TiO2-MoS2-PMMA Nanocomposites for an Efficient Water Remediation.

An improvement of water supply and sanitation and better management of water resources, especially in terms of water reuse, is one of the priorities of the European Green Deal. In this context, it is crucial to find new strategies to recycle wastewater efficiently in a low-cost and eco-friendly manner. The immobilization of inorganic nanomaterials on polymeric matrices has been drawing a lot of attention in recent years due to the extraordinary properties characterizing the as-obtained nanocomposites. The hybrid materials, indeed, combine the properties of the polymers, such as flexibility, low cost, mechanical stability, high durability, and ease of availability, with the properties of the inorganic counterpart. In particular, if the inorganic fillers are nanostructured photocatalysts, the materials will be able to utilize the energy delivered by light to catalyze chemical reactions for efficient wastewater treatment. Additionally, with the anchoring of the nanomaterials to the polymers, the dispersion of the nanomaterials in the environment is prevented, thus overcoming one of the main limits that impede the application of nanostructured photocatalysts on a large scale. In this work, we will present nanocomposites made of polymers, i.e., polymethyl methacrylate (PMMA), and photocatalytic semiconductors, i.e., TiO2 nanoparticles (Evonik). MoS2 nanoflakes were also added as co-catalysts to improve the photocatalytic performance of the TiO2. The hybrid materials were prepared using the sonication and solution casting method. The nanocomposites were deeply characterized, and their remarkable photocatalytic abilities were evaluated by the degradation of two common water pollutants: methyl orange and diclofenac. The relevance of the obtained results will be discussed, opening the route for the application of these materials in photocatalysis and especially for novel wastewater remediation.

Association of PD-L1 expression with driver gene mutations and clinicopathological characteristics in non-small cell lung cancer: A real-world study of 10 441 patients.

BACKGROUND: Programmed death ligand-1 (PD-L1) expression is a well-known predictive biomarker of response to immune checkpoint blockade in non-small cell lung cancer (NSCLC). However, there is limited evidence of the relationship between PD-L1 expression, clinicopathological features, and their association with major driver mutations in NSCLC patients in Latin America. METHODS: This retrospective study included patients from Argentina with advanced NSCLC, and centralized evaluation of PD-L1 expression concurrently with genomic alterations in the driver genes EGFR, ALK, ROS1, BRAF, and/or KRAS G12C in FFPE tissue samples. RESULTS: A total of 10 441 patients with advanced NSCLC were analyzed. Adenocarcinoma was the most frequent histological subtype (71.1%). PD-L1 expression was categorized as PD-L1 negative (45.1%), PD-L1 positive low-expression 1%-49% (32.3%), and PD-L1 positive high-expression ≥50% (22.6%). Notably, current smokers and males were more likely to have tumors with PD-L1 tumor proportion score (TPS) ≥50% and ≥ 80% expression, respectively (p < 0.001 and p = 0.013). Tumors with non-adenocarcinoma histology had a significantly higher median PD-L1 expression (p < 0.001). Additionally, PD-L1 in distant nodes was more likely ≥50% (OR 1.60 [95% CI: 1.14-2.25, p < 0.01]). In the multivariate analysis, EGFR-positive tumors were more commonly associated with PD-L1 low expression (OR 0.62 [95% CI: 0.51-0.75], p < 0.01), while ALK-positive tumors had a significant risk of being PD-L1 positive (OR 1.81 [95% CI: 1.30-2.52], p < 0.01). CONCLUSIONS: PD-L1 expression was associated with well-defined clinicopathological and genomic features. These findings provide a comprehensive view of the expression of PD-L1 in patients with advanced NSCLC in a large Latin American cohort.

EVA Films Loaded with Layered Double Hydroxide (LDH) Modified with Methacrylic Anion: Effect of the Nanohybrid Filler on the Photodegradation Phenomena.

The photo-oxidative studies of ethylene vinyl acetate copolymer (EVA) matrix, filled with Layered Double Hydroxide (LDH) modified with methacrylic anion (MA), were herein reported, together with gas permeation tests. The formulation of nano-hybrid LDHs was characterized using X-ray diffractometry (XRD) and thermogravimetric analysis (TGA), demonstrating the partial intercalation of the 30% of MA anion between the LDH's galleries. The as-modified filler was introduced into an EVA matrix by mechanical milling, producing free-standing films subjected to accelerated aging. Fourier transform infrared spectroscopy (FT-IR) results suggested that the nanohybrid presence determined a stabilizing effect up to 45 days of UV irradiation, especially if compared to the EVA/LDH references for all formulated EVA hybrid nanocomposites. Conversely, the presence of nanohybrid in the matrix did not significantly change the thermal stability of EVA samples. The dispersion of modified MA-LDH in the EVA matrix produces defect-free samples in the whole range of investigated loadings. The samples show a slight decrease in gas permeability, coupled with a substantial stabilization of the original CO2/O2 selectivity, which also proves the integrity of the films after 30 days of UV irradiation.

