Environmental Biocatalysis

The use of biocatalysts for the industrial synthesis of chemicals has been attracting much attention as an environmental friendly synthetic method. Various plants, such as apple (Malus pumila), carrot (Daucus carota), cucumber (Cucumis sativus), onion (Allium cepa), potato (Solanum tuberosum), radish (Raphanus sativus) and sweet potato (Ipomoea batatas) were used as biocatalysts. Enzymes that plants produce are able to perform reactions under mild conditions (pH and temperature), with remarkable chemo-, regio-, and stereoselectivity. