Characterization and genome analysis of a broad host range lytic phage vB_SenS_TUMS_E19 against Salmonella enterica and its efficiency evaluation in the liquid egg.

Salmonella enterica serovars are zoonotic bacterial that cause foodborne enteritis. Due to bacteria's antibiotic resistance, using bacteriophages for biocontrol and treatment is a new therapeutic approach. In this study, we isolated, characterized, and analyzed the genome of vB_SenS_TUMS_E19 (E19), a broad host range Salmonella bacteriophage, and evaluated the influence of E19 on liquid eggs infected with Salmonella enterica serovar Enteritidis. Transmission electron microscopy showed that the isolated bacteriophage had a siphovirus morphotype. E19 showed rapid adsorption (92% in 5 min), a short latent period (18 min), a large burst size (156 PFU per cell), and a broad host range against different Salmonella enterica serovars. Whole-genome sequencing analysis indicated that the isolated phage had a 42 813 bp long genome with 49.8% G + C content. Neither tRNA genes nor those associated with antibiotic resistance, virulence factors, or lysogenic formation were detected in the genome. The efficacy of E19 was evaluated in liquid eggs inoculated with S. Enteritidis at 4 and 25 °C, and results showed that it could effectively eradicate S. Enteritidis in just 30 min and prevented its growth up to 72 h. Our findings indicate that E19 can be an alternative to a preservative to control Salmonella in food samples and help prevent and treat salmonellosis.

Development of non-destructive methods for the assessment of authenticity of sports whey protein supplements.

In the category of sports supplements, whey protein powder is one of the popular supplements for muscle building applications. Therefore, verification of the sport supplements as authentic products has become a universal concern. This work aimed to propose vibrational spectroscopy including near infrared (NIR) and infrared (IR) as rapid and non-destructive testing tools for the detection and quantification of maltodextrin, milk powder and milk whey powder in whey protein supplements. Initially, principal component analysis was applied to data for pattern recognition and the results displayed a fine pattern of discrimination. Partial least square discrimination analysis (PLS-DA) and K-nearest neighbours (KNN) were exploited as supervised method modelling classification. This process was done in order to respond to two vital questions whether the sample is adulterated or not and what is the kind of adulteration. PLS-DA showed better classification results rather than KNN according to the figure of merits of the model. Partial least square regression (PLSR) was employed on pre-treated spectra to quantify the amount of adulteration in sport whey supplements. Eventually, it seems vibrational spectroscopy could be implemented as a simple, and low-cost analysis method for the detection and quantification of mentioned adulterants in whey protein supplements.

Design of experiments approach for the development of a validated method to determine the exenatide content in poly(lactide-co-glycolide) microspheres.

Due to the lack of pharmacopeia guidelines for injectable microspheres based on poly (D, L-lactide-co-glycolide) (PLGA), an internal method validation is a critical prerequisite for quality assurance. One of the essential issues of developing peptide-based drugs loaded PLGA microspheres is the precise determination of the amount of peptide drug entrapped in the microspheres. The aim of this study is the development and optimization of a method for measuring the drug content loading of PLGA microspheres using exenatide as a model peptide drug. Exenatide-loaded PLGA microspheres were prepared by a double emulsion solvent evaporation method. The extraction method to determine exenatide content in microspheres was optimized using Design of Experiments (DoE) approach. After the initial screening of six factors, using Fractional Factorial design (FFD), four of them, including type of organic solvent, buffer/organic solvent ratio (v/v), shaking time and pH, exhibited significant effects on the response, namely the exenatide loading, and a Box-Behnken design (BBD) was subsequently applied to obtain its optimum level. The optimum level for organic solvent volume, buffer/organic solvent ratio, shaking time, and pH were 4 ml, 1, 5.6 hrs, and pH 6, respectively. The exenatide content in microspheres under these conditions was 6.4 ± 0.0 (%w/w), whereas a value of 6.1% was predicted by the derived equation. This excellent agreement between the actual and the predicted value demonstrates that the fitted model can thus be used to determine the exenatide content.

Isolation, characterization, and genome analysis of a broad host range Salmonella phage vB_SenS_TUMS_E4: a candidate bacteriophage for biocontrol.

Salmonella enteritidis is one of the most important foodborne pathogens that cause numerous outbreaks worldwide. Some strains of Salmonella have become progressively resistant to antibiotics, so they could represent a critical threat to public health and have led to the use of alternative therapeutic approaches like phage therapy. In this study, a lytic phage, vB_SenS_TUMS_E4 (E4), was isolated from poultry effluent and characterized to evaluate its potential and efficacy for bio-controlling S. enteritidis in foods. Transmission electron microscopy revealed that E4 has a siphovirus morphotype, with an isometric head and non-contractile tail. Determining the host range showed that this phage can effectively infect different motile as well as non-motile Salmonella enterica serovars. The biological characteristics of E4 showed that it has a short latent period of about 15 min and a large burst size of 287 PFU/cell, and is also significantly stable in a broad range of pHs and temperatures. The E4 whole genome contains 43,018 bp and encodes 60 coding sequences (CDSs) but no tRNA genes. Bioinformatics analysis revealed that the genome of E4 lacks any genes related to lysogeny behavior, antibiotic resistance, toxins, or virulence factors. The efficacy of phage E4 as a bio-control agent was assessed in various foodstuffs inoculated with S. enteritidis at 4°C and 25°C, and the resulting data indicated that it could eradicate S. enteritidis after a very short time of 15 min. The findings of the present study showed that E4 is a hopeful candidate as a bio-control agent against S. enteritidis and has the potential to be used in various foodstuffs.

Isolation, characterization, and genomic analysis of vB_PaeS_TUMS_P81, a lytic bacteriophage against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that can lead to nosocomial infections which are in turn life threatening. The increase in antibiotic resistance, at an alarming rate, has resulted in a pressing need for alternative therapeutic approaches such as phage therapy, which hold promise according to several studies. This study featured the isolation and characterization of vB_PaeS_TUMS_P81, a new lytic Pseudomonas phage. The whole-genome sequencing indicated that it has a genome of 73,167 bp containing 93 predicted coding sequences. Genes involved in virulence or lysogeny pathway were nowhere to be found in the genome, so it is potentially safe when it comes to therapeutic applications. Genomic and phylogenetic analysis indicated that vB_PaeS_TUMS_P81 is a member of the genus Litunavirus, belonging to Schitoviridae family. The present study lays the groundwork for further research on treatment of P. aeruginosa infections.

Isolation, characterization, and genomic analysis of vB_PaeP_TUMS_P121, a new lytic bacteriophage infecting Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause life-threatening nosocomial infections. The alarming increase in antibiotic resistance has led to an urgent need for alternative therapeutic approaches, such as phage therapy, which has shown promising results in many studies. In this study, P121, a new lytic Pseudomonas phage, was isolated and characterized. Whole-genome sequencing showed that it has a genome of 73,001 bp that contains 91 predicted coding sequences. No genes involved in virulence or lysogeny were found in the genome, thus making it potentially safe for therapeutic applications. Genomic and phylogenetic analysis indicated that P121 is a member of the genus Litunavirus, family Schitoviridae. The present study provides some basic information for further research on treatment of P. aeruginosa infections.

Bisphenol-A in biological samples of breast cancer mastectomy and mammoplasty patients and correlation with levels measured in urine and tissue.

Endocrine disrupting chemicals (EDCs) are organic compounds that have estrogenic activity and can interfere with the endocrine system. Bisphenol-A (BPA) is one of these compounds which possess a potential risk for breast cancer. The aim of this research was to evaluate BPA concentration in both the urine and breast adipose tissue samples of breast cancer mastectomy and mammoplasty patients and study correlations of BPA levels in breast adipose tissue with urine samples in the both groups. Urine and breast adipose tissue samples from 41 breast cancer mastectomy and 11 mammoplasty patients were taken. BPA concentrations were detected using an ELISA assay. Urinary BPA concentrations were significantly higher in cancerous patients (2.12 ± 1.48 ng/ml; P < 0.01) compared to non-cancerous (0.91 ± 0.42 ng/ml). Likewise, tissue BPA concentrations in cancerous patients (4.20 ± 2.40 ng/g tissue; P < 0.01) were significantly higher than non- cancerous (1.80 ± 1.05 ng/g tissue). Urinary BPA concentrations were positively correlated with breast adipose tissue BPA in the case group (P < 0.001, R = 0.896). We showed that BPA was present in urine and breast adipose tissue samples of the studied populations. With regard to higher BPA mean concentration in cancerous patients than non-cancerous individuals in this study, BPA might increase the risk of breast cancer incidence.