Synthesis and Photochemical Properties of Monolithic TiO2 Nanowires Diode.

In this paper, the structural and photochemical properties of a monolithic photochemical diode are discussed. The present structure is composed, from the top to the bottom, of a TiO2 nanowire layer, a TiO2 film, a Ti foil, and a porous layer made of Pt nanoparticles. The synthesis of the nanowires was simply carried out by Au-catalysed-assisted process; the effects of the annealing temperature and time were deeply investigated. Morphological and structural characterizations were performed by scanning electron microscopy and Raman spectroscopy. The analyses showed the rutile structure of the TiO2 nanowires. The photocatalytic properties were studied through the degradation of methylene blue (MB) dye under UV light irradiation. The nanowires induced an enhancement of the photo-degradation rate, compared to TiO2 in a bulk form, due to an increase in the surface area. Moreover, the presence of a nano-porous Pt layer deposited on the rear side of the samples provided a further increase in the MB degradation rate, related to the scavenging effect of Pt nanoparticles. The overall increment of the photo-activity, due to the nano-structuration of the TiO2 and to the presence of the Pt layer, resulted a factor 7, compared to the bulk reference. In addition, photovoltage measurements allowed to assess the effects of TiO2 nano-structuration and Pt nanoparticles on the electron accumulation.

Innovative Polymeric Hybrid Nanocomposites for Application in Photocatalysis.

The immobilization of inorganic nanomaterials on polymeric substrates has been drawing a lot of attention in recent years owing to the extraordinary properties of the as-obtained materials. The hybrid materials, indeed, combine the benefits of the plastic matter such as flexibility, low-cost, mechanical stability and high durability, with them deriving from their inorganic counterparts. In particular, if the inorganic fillers are nanostructured photocatalysts, the originated hybrid systems will be able to utilize the energy delivered by light, catalysing chemical reactions in a sustainable pathway. Most importantly, since the nanofillers can be ad-hoc anchored to the macromolecular structure, their release in the environment will be prevented, thus overcoming one of the main restrictions that impedes their applications on a large scale. In this review, several typologies of hybrid photocatalytic nanomaterials, obtained by using both organic and inorganic semiconductors and realized with different synthetic protocols, were reported and discussed. In the first part of the manuscript, nanocomposites realized by simply blending the TiO2 or ZnO nanomaterials in thermoplastic polymeric matrices are illustrated. Subsequently, the atomic layer deposition (ALD) technique is presented as an excellent method to formulate polymeric nanocomposites. Successively, some examples of polyporphyrins hybrid systems containing graphene, acting as photocatalysts under visible light irradiation, are discussed. Lastly, photocatalytic polymeric nanosponges, with extraordinary adsorption properties, are shown. All the described materials were deeply characterized and their photocatalytic abilities were evaluated by the degradation of several organic water pollutants such as dyes, phenol, pesticides, drugs, and personal care products. The antibacterial performance was also evaluated for selected systems. The relevance of the obtained results is widely overviewed, opening the route for the application of such multifunctional photocatalytic hybrid materials in wastewater remediation.

Toxicity assessment of two-dimensional nanomaterials molybdenum disulfide in Gallus gallus domesticus.

Recently two-dimensional nanomaterials, such as graphene and molybdenum disulfide (MoS2), have received much attention as adsorbent materials for the effective removal of organic contaminants. MoS2 is attracting attention, not only for its chemical-physical properties, but also for its wide availability in nature as a constituent of molybdenite. The aim of this investigation was to assess the effects of different MoS2 concentrations (5 × 10-1, 5 × 10-2 and 5 × 10-3 mg/ml) on the embryonated eggs of Gallus gallus domesticus, according to Beck method. We evaluated the toxic effect of the MoS2 powder purchased at Sigma-Aldrich indicated as "received" and MoS2 powder treated via mechanical milling indicated as "ball mille". Subsequently, the embryos were sacrificed at different times of embryonic development (11th, 15th and 19th day after incubation) in order to evaluate their embryotoxic and teratogenic effects. The alterations of the embryonic development were studied by morphological and immunohistochemical analysis of the tissues. The results obtained have shown the toxicity of both powders of MoS2 with a high percentage of deaths and growth delays. Moreover, the immunohistochemical analysis performed on several tissue sections showed a strong positivity to the anti-metallothionein1 antibody only for the erythrocytes.

