Laccases as Effective Tools in the Removal of Pharmaceutical Products from Aquatic Systems.

This review aims to provide a comprehensive overview of the application of bacterial and fungal laccases for the removal of pharmaceuticals from the environment. Laccases were evaluated for their efficacy in degrading pharmaceutical substances across various categories, including analgesics, antibiotics, antiepileptics, antirheumatic drugs, cytostatics, hormones, anxiolytics, and sympatholytics. The capability of laccases to degrade or biotransform these drugs was found to be dependent on their structural characteristics. The formation of di-, oligo- and polymers of the parent compound has been observed using the laccase mediator system (LMS), which is advantageous in terms of their removal via commonly used processes in wastewater treatment plants (WWTPs). Notably, certain pharmaceuticals such as tetracycline antibiotics or estrogen hormones exhibited degradation or even mineralization when subjected to laccase treatment. Employing enzyme pretreatment mitigated the toxic effects of degradation products compared to the parent drug. However, when utilizing the LMS, careful mediator selection is essential to prevent potential increases in environment toxicity. Laccases demonstrate efficiency in pharmaceutical removal within WWTPs, operating efficiently under WWTP conditions without necessitating isolation.

Identification and evaluation of antiviral activity of novel compounds targeting SARS-CoV-2 virus by enzymatic and antiviral assays, and computational analysis.

The viral genome of the SARS-CoV-2 coronavirus, the aetiologic agent of COVID-19, encodes structural, non-structural, and accessory proteins. Most of these components undergo rapid genetic variations, though to a lesser extent the essential viral proteases. Consequently, the protease and/or deubiquitinase activities of the cysteine proteases Mpro and PLpro became attractive targets for the design of antiviral agents. Here, we develop and evaluate new bis(benzylidene)cyclohexanones (BBC) and identify potential antiviral compounds. Three compounds were found to be effective in reducing the SARS-CoV-2 load, with EC50 values in the low micromolar concentration range. However, these compounds also exhibited inhibitory activity IC50 against PLpro at approximately 10-fold higher micromolar concentrations. Although originally developed as PLpro inhibitors, the comparison between IC50 and EC50 of BBC indicates that the mechanism of their in vitro antiviral activity is probably not directly related to inhibition of viral cysteine proteases. In conclusion, our study has identified new potential noncytotoxic antiviral compounds suitable for in vivo testing and further improvement.

Optimization of an Inclusion Body-Based Production of the Influenza Virus Neuraminidase in Escherichia coli.

Neuraminidase (NA), as an important protein of influenza virus, represents a promising target for the development of new antiviral agents for the treatment and prevention of influenza A and B. Bacterial host strain Escherichia coli BL21 (DE3)pLysS containing the NA gene of the H1N1 influenza virus produced this overexpressed enzyme in the insoluble fraction of cells in the form of inclusion bodies. The aim of this work was to investigate the effect of independent variables (propagation time, isopropyl ß-d-1-thiogalactopyranoside (IPTG) concentration and expression time) on NA accumulation in inclusion bodies and to optimize these conditions by response surface methodology (RSM). The maximum yield of NA (112.97 ± 2.82 U/g) was achieved under optimal conditions, namely, a propagation time of 7.72 h, IPTG concentration of 1.82 mM and gene expression time of 7.35 h. This study demonstrated that bacterially expressed NA was enzymatically active.

The production of laccases by white-rot fungi under solid-state fermentation conditions.

Laccases (E.C. 1.10.3.2) produced by white-rot fungi (WRF) can be widely used, but the high cost prevents their use in large-scale industrial processes. Finding a solution to the problem could involve laccase production by solid-state fermentation (SSF) simulating the natural growth conditions for WRF. SSF offers several advantages over conventional submerged fermentation (SmF), such as higher efficiency and productivity of the process and pollution reduction. The aim of this review is therefore to provide an overview of the current state of knowledge about the laccase production by WRF under SSF conditions. The focus is on variations in the up-stream process, fermentation and down-stream process and their impact on laccase activity. The variations of up-stream processing involve inoculum preparation, inoculation of the medium and formulation of the propagation and production media. According to the studies, the production process can be shortened to 5-7 days by the selection of a suitable combination of lignocellulosic material and laccase producer without the need for any additional components of the culture medium. Efficient laccase production was achieved by valorisation of wastes as agro-food, municipal wastes or waste generated from wood processing industries. This leads to a reduction of costs and an increase in competitiveness compared to other commonly used methods and/or procedures. There will be significant challenges and opportunities in the future, where SSF could become more efficient and bring the enzyme production to a higher level, especially in new biorefineries, bioreactors and biomolecular/genetic engineering.

