Nowadays, major problems worldwide derive from the presence of pharmaceutically active compounds in the wastewater of hospitals and healthcare facilities. To address this issue, biological transformation of diclofenac using native and immobilized laccase is described in our study. Laccase stabilization is demonstrated, using two different types of immobilization: cross-linked enzyme aggregates (CLEAs) and magnetic cross-linked enzyme aggregates (mCLEAs). High diclofenac (DCF) removal capacity of CLEAs laccase and mCLEAs laccase in comparison to native laccase is determined. Under optimal conditions, the removal capacity of native laccase is 11.5 ± 0.3 µg DCF/g laccase, of CLEAs 15.6 ± 0.4 µg DCF/glaccase and of mCLEAs 13.6 ± 0.4 µg DCF/glaccase. Our immobilized laccase also possesses very good reusability, since the half-life of the immobilized laccase in the form of CLEAs is reached after the second cycle of biocatalyst reuse, while the half-life of the mCLEAs laccase can be detected at the fourth cycle. The improved stability of immobilized laccase in supercritical carbon dioxide is shown. The promising results of DCF removal using CLEAs laccase and mCLEAs laccase show that both immobilized forms of laccase have the potential to be used in cleaner industrial technologies, e.g. wastewater treatment.
COBISS.SI-ID: 27965955
Opioids are molecules whose binding to specialized G-Protein Coupled Receptors (GPCRs) triggers a signaling cascade that leads to the downregulation of pain pathways. Binding of an opioid to the membrane-embedded GPCR occurs when the opioid molecule is protonated, which provides a potential strategy to design nontoxic opioids that are protonated and bind to the GPCR only at the low pH of injured or inflamed tissue. Excellent model systems to study protonation-dependent binding of opioids to GPCRs are fentanyl, which is protonated and binds to the GPCR at both physiological and low pH, and the fluorinated fentanyl derivative NFEPP, which is protonated and binds to the GPCR only at low pH. The molecular mechanisms of fentanyl and NFEPP binding to the GPCR are largely unknown. To enable atomistic studies of opioid binding to GPCRs, we have carried out extensive quantum mechanical and classical mechanical computations to derive a potential energy function for fentanyl and NFEPP and present force field parameters for both opioid molecules. We find that fluorination alters the electronic ground state properties of fentanyl. As a consequence, fentanyl and NFEPP have distinct torsional and electrostatic properties likely to impact how they bind to receptors.
COBISS.SI-ID: 19582723
The enzyme transglutaminase (TGM) has a very important role in applications in various industries. Potentially, a substrate for microbial TGM may be any proteinaceous molecule with glutamine and lysine residues, which broadens the importance of microbial TGM for clean industrial applications or for the use of biosensors. Because stabilization and reusability of an enzyme is an economically important goal for enzymatic processes, the purpose of this study was immobilization of TGM onto magnetic nanoparticles (MNPs) synthesized by the co-precipitation of Fe2+ in Fe3+ ions and modified with carboxymethyl-dextran (CMD). Synthesized nanoparticles were firstly activated with a cross-linking agent (glutaraldehyde (GA) and/or pentaethylenehexamine (PEHA)) and then used for covalent immobilization of the enzyme. Activity of immobilized TGM was determined and compared with activity of the free enzyme. Under optimal immobilization conditions, the enzyme was hyperactivated and showed 99% immobilization efficiency and 110% residual activity. The immobilized TGM showed excellent thermal stability at 50?°C and 70?°C in comparison with non-immobilized enzyme and very good reusability and can be used for cleaner production in different real life industrial applications, such as in leather, textile and wool industry.
COBISS.SI-ID: 22565910
Rapid methods for diagnosis of bacterial infections are urgently needed to reduce inappropriate use of antibiotics, which contributes to antimicrobial resistance. In many rapid diagnostic methods, DNA oligonucleotide probes, attached to a surface, bind to specific nucleotide sequences in the DNA of a target pathogen. Typically, each probe binds to a single target sequence; i.e., target–probe binding is monovalent. Here we show using computer simulations that the detection sensitivity and specificity can be improved by designing probes that bind multivalently to the entire length of the pathogen genomic DNA, such that a given probe binds to multiple sites along the target DNA. Our results suggest that multivalent targeting of long pieces of genomic DNA can allow highly sensitive and selective binding of the target DNA, even if competing DNA in the sample also contains binding sites for the same probe sequences. Our results are robust to mild fragmentation of the bacterial genome. Our conclusions may also be relevant for DNA detection in other fields, such as disease diagnostics more broadly, environmental management, and food safety.
COBISS.SI-ID: 23106326
SARS-CoV-2, or severe acute respiratory syndrome coronavirus 2, represents a new strain of Coronaviridae. We performed a virtual screening study in order to identify potential inhibitors of the SARS-CoV-2 main viral protease (3CLpro or Mpro). For this purpose, we developed a novel approach using ensemble docking high-throughput virtual screening directly coupled with subsequent Linear Interaction Energy (LIE) calculations to maximize the conformational space sampling and to assess the binding affinity of identified inhibitors. A large database of small commercial compounds was prepared, and top-scoring hits were identified with two compounds singled out, namely 1-[(R)-2-(1,3-benzimidazol-2-yl)-1-pyrrolidinyl]-2-(4-methyl-1,4-diazepan-1-yl)-1-ethanone and [({(S)-1-[(1H-indol-2-yl)methyl]-3-pyrrolidinyl}methyl)amino](5-methyl-2H-pyrazol-3-yl)formaldehyde. Moreover, we obtained a favorable binding free energy of the identified compounds, and using contact analysis we confirmed their stable binding modes in the 3CLpro active site. These compounds will facilitate further 3CLpro inhibitor design.
COBISS.SI-ID: 42014467