Mechanical milling: a sustainable route to induce structural transformations in MoS2 for applications in the treatment of contaminated water.

Two-dimensional (2D) nanomaterials have received much attention in recent years, because of their unusual properties associated with their ultra-thin thickness and 2D morphology. Besides graphene, a new 2D material, molybdenum disulfide (MoS2), has attracted immense interest in various applications. On the other hand, ball-milling process provides an original strategy to modify materials at the nanometer scale. This methodology represents a smart solution for the fabrication of MoS2 nanopowders extremely-efficient in adsorbing water contaminants in aqueous solution. This work reports a comprehensive morphological, structural, and physicochemical investigation of MoS2 nanopowders treated with dry ball-milling. The adsorption performances of the produced nanopowders were tested using methylene blue (MB) dye and phenol in aqueous solution. The adsorption capacity as a function of ball-milling time was deeply studied and explained. Importantly, the ball-milled MoS2 nanopowders can be easily and efficiently regenerated without compromising their adsorption capacity, so to be reusable for dye adsorption. The eventual toxic effects of the prepared materials on microcrustacean Artemia salina were also studied. The present results demonstrate that ball-milling of MoS2 offers a valid method for large-scale production of extremely efficient adsorbent for the decontamination of wastewaters from several pollutants.

Hybrid nickel-free graphene/porphyrin rings for photodegradation of emerging pollutants in water.

A novel hybrid photoactive material based on graphene foam (G) coupled with porphyrin-based polymers (Porph rings) was formulated by using a time-saving procedure to remove nickel from the final device. Specifically, Porph rings were spin coated onto the G platform with the double function of a visible-light photocatalyst and protective agent during nickel etching. The characterization of G-Porph rings was assessed by Scanning Electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and photoluminescence (PL). The novel material showed photocatalytic ability in degrading different classes of pollutants such as the herbicide 2,4 dichlorophenoxyacetic acid (2,4-D), polyethylene glycol (PEG) as an ingredient of care and health products, and also the methylene blue (MB) dye. UV-Vis spectroscopy, total organic carbon (TOC) and soft mass spectrometry techniques were used to monitor the photocatalytic process. The best performance in terms of photocatalytic efficiency was exhibited versus PEG and MB degradation. Furthermore, to determine the individual contribution of Reactive Oxygen Species (ROS) produced, free radical and hole scavenging tests were also carried out. Finally, a detailed map of the photocatalytic degradation mechanisms was proposed, reporting also the calculation of Porph rings' Highest Occupied Molecular Orbital (HOMO) and Lowest Occupied Molecular Orbital (LUMO) energy level values.

LINE-1 hypermethylation in white blood cell DNA is associated with high-grade cervical intraepithelial neoplasia.

BACKGROUND: Long Interspersed Nuclear Elements-1 (LINEs-1) methylation from white blood cells (WBCs) DNA has been proposed as biomarker associated with different types of cancer. The aim of the present study was to investigate the degree of WBCs LINE-1 methylation, according to high-risk Human Papilloma Virus (hrHPV) status in a healthy population, and the association with high-grade Cervical Intraepithelial Neoplasia (CIN2+) in hrHPV positive women. METHODS: Women with abnormal cervical cells were enrolled and classified by histological diagnosis and hrHPV infection. A structured questionnaire was used to obtain information on socio-demographic variables and lifestyle factors. LINE-1 methylation level in WBCs was measured by pyrosequencing-based methylation analysis after bisulfite conversion. RESULTS: Among 252 women diagnosed with normal cervical epithelium, with regard to LINE-1 methylation level no significant difference was observed between hrHPV positive and hrHPV negative women, also adjusting for known risk factors of infection. The association between WBCs LINE-1 methylation and CIN2+ status was analyzed in hrHPV positive women. The median value of LINE-1 methylation levels was higher in cases (CIN2+) than in controls (75.00% versus 73.17%; p = 0.002). For a one-unit increase in LINE-1 methylation level, the odds of being diagnosed with CIN2+ increased by 10%, adjusting for known factors related to LINE-1 methylation (adjOR: 1.10; 95% CI:1.01-1.20; p = 0.032). The Receiver-Operating Characteristic (ROC) curve analysis identified the cut-off value of 73.8% as the best threshold to separate cases from controls (sensitivity: 63.4% and specificity: 61.8%). CONCLUSIONS: LINE-1 methylation status in WBCs DNA may represent a cost-effective and tissue-accessible biomarker for high-grade CIN in hrHPV positive women. However, LINE-1 hypermethylation cannot be considered specific for cervical cancer (CC) and a model based solely on LINE-1 methylation levels has limited performance. Further investigations are necessary to propose and validate a novel methylation biomarker panel, based on LINE-1 methylation and other differentially methylated regions, for the screening of women at risk of CC.

Novel synthesis of ZnO/PMMA nanocomposites for photocatalytic applications.

The incorporation of nanostructured photocatalysts in polymers is a strategic way to obtain novel water purification systems. This approach takes the advantages of: (1) the presence of nanostructured photocatalyst; (2) the flexibility of polymer; (3) the immobilization of photocatalyst, that avoids the recovery of the nanoparticles after the water treatment. Here we present ZnO-polymer nanocomposites with high photocatalytic performance and stability. Poly (methyl methacrylate) (PMMA) powders were coated with a thin layer of ZnO (80 nm thick) by atomic layer deposition at low temperature (80 °C). Then the method of sonication and solution casting was performed so to obtain the ZnO/PMMA nanocomposites. A complete morphological, structural, and chemical characterization was made by scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) analyses. The remarkable photocatalytic efficiency of the nanocomposites was demonstrated by the degradation of methylene blue (MB) dye and phenol in aqueous solution under UV light irradiation. The composites also resulted reusable and stable, since they maintained an unmodified photo-activity after several MB discoloration runs. Thus, these results demonstrate that the proposed ZnO/PMMA nanocomposite is a promising candidate for photocatalytic applications and, in particular, for novel water treatment.

Purification and characterization of novel cationic peroxidases from Asparagus acutifolius L. with biotechnological applications.

Four novel basic peroxidases, named AaP-1, AaP-2, AaP-3, and AaP-4, were purified from Asparagus acutifolius L. seeds by cation-exchange and gel filtration chromatographies. The four proteins showed a similar electrophoretic mobility of 46 kDa while, by MALDI-TOF MS, different Mr values of 42758.3, 41586.9, 42796.3, and 41595.5 were determined for AaP-1, AaP-2, AaP-3, and AaP-4, respectively. N-terminal sequences of AaPs 1-4 up to residue 20 showed a high percentage of identity with the peroxidase from Glycine max. In addition, AaP-1, AaP-2, AaP-3, and AaP-4 were found to be glycoproteins, containing 21.75, 22.27, 25.62, and 18.31 % of carbohydrates, respectively. Peptide mapping and MALDI-TOF MS analysis of AaPs 1-4 showed that the structural differences between AaP-1 and AaP-2 and AaP-3 and AaPs-4 were mainly due to their glycan content. We also demonstrate that AaPs were able to remove phenolic compounds from olive oil mill wastewaters with a higher catalytic efficiency with respect to horseradish peroxidase, thus representing candidate enzymes for potential biotechnological applications in the environmental field.

Inactivating effects of lignin-derived compounds released during lignocellulosic biomass pretreatment on the endo-glucanase catalyzed hydrolysis of carboxymethylcellulose: A study in continuous stirred ultrafiltration-membrane reactor.

This study focusses on the reversible/irreversible damage that selected phenolic compounds, released during steam-explosion pretreatment, mandatory for cellulose accessibility, causes on both stability and activity of a commercial cellulase (half-life=173h) during carboxymethyl-cellulose hydrolysis. Long-term experiments performed in continuous stirred UF-membrane bioreactors, operating at steady-state regime, in controlled operational conditions, allowed evaluating the inactivation-constant in the phenol presence (kd1) and after its removal (kd2) from the reactor feed. p-Hydroxybenzoic acid (1 and 2g L(-1)) are the extreme limits in the inactivating effect with enzyme half-lives 99.02 and 14.15h, respectively. The inactivation reversibility was assessed for vanillic acid, p-hydroxybenzoic acid, syringaldehyde, p-coumaric acid, being kd1>kd2. p-Hydroxybenzaldehyde and protocatechuic acid irreversibly affected cellulase stability increasing its inactivation with kd2>kd1. p-Hydroxybenzaldehyde, 1g L(-1), syringaldehyde, and vanillin, at 2gL(-1), had similar kd1÷kd2.

Sucrose hydrolysis by invertase using a membrane reactor: effect of membrane cut-off on enzyme performance

Sucrose hydrolysis by invertase [EC.3.2.1.26] produces inverted sugar syrup, an ingredient mainly used in the food industry. To properly catalyze hydrolysis, the enzyme should be reused after this reaction. It is advisable to maintain constant activity over a considerable period. Thus, sucrose hydrolysis was performed in a membrane bioreactor - a continuously stirred tank reactor coupled with an ultrafiltration membrane (UFM) which provides good diffusion and high activity per unit volume. Molecular weight cut-off for soluble invertase UFMs was up to 100kDa. This study focused on the role of UFM invertase cut-off as it is the main element in the process. We demonstrated that both the cut-off and chemical nature of the UFM affected specific invertase activity.

A hidrólise da sacarose através da invertase [EC.3.2.1.26] gera xarope de açúcar invertido, que é usado principalmente como ingrediente na indústria alimentícia. Para ter-se uma hidrólise satisfatória, a enzima deve ser reaproveitada após a reação. É desejável que sua atividade seja mantida constante durante um período considerável de reação. A hidrólise da sacarose foi realizada em um reator com membrana (RM) - que é um reator continuamente agitado acoplado a uma membrana de ultrafiltração (MUF) -, porque apresenta efeitos mínimos de difusão e elevada atividade por unidade de volume. O corte molecular da MUF utilizada para reter a invertase solúvel dentro do MR foi de até 100kDa. Como a invertase é o elemento principal deste processo, este trabalho foi focado no papel do corte molecular da MUF na sua atividade. O corte molecular e a natureza química da membrana-UF mostraram afetar a atividade específica da invertase.

Nitrile, amide and temperature effects on amidase-kinetics during acrylonitrile bioconversion by nitrile-hydratase/amidase in situ cascade system.

In this study the amidase kinetics of an in situ NHase/AMase cascade system was explored as a function of operational parameters such as temperature, substrate concentration and product formation. The results indicated that controlling amidase inactivation, during acrylonitrile bioconversion, makes it possible to recover the intermediate product of the two-step reaction in almost a pure form, without using purified enzyme. It has been demonstrated, in long-term experiments performed in continuous stirred UF-membrane bioreactors, that amidase is kinetically controlled by its proper substrate, depending on the structure, and by acrylonitrile. Using acrylamide, AMase-stability is temperature dependent (5°C, kd=0.008 h(-1); 30°C kd=0.023 h(-1)). Using benzamide, amidase is thermally stable up to 50°C and no substrate inhibition/inactivation occurs. With acrylonitrile, AMase-activity and -stability remain unchanged at concentrations <200 mM but at 200 mM, 35°C, after 70 h process, 90% irreversible inactivation occurs as no AMase-activity on benzamide revives.

Heterologous expression, purification and characterization of nitrilase from Aspergillus niger K10.

BACKGROUND: Nitrilases attract increasing attention due to their utility in the mild hydrolysis of nitriles. According to activity and gene screening, filamentous fungi are a rich source of nitrilases distinct in evolution from their widely examined bacterial counterparts. However, fungal nitrilases have been less explored than the bacterial ones. Nitrilases are typically heterogeneous in their quaternary structures, forming short spirals and extended filaments, these features making their structural studies difficult. RESULTS: A nitrilase gene was amplified by PCR from the cDNA library of Aspergillus niger K10. The PCR product was ligated into expression vectors pET-30(+) and pRSET B to construct plasmids pOK101 and pOK102, respectively. The recombinant nitrilase (Nit-ANigRec) expressed in Escherichia coli BL21-Gold(DE3)(pOK101/pTf16) was purified with an about 2-fold increase in specific activity and 35% yield. The apparent subunit size was 42.7 kDa, which is approx. 4 kDa higher than that of the enzyme isolated from the native organism (Nit-ANigWT), indicating post-translational cleavage in the enzyme's native environment. Mass spectrometry analysis showed that a C-terminal peptide (Val327 - Asn356) was present in Nit-ANigRec but missing in Nit-ANigWT and Asp298-Val313 peptide was shortened to Asp298-Arg310 in Nit-ANigWT. The latter enzyme was thus truncated by 46 amino acids. Enzymes Nit-ANigRec and Nit-ANigWT differed in substrate specificity, acid/amide ratio, reaction optima and stability. Refolded recombinant enzyme stored for one month at 4°C was fractionated by gel filtration, and fractions were examined by electron microscopy. The late fractions were further analyzed by analytical centrifugation and dynamic light scattering, and shown to consist of a rather homogeneous protein species composed of 12-16 subunits. This hypothesis was consistent with electron microscopy and our modelling of the multimeric nitrilase, which supports an arrangement of dimers into helical segments as a plausible structural solution. CONCLUSIONS: The nitrilase from Aspergillus niger K10 is highly homologous (≥86%) with proteins deduced from gene sequencing in Aspergillus and Penicillium genera. As the first of these proteins, it was shown to exhibit nitrilase activity towards organic nitriles. The comparison of the Nit-ANigRec and Nit-ANigWT suggested that the catalytic properties of nitrilases may be changed due to missing posttranslational cleavage of the former enzyme. Nit-ANigRec exhibits a lower tendency to form filaments and, moreover, the sample homogeneity can be further improved by in vitro protein refolding. The homogeneous protein species consisting of short spirals is expected to be more suitable for structural studies.