Potential of chitosan/alginate nanoparticles as a non-viral vector for gene delivery: Formulation and optimization using D-optimal design.

Chitosan/alginate (Chi/Alg) nanoparticles as a non-viral vector for the Smad4 encoding plasmid were optimized utilizing D-optimal design based on the nanoparticles/plasmid ratio, Chi/Alg MW, and preparation method type. Following the optimization and validation of the best formula, morphology studies and FTIR measurements were performed to evaluate the optimized Chi/Alg/S NPs. Toxicity (MTT assay) and transfection studies were performed for the best formula in comparison with Lipofectamine 2000, and Polyethyleneimine (PEI) and evaluated using Green Fluorescence Protein (GFP) assay, Flow cytometry, and RT-PCR. The model predicted a particle size of 111 nm, loading efficacy (LE) of 43%, cumulative release (CMR) of 39%, the ζ-potential of +50 mV, and PDI of 0.13. The predicted point condition was as follows: NP ratio = 13, Chi/Alg MW ratio = 2.35, and preparation method type = 1. Microscopic findings revealed that the shape of nanoparticles was spherical. The Chi/Alg/S nanoparticles showed no toxicity and transfection efficacy of 29.9% was observed in comparison with Lipofectamine (35.5%) and PEI (30.9%).

Development of an enzyme-enhancer system to improve laccase biological activities.

The present investigation reports an in-vitro study using combination of laccase and an enhancer capable of inhibiting the growth of pathogenic microorganisms, preventing biofilm formation, and whitening teeth. Laccase-cinnamic acid system remarkably inhibited the growth of Aggregatibacter actinomycetemcomitans, Candida albicans, S. aureus, and Streptococcus mutans whilst showed no significant effects on Gram-negative bacteria. Data presented that cinnamic acid (10 mM) with laccase (0.125 U ml-1) led to a maximum decrease of about 90%, in S. mutans biofilm formation. The confocal laser scanning microscopy showed considerable detachment of S. mutans cells from glass substratum. The combined laccase-cinnamic acid system could remove teeth discoloration caused by coffee. SEM of the teeth surface exhibited no damages such as surface cracking or fracture. Liquid chromatography-tandem mass spectrometry (LC-MS) and cyclic voltammetry (CV) studies showed that laccase can catalyze the one-electron oxidation of cinnamic acid to the respective radical. This radical can then undergo several fates, including recombination with another radical to form a dimeric species, dismutation of the radical back to cinnamic acid or decarboxylation to give various reduced oxygen species. Therefore, the redox potential values of phenolic monomers/oligomers are related with their biological activities.

Optimization of chitosan-based polyelectrolyte nanoparticles for gene delivery, using design of experiment: in vitro and in vivo study.

Gene therapy is a novel approach for cancer treatment and investigation for suitable gene delivery systems is remarkable. Here, preparation of a polyelectrolyte complex containing polysaccharides: trimethyl chitosan (TMC) as the positive and hyaluronate (HA), dextran sulfate and alginate as the negative part was studied. The optimized nanoparticles (TMC: between 0.2 and 0.47 mg/ml, HA: 0.35 mg/ml (≈131 nm, nearly full gene loading)) were obtained via primary screening followed by the D-optimal method. In vitro cellular study on the MCF7 cell line confirmed the non-toxicity and high cellular uptake (>90%) of prepared nanoparticles. Notably, in vivo study indicated noticeable tumor uptake of nanoparticles while low accumulation in vital organs such as heart, liver and lungs. Moreover, although a qualitative variable was considered, the applied method restricted the number of runs by selecting spots from the spherical atmosphere. The prepared nanoparticles could be suggested as an efficient and safe delivery system for cancer gene delivery.

Response Surface Methodology for Statistical Optimization of Chitosan/Alginate Nanoparticles as a Vehicle for Recombinant Human Bone Morphogenetic Protein-2 Delivery.

PURPOSE: In this study, chitosan/alginate nanoparticles are prospected as a carrier for controlled release of recombinant human bone morphogenetic protein-2 (rhBMP-2). MATERIALS AND METHODS: The rhBMP-2-loaded chitosan/alginate nanoparticles (Cs/Alg/B NPs) were prepared using the ionic gelation (IG) method. The current research was conducted to optimize the effective factors for entrapping rhBMP-2 in Cs/Alg NPs using response surface methodology (RSM) and the Box-Behnken design (BBD). The variables were the Cs/Alg molecular weight (Mw) ratios (1-3), pH (4.8-5.5), stirring rates (900-1300 rpm) and the responses included size, ζ-potential, polydispersity index (PDI), loading efficacy (LE), cumulative release (CR), and morphological degradation time (MDE). Then, the morphological properties of optimum formulation were studied for post-characterization. In the next step, the MTT assay for the optimized run was done for 24 and 48 hours. RESULTS: The results revealed that the optimum conditions for the mentioned variables were stirring rate=1100 rpm, pH=5.15, and Cs/Alg Mw ratio=1.75 based on numerical optimization. It was shown that the average particle size and loading efficacy at optimum conditions were 253 nm and 67%, respectively. Other responses were as follows: CR=66%, ζ-potential=+35mV, PDI=0.5, and MDT=7 days. CONCLUSION: The results have suggested that the statistical optimization of rhBMP-2 offers the possibility of preparing Cs/Alg/B NPs with a favorable size, controlled release characteristics, and high loading efficiency. It is expected that the acquired optimum conditions will be useful for efficient rhBMP-2 delivery.

Second-order universal calibration.

Quantification and qualification of an analyte of interest in pharmaceutical tablets from different manufacturers/companies are a hard task because of the potential presence of various interfering molecules. Indeed, the composition of the tablets covers a wide range of interferents which can be even unknown. As a consequence, we propose to determine the concentration of an analyte of interest regardless of the interferents using the concept of universal calibration. Universal calibration paves the way to the quantification of a specific chemical entity in samples with various compositions and different interferents. This is possible by the trilinear structure of analyte's signal. In fact, the second-order advantage resulting from the second-order universal calibration models is exploited. However, a new second-order calibration strategy was conducted in this work using Trilinear Factor Extraction (TFE). A simulated data set was exemplified to highlight the ability of the proposed procedure in order to accurate extraction of the analyte's concentration profile. Additionally, two real data sets were also explored in order to test the TFE method. In the first case, Acetaminophen was quantified using fluorescence spectroscopy in tablets with different formulations from 6 companies. In the second experimental data, a peptide (Valine-Tyrosine-Valine) was successfully quantified in different samples using spectrofluorimetric data. Finally, these real data sets were analyzed by Multivariate Curve resolution - Alternating Least-Squares (MCR-ALS) under non-negativity and trilinearity constraints for the sake of comparison. The calculated Root Mean Square Error of Predictions (RMSEP) of Acetaminophen were 0.028 and 0.026 for the MCR-ALS and TFE models, respectively. On the other hand, for the second experimental data set, the RMSEP were 0.216 and 0.165, respectively. Finally, based on a paired t-test, the results of MCR-ALS and TFE were not significantly different.

Enhanced Production and Characterization of a Highly Stable Extracellular Protease from an Extreme Halophilic Isolate Salicola marasensis.

Owing to their superior catalytic activity in the extreme conditions, extremozymes have found the potential biotechnological applications for industrial purposes. A robust extracellular protease activity was detected in the culture broth of Salicola marasensis, an extreme halophilic bacterium, after a 48 h-incubation. The effect of different media ingredients in a liquid state fermentation was followed with the aim of improving the enzyme production yield. Fractional factorial and Box-Behnken designs were applied to get a 3.4 fold (from 6.0 to 20.3 U mL-1) improvement of protease production. The distinguishing features of this enzyme were stability at a wide range of pH (5.0-11.0) and temperature (25-60 °C), significant compatibility towards organic solvents, metal ions, chemicals, and surfactants, and hydrolysis of a variety of substrates. The properties of this enzyme can be of tremendous help in terms of the halophilic proteolytic extract's industrial applications.

Determination of Biological Activity of Recombinant Reteplase Using Clot Lysis Time and Activated Partial Thromboplastin Time (APTT) Lysis Methods: A Comparative Study.

Recombinant plasminogen activator (reteplase) is a third generation thrombolytic agent which has been used on coronary artery thrombosis and acute myocardial infarction. Clot lysis assay is usually considered as a unique method to evaluate biological activity of reteplase. In this study biological activity of reteplase was determined by APTT (activated partial thromboplastin time) lysis method. Validity of this method was evaluated in comparison with reference method, clot lysis time assay. Results of APTT lysis test showed good reproducibility (relative standard deviation (RSD) 3-5% for within day analysis and 4-7% for between day analysis), and accuracy (101.3-102.7%). APTT lysis responses were linear in range of 0.001-0.1 mg/mL reteplase. Therefore, APTT lysis method is applicable for biological activity determination of reteplase. Although more comprehensive studies are required to approve this test as a reference method, APTT lysis method seems to be valuable to receive more attention due to advantages of technical simplicity, sensitivity, applicability, and cost efficiency.

Applications and opportunities of experimental design for the dispersive liquid-liquid microextraction method - A review.

Nowadays, the trend to simplify and miniaturize sample preparation methods has resulted in the development of effective and low-cost microextraction techniques that utilize a very small volume of the extracting phase. Among them, the liquid-liquid microextraction (LLME) method is a simple and effective sample pre-treatment technique applicable to numerous analytical methods. A related miniaturized and environmentally friendly extraction technique, dispersive liquid-liquid microextraction (DLLME), has been developed within the last decade and shows a very high enrichment factor and very low solvent consumption compared to other liquid- or even solid-phase extraction methods. The inclusion of several effective parameters in DLLME and its variants has increased the need for optimization to obtain the best possible extraction results. In fact, experimental design and optimization of performance conditions are the most important applications of chemometrics in analytical chemistry. Thus, design of experiments (DoE) helps us to determine the best model of the relationship between variables, as well as the optimal experimental conditions. Here, a comprehensive review of recent advancements in the use of DoE methodologies including full factorial, fractional factorial, Plackett-Burman, orthogonal array, central composite, Box-Behnken, Doehlert, and D-optimal designs to optimize DLLME applications is provided. In addition, the preponderance and drawbacks of each optimal method are discussed. The overall purpose of this review is to present a general overview of the different DoEs that are currently used to optimize DLLME for various matrices and analytes.

Optimization of rPDT fusion protein expression by Escherichia coli in pilot scale fermentation: a statistical experimental design approach.

High yield recombinant protein production is highly desirable for biotechnological purposes. In the design of recombinant expression conditions, a number of essential central elements such as expression strain, type of medium, bioprocess optimization, and mathematical modeling should be considered. Well-designed industrial scale production of one recombinant protein with optimized influential parameters and yield can address the cost and production reproducibility issues. In the present study, statistical experimental design methodology was used to investigate the effect of fermentation conditions (dissolved oxygen, IPTG, and temperature) on rPDT production by Escherichia coli. rPDT is a recombinant fusion protein consisting of three different protein domains including the N-terminal 179 amino acid fragment of the S1 subunit of pertussis toxin, the full-length genetically detoxified diphtheria toxin (CRM197), and the 50 kDa tetanus toxin fragment C. A 15 Box-Behnken design augmented with center points revealed that IPTG and DO at the center point and low temperature will result in high yield. The optimal condition for rPDT production were found to be 100 µM IPTG, DO 30% and temperature 20 °C.

Characterization and Classification of Iranian Honey Based on Physicochemical Properties and Antioxidant Activities, with Chemometrics Approach.

In the present study, the physicochemical properties and antioxidant activities of different Iranian honey samples are investigated using various multivariate techniques in order to develop a quality control model. Forty-eight Iranian honey samples were tested for 15 physicochemical and antioxidant parameters. The parameters for which the samples were tested included color intensity, moisture, electrical conductivity, pH, free acidity, diastase activity, hydroxymethylfurfural content, proline level, total phenolic content, antioxidant activity, and radical scavenging activity. The study attempted to differentiate honey samples based on origin and composition. In the study, the Iranian honey samples were classified according to their respective physiochemical properties and antioxidant activities using principal component analysis and hierarchical cluster analysis. Furthermore, the relationships between the geographical and botanical origins were determined for the samples used in the study.

