblia
PublikacjaThe oligoHis-tagged versions of glucosamine-6- phosphate deaminase from Giardia lamblia (GlmNagB-HisN, GlmNagB-HisC) were constructed and purified to hear homogeneity, and their kinetic and structural properties were compared to those of the wild-type enzyme (GlmNagB). Introduction of the oligoHis tag at the GlmNagB C-terminus resulted in almost complete loss of the catalytic activity, while the catalytic properties of GlmNagB-HisN...
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Enzyme immobilization on magnetic nanoparticles
PublikacjaImmobilized enzymes have been commonly used in many branches of industry, biotechnology, medicine and also in analytical chemistry. Their use provides many advantages in comparison with free enzymes including repeated, easy recovery of the enzyme and improvement in enzyme stability. Enzymes are strong and specific catalysts, but are also expensive and fragile. Their immobilization and stabilization may reduce reaction costs, which...
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A new cold-active β-galactosidase from Arthrobacter sp. S3* - gene cloning, overexpression, purification and properties
PublikacjaA psychrotrophic bacterium producing a cold-active β-galactosidase was isolated from Spitsbergen soil and classified as Arthrobacter sp. S3*. The gene encoding β-galactosidase was isolated from the genomic DNA library, sequenced, cloned, expressed in Escherichia coli, purified by ion exchange chromatography and characterized. The Arthrobaster sp. S3* β-galactosidase is a homotrimeric enzyme composed of 74,4 kDa subunits. It is...
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B-GALACTOSIDASE ARTHROBACTER SP. 32cB - OBTAINING THE GENE SEQUENCE, CONSTRUCTION OF THE EXPRESSION SYSTEM, BIOSYNTHESIS AND BIOCHEMICAL CHARACTERIZATION OF THE ENZYME
PublikacjaINTRODUCTION: β-Galactosidase is an enzyme which catalyzes the hydrolysis of O glycosidic bond in β-galactosides. Another activity of β galactosidase is a transglycosylation activity. The main industrial use of this protein is the hydrolysis of lactose in milk in a cooling conditions. Synthesis of galactooligosaccharides, which are mostly used as a prebiotics added to some foods or available as dietary supplements, is only one...
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Cavitation-based technologies for pretreatment and processing of food wastes: Major applications and mechanisms – A review
PublikacjaConversion of food wastes to valuable products is an important topic for sustainable development. Feedstock hydrolysis is a stage strongly affecting the anaerobic digestion process, and resistance of food waste towards hydrolysis causes a decrease in product yield. Such as biomethane, biohydrogen, biohythane, VFAs, and lactic acids. Moreover, mass transfer is a serious limitation of transesterification for the production of biodiesel....
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Extracellular proteolytic activity of Deinococcus geothermalis
PublikacjaProduction of extracellular protease by extremophilic bacteria Deinococcus geothermalis cultivated in liquid media containing 0.1% (w/v) of peptone K, 0.1% yeast extract and 0.2% marine salt reached a maximum in 14 h of the cell growth at 45°C and pH 8.0. The enzyme was purified by a two-step procedure using fractionation by a graded ammonium sulphate precipitation technique and gel filtration on Sephadex G-100 column. Protease...
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Cloning, expression, and biochemical characterization of a coldactive GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil
PublikacjaAn estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa.Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli...
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Docking simulations, Molecular properties and ADMET studies of novel Chromane6,7diol analogues as potential inhibitors of Mushroom tyrosinase
PublikacjaResearch on inhibition of tyrosinase enzyme has attained significant value, because tyrosinase inhibitors have potential applications in medicine, cosmetics (as whitening agents) and in agriculture (as bioinsecticides). Determination and elucidation of new tyrosinase inhibitors are not only beneficial for medical purposes, but their promising applications in improving food quality and nutritional...
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Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery
PublikacjaIn the presented study, a variety of hybrid and single nanomaterials of various origins were tested as novel platforms for horseradish peroxidase immobilization. A thorough characterization was performed to establish the suitability of the support materials for immobilization, as well as the activity and stability retention of the biocatalysts, which were analyzed and discussed. The physicochemical characterization of the obtained...
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Inhibitors of glucosamine-6-phosphate synthase as potential antimicrobials or antidiabetics – synthesis and properties
PublikacjaGlucosamine-6-phosphate synthase (GlcN-6-P synthase) is known as a promising target for antimicrobial agents and antidiabetics. Several compounds of natural or synthetic origin have been identified as inhibitors of this enzyme. This set comprises highly selective L-glutamine, amino sugar phosphate or transition state intermediate cis-enolamine analogues. Relatively low antimicrobial activity of these inhibitors, poorly penetrating...
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INFOGEST inter-laboratory recommendations for assaying gastric and pancreatic lipases activities prior to in vitro digestion studies
PublikacjaIn vitro digestion studies often use animal digestive enzyme extracts as substitutes of human gastric and pancreatic secretions. Pancreatin from porcine origin is thus commonly used to provide relevant pancreatic enzymes such as proteases, amylase and lipase. Rabbit gastric extracts (RGE) have been recently introduced to provide gastric lipase in addition to pepsin. Before preparing simulated gastric and pancreatic extracts with...
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Characterization of glucoamylase immobilized on magnetic nanoparticles
PublikacjaMagnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3-aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si-O-Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support...