The TLC-Bioautography as a Tool for Rapid Enzyme Inhibitors detection - A Review.

Microorganisms and plants can be important sources of many compounds with potential pharmaceutical applications. Extraction of these matrices is one of the ways of identifying the presence of inhibitory active substances against enzymes whose high activity leads to serious human diseases including cancer, Parkinson's or Crohn's diseases. The isolation and purification of inhibitors are time-consuming and expensive steps in the analysis of the crude extract and therefore, it is necessary to find a fast, efficient, and inexpensive method for screening extracts of interest. TLC-Bioautography combines the separation of the extract on a thin layer with its subsequent biological analysis. TLC-Bioautography methods have been developed for several classes of enzymes including oxidoreductases, hydrolases and isomerases, and there is a potential for developing functional methods for other classes of enzymes. This review summarizes known TLC-Bioautography methods and their applications for determining the presence of enzyme inhibitors in extracts and compares the effectiveness of different methodological approaches. It also indicates the current state and perspective of the development of TLC-Bioautography and its possible future applications.

Purification of viral neuraminidase from inclusion bodies produced by recombinant Escherichia coli.

Neuraminidase (NA) is one of the targets for the development of new antivirals against the influenza virus. The recombinant Escherichia coli cells, namely the strains BL21(DE3)pLysS and ArcticExpress(DE3) were used to produce the influenza virus neuraminidase. Although the different conditions of induction were tested, the accumulation of over-expressed NA in insoluble fraction occurred independently of these conditions. The level of over-expressed protein represents 26.15 % of the total cellular proteins. Therefore, the aim of these study was to design the procedure for isolation of recombinant neuraminidase from IBs and subsequently its solubilization and refolding to its native and active form. The highest purity of IBs (86 %) was achieved after repeatedly washing for at least five times with 2 M urea. The best solubilizing agent for releasing NA from IBs was the solution of 8 M urea at pH 8.0 with 94.8 ± 0.4 mg/L released proteins. The most appropriate buffer for refolding of solubilized NA was found to be 50 mM Tris-HCl at pH 7.5 (102 ± 24.2 mg proteins) and the addition of glycerol or arginine had no stimulating effect on protein recovery. The determination of non-glycosylated activity of refolded NA monomer (Km = 0.51 g/L; Vmax = 9.73 U/mg; kcat = 8.76 s-1) using fetuin as a substrate in the coupled enzyme reaction system was the highlight of this work. This procedure provides a way to produce active form of NA monomer by recombinant E. coli cells.

Ultrasonic-assisted extraction of polyphenols and antioxidants from Picea abies bark.

Spruce bark represents a reservoir of bioactive compounds. The aim of this study was to investigate the effect of independent variables (temperature, liquid to solid ratio, time and methanol content) and their interaction within the extraction process by the response surface methodology (RSM). The effect of conventional (solvent extraction; SE) and modern (ultrasound-assisted extraction; UAE) methods for the extraction of antioxidants (antioxidant capacity; AC) and polyphenols (total polyphenol content; TPC) was compared. Maximum yields of AC and TPC by SE and UAE were obtained at modified optimal conditions of 63 °C, methanol content of 53 % (v/v) and 38 mL of extraction solvent per gram of dry material. Two-step extraction process consisting of the fast washing and slow diffusion steps was suitable described by Peleg and Patricelli mathematic models. The HPLC fingerprints of both extracts did not show significant differences while the content of phenolic compounds extracted by UAE was 1.1- to 7.1-times higher than that obtained by SE, quantified by HPLC.

TLC-Bioautography as a fast and cheap screening method for the detection of α-chymotrypsin inhibitors in crude plant extracts.

TLC-Bioautography is a fast and effective method for assessing the inhibitory effect of compounds present in plant extracts against microbial species. However, this method has a hidden, currently underutilized potential for evaluating the presence of inhibitory compounds against selected enzymes. The aim of this work was to design a functional TLC-Bioautography method for the evaluation of protease inhibitors present in plant extracts. The method is based on the hydrolysis of Nα-benzoyl-DL-arginine-p-nitroanilide hydrochloride (BApNA) by α-chymotrypsin as a representative serine protease to produce coloured para-nitroaniline (pNA). Derivatization of pNA with both sodium nitrite and N-(1-naphthyl) ethylenediamine (NPED) leads to the formation of a pink azo dye. This step improves the resolution of active compounds on the chromatogram, which appear as light spots on a pink background. The developed method was tested for the analysis of protease inhibitors in different plant materials such as grape pomace from Vitis vinifera, Picea abies bark, Hippophae rhamnoides berries, Hordeum sativum bran, Triticum aestivum bran and Avena sativa bran. Plant extracts, which could not be analysed by a commonly used spectrophotometric method due to interference, were assessed by this method.

Diversity of sialidases found in the human body - A review.

Sialidases are enzymes essential for numerous organisms including humans. Hydrolytic sialidases (EC 3.2.1.18), trans-sialidases and anhydrosialidases (intramolecular trans-sialidases, EC 4.2.2.15) are glycoside hydrolase enzymes that cleave the glycosidic linkage and release sialic acid residues from sialyl substrates. The paper summarizes diverse sialidases present in the human body and their potential impact on development of antiviral compounds - inhibitors of viral neuraminidases. It includes a brief overview of catalytic mechanisms of action of sialidases and describes the origin of sialidases in the human body. This is followed by description of the structure and function of sialidase families with a special focus on the GH33 and GH34 families. Various effects of sialidases on human body are also briefly described. Modulation of sialidase activity may be considered a useful tool for effective treatment of various diseases. In some cases, it is desired to completely suppress the activity of sialidases by suitable inhibitors. Specific sialidase inhibitors are useful for the treatment of influenza, epilepsy, Alzheimer's disease, diabetes, different types of cancer, or heart defects. Challenges and future directions are shortly depicted in the final part of the paper.

Decolourization and detoxification of monoazo dyes by laccase from the white-rot fungus Trametes versicolor.

The decolourization and detoxification of azo dyes (Orange 2, Acid Orange 6) by fungal laccase from Trametes versicolor were evaluated. For laccase catalysed reaction, the azonaphthol Orange 2, with 72.8% decolourization, was degraded more rapidly than the azobenzene Acid Orange 6, with 45.3%. The presence of hydroxyl group at o-position to azo bond in the structure of Orange 2 was more preferable than the presence of two hydroxyl groups at o- and p-positions to azo bond in Acid Orange 6. Although the laccase treatment was more effective for the Orange 2 decolourization, the toxicity of both monoazo dye solutions became less toxic for the prokaryote growth. The phytotoxicity of Orange 2 and Acid Orange 6 solutions after laccase treatment was decreased in the range of 41.2-64.3 %. Also, the photoxicity, as measured by the production of chlorophylls a and b by Chlorella vulgaris and Microcystis aeruginosa, was decreased by laccase treatment of selected monoazo dyes. Our results show that different dyes can be decolorized and detoxified by laccase from T. versicolor in a single step.

Purification and characterization of extracellular laccase produced by Ceriporiopsis subvermispora and decolorization of triphenylmethane dyes.

Laccases of white-rot fungi provide a promising future as a tool to be used in the field of biodegradation of synthetic dyes with different chemical structures. The aim of this study was production, characterization, and application of laccases from the white-rot fungus Ceriporiopsis subvermispora ATCC 90467 for decolorization of triphenylmethane dyes that could remain persistent in wastewater. Laccase was purified from a C. subvermispora culture by a four-step method resulting high specific activity of 2,571 U g-1 , 88-fold higher than crude laccase. Purified laccase (molecular weight 45 kDa) had the optimum activity at pH 2.0 and the optimum temperature 50 °C using ABTS as chromogenic substrate. Laccases efficiently decolorized triphenylmethane dyes such as Malachite Green (87.8%), Bromocresol Purple (71.6%), and Methyl Violet (68.1%) without redox mediator. However, decolorization percentage of hardly degradable triphenylmethane dyes such as Phenol Red, Bromophenol Blue, and Brilliant Blue R-250 was increased the presence of some low-molecular weight compounds (natural or synthetic redox mediators). Purified laccases were resistant to Mg2+ , Ca2+ , Ba2+ , Mn2+ , Fe2+ , Cu2+ , Zn2+ , and Sn2+ (10 mmol L-1 ). These findings suggest that laccases from C. subvermispora are able to decolorize triphenylmethane dyes without the negative influence of metal ions that can be found in wastewater.