Ocular implant containing bevacizumab-loaded chitosan nanoparticles intended for choroidal neovascularization treatment.

Choroidal neovascularization (CNV) is among the leading causes of blindness worldwide. Bevacizumab has demonstrated promising effects on CNV treatment; however, frequent intravitreal injection is its major drawback. Current study aimed to address this issue by developing a sustained release formulation through nanoparticles of bevacizumab imbedded in an ocular implant. Bevacizumab-loaded chitosan nanoparticles were prepared by ionic gelation method and inserted in the matrix of hyaluronic acid and zinc sulfate. Despite the common approaches in using ultraviolet (UV)-spectrophotometry, microprotein-Bradford, and bicinchoninic acid (BCA), assay for protein assessment, our results revealed a remarkable UV-Vis absorption overlap of protein and chitosan during these analysis and thus enzyme-linked immunosorbent assay was employed for the antibody concentration assay. The size of optimized nanoparticles obtained through statistical analysis based on design of experiments was 78.5 ± 1.9 nm with polydispersity index of 0.13 ± 0.05 and the entrapment-efficiency and loading-efficiency were 67.6 ± 6.7 and 15.7 ± 5.7%, respectively. The scanning electron microscopy and confocal microscopy images revealed a homogenous distribution of nanoparticles in the implant matrix and the release test results indicated an appropriate extended release of bevacizumab from the carrier over two months. In conclusion, the prepared system provided a sustained release bevacizumab delivery formulation which can introduce a promising ocular drug delivery system intended for posterior segment disease. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2261-2271, 2018.

Prolonged injectable formulation of Nafarelin using in situ gel combination delivery system.

The principal purpose of the present study was to prepare and characterize a complex drug delivery system consisting of Nafarelin-poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) nanoparticles (NPs) in combination with sodium alginate/poloxamer 407 in situ gel. Nafarelin-loaded PHBV NPs were prepared via double emulsion solvent evaporation technique. Box-Behnken Response Surface Methodology was utilized to optimize NPs. Mean particle size, polydispersity index (PDI), entrapment efficiency (EE), and drug loading (DL) of the optimized NPs were measured. Incorporation of Nafarelin within NPs was proven by differential scanning calorimetry (DSC). The combination delivery system (CDS) was prepared by adding Nafarelin-loaded PHBV NPs to sodium alginate/poloxamer 407 solution followed by physical mixing. Morphological properties of Nafarelin-loaded PHBV NPs and CDS were evaluated by SEM. Rheological properties were employed to investigate the effects of alginate concentration on sol-gel transition temperature. The release profile of Nafarelin from both PHBV NPs and CDS were individually assessed. The cumulative release percentage from CDS was significantly lower than Nafarelin released from PHBV NPs. Based on the favorable results in this study, the CDS consisting of sodium alginate/poloxamer 407 loaded with PHBV NPs could be a promising candidate for designing a long-lasting formulation of Nafarelin.

Delignification and detoxification of peanut shell bio-waste using an extremely halophilic laccase from an Aquisalibacillus elongatus isolate.

Lignocellulose bioconversion is a harsh process requiring the use of surfactants and organic solvents. Consequently, the incorporation of laccases in this bioconversion requires the bioprospecting of enzymes that can remain stable under extreme conditions. An extracellular, highly stable laccase was produced by the halophilic isolate Aquisalibacillus elongatus in submerged liquid culture fermentation. Statistical and non-statistical strategies gave the highest enzymatic activity (8.02 U mL-1) following addition of glucose (1.7 g L-1), copper sulfate (0.8 g L-1), urea (15 g L-1), and CaCl2 (0.8 g L-1). The enzyme, after purification using a synthetic affinity support, delignified a peanut shell substrate by 45%. A pH of 8.0 and a temperature of 35 °C were optimal for delignification of this bio-waste material. Addition of [Bmim][PF6], 1,4-dioxane, acetone, and HBT promoted this bio-waste delignification. Bio-treatment in the presence of 50% [Bmim][PF6] gave a maximal lignin removal of 87%. The surfactants tested had no significant effects on the delignification yield. The laccase also detoxified the toxic phenols found in peanut shell waste. The high catalytic efficiency of this enzyme against a lignocellulosic sample under extreme conditions suggests the suitability of this laccase for industrial applications.