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Synergic Effect of Naphthylated Carbon Nanotubes and Gold Nanoparticles on Catalytic Performance of Hybrid Films Containing Bilirubin Oxidase for the Dioxygen Reduction
PublikacjaTo improve the direct electron transfer from a multicopper oxidase MvBOd to the glassy carbon electrode surface the naphthylated multiwall carbon nanotubes (Napht-MWCNTs) are ombined with gold nanoparticles (AuNPs) to form a conductive network at the electrode and to increase its working surface. Such approach allowed to eliminate the necessity of employing mediators to reach elevated current values. The enzyme stability was improved...
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Properties and applications of thermostable proteases sourced from Deinococcus geothermalis.
PublikacjaThe growing interest of extremophiles results from the fact that their enzymes arestable and active under harsh environment conditions. These type of biocatalysts are attractivedue to the fact that can be used in industrial processes that were previously regarded asincompatible with biological materials. Among extremozymes the largest group constitutethermozymes. Currently it is estimated that approximately 40% of enzymes used...
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Role of cholesterol in substrate recognition by -secretase
Publikacja-Secretase is an enzyme known to cleave multiple substrates within their transmembrane domains, with the amyloid precursor protein of Alzheimer’s Disease among the most prominent examples. The activity of -secretase strictly depends on the membrane cholesterol content, yet the mechanistic role of cholesterol in the substrate binding and cleavage remains unclear. In this work, we used all-atom molecular dynamics simulations to examine...
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Comparison of oil yield and quality obtained by different extraction procedures from salmon (Salmo salar) processing byproducts
PublikacjaThe content and composition of lipids in different byproducts (skins, heads, and backbones) from mechanically processed farmed Atlantic salmon were determined and compared with that obtained from wild salmon. Three different procedures were used to establish the optimal conditions of oil extraction (at high temperature −95°C, “cold” extraction at temperature not exceeding 15°C and enzyme assisted with Alcalase). “Cold” extraction...
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Inspired by Nature: The Use of Plant-derived Substrate/Enzyme Combinations to Generate Antimicrobial Activity in situ
PublikacjaThe last decade has witnessed a renewed interest in antimicrobial agents. Plants have received particular attention and frequently rely on the spontaneous enzymatic conversion of an inactive precursor to an active agent. Such two-component substrate/enzyme defence systems can be reconstituted ex vivo. Here, the alliin/alliinase system from garlic seems to be rather effective against Saccharomyces cerevisiae, whilst the glucosinolate/myrosinase...
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In silico design of telomerase inhibitors.
PublikacjaTelomerase is a reverse transcriptase enzyme involved in DNA synthesis at the end of linear chromosomes. Unlike in most other cells, telomerase is reactivated most cancerous cells and, therefore, has become a promising new anticancer target. Despite extensive research, direct telomerase inhibitors have yet not been introduced to the clinics because of the complexity of this enzyme. Structures of this protein from simple organisms...
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Oxygen biosensor based on carbon nanotubes directly grown on graphitic substrate
PublikacjaFilms of carbon nanotubes were synthesized on the graphitic substrates by CVD method in the presence of catalyst. Two different catalysts were used, that is nickel and iron, supported on graphitic substrates. The as-obtained nanotubes were analyzed by SEM imaging with EDX and Raman spectroscopy. The results revealed their multi-walled morphology. Free radical reaction was carried out in order to chemically functionalize synthesized...
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Modulation of UDP-glucuronidation by acridinone antitumor agents C-1305 and C-1311 in HepG2 and HT29 cell lines, despite slight impact in noncellular systems.
PublikacjaBackground Among the studied antitumor acridinone derivatives developed in our laboratory, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) exhibited cytotoxic and antitumor properties against several cancer types and were selected to be evaluated in preclinical and early-phase clinical trials. In the present work, we investigated the impact of C-1305...
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Comparative molecular dynamics study of dimeric and monomeric forms of HIV-1 protease in ligand bound and unbound state
PublikacjaHuman immunodeficiency virus type 1 protease (HIV-1 PR) is a viral-encoded enzyme that forms a homodimer. HIV-1 PR is essential for replication and assembly of the virus and inactivation of HIV-1 PR enzyme causes production of immature, noninfectious viral particles and thus HIV-1 PR is an attractive target in anti-AIDS drug design. In our current work, we performed molecular dynamics (MD) calculations (500 ns) for two different...
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Active Site Architecture and Reaction Mechanism Determination of Cold Adapted beta-D-galactosidase from Arthrobacter sp. 32cB
PublikacjaArthbetaDG is a dimeric, cold-adapted beta-D-galactosidase that exhibits high hydrolytic and transglycosylation activity. A series of crystal structures of its wild form, as well as its ArthbetaDG_E441Q mutein complexes with ligands were obtained in order to describe the mode of its action. The ArthbetaDG_E441Q mutein is an inactive form of the enzyme designed to enable observation of enzyme interaction with its substrate. The...
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Structure of EstA esterase from psychrotrophic Pseudoalteromonas sp. 643A covalently inhibited by monoethylphosphonate.
PublikacjaThe crystal structure of the esterase EstA from the cold-adapted bacteriumPseudoalteromonas sp. 643A was determined in a covalently inhibited form at aresolution of 1.35 A˚. The enzyme has a typical SGNH hydrolase structureconsisting of a single domain containing a five-stranded beta-sheet, with threehelices at the convex side and two helices at the concave side of the sheet, and isornamented with a couple of very short helices...