Continuous hydrolysis of 4-cyanopyridine by nitrilases from Fusarium solani O1 and Aspergillus niger K10.

The operational stabilities of nitrilases from Aspergillus niger K10 and Fusarium solani O1 were examined with 4-cyanopyridine as the substrate in continuous-stirred membrane reactors (CSMRs). The former enzyme was fairly stable at 30 degrees C with a deactivation constant (k (d)) and enzyme half-life of 0.014 h(-1) and 50 h, respectively, but the latter exhibited an even higher stability characterized by k (d) = 0.008 h(-1) and half-life of 87 h at 40 degrees C. Another advantage of this enzyme was its high chemoselectivity, i.e., selective transformation of nitriles into carboxylic acids, while the amide formed a high ratio of A. niger K10 nitrilase product. High conversion rates (>90%) were maintained for about 52 h using the nitrilase from F. solani O1 immobilized in cross-linked enzyme aggregates (CLEAs). The purity of isonicotinic acid was increased from 98% to >99.9% by using two CSMRs connected in series, the first one containing the F. solani O1 nitrilase and the second the amidase from Rhodococcus erythropolis A4 (both enzymes as CLEAs), the amidase hydrolyzing the by-product isonicotinamide.

Fungal nitrilases as biocatalysts: Recent developments.

Of the numerous putative fungal nitrilases available from protein databases only a few enzymes were purified and characterized. The purified nitrilases from Fusarium solani, Fusarium oxysporum f. sp. melonis and Aspergillus niger share a preference for (hetero)aromatic nitriles, temperature optima between 40 and 50 degrees C and pH optima in the slightly alkaline region. On the other hand, they differ in their chemoselectivity, i.e. their tendency to produce amides as by-products. The production of fungal nitrilases is increased by up to three orders of magnitude on the addition of 2-cyanopyridine to the culture media. The whole-cell and subcellular biocatalysts were immobilized by various methods (LentiKats(R); adsorption on hydrophobic or ion exchange resins; cross-linked enzyme aggregates). Operational stability was examined using continuous stirred membrane bioreactors. Fungal nitrilases appear promising for biocatalytic applications and biodegradation of nitrile environmental contaminants.

Investigating the kinetics of the enzymatic depolymerization of polygalacturonic acid in continuous UF-membrane reactors.

A stirred tank membrane reactor is used to study the kinetics of polygalacturonic acid (PGA) enzymatic hydrolysis. The reactor operates in semicontinuous configuration: the native biopolymer is loaded at the initial time and the system is continuously fed with the buffer. The effect of retention time (from 101 to 142 min) and membrane molecular weight cutoff (from 1 to 30 kDa) on the rate of permeable oligomers production is investigated. Reaction products are clustered in two different classes, those sized below the membrane cutoff and those above. The reducing power measured in the permeate is used as an estimate of total product concentration. The characteristic breakdown times range from 40 to 100 min. The overall kinetics obeys a first-order law with a characteristic time estimated to 24 min. New mathematical data handling are developed and illustrated using the experimental data obtained. Finally, the body of the experimental results suggests useful indications (reactor productivity, breakdown induction period) for implementing the bioprocess at the industrial scale.

Purification and characterization of a nitrilase from Aspergillus niger K10.

Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg(-1)) at 45 degrees C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of D: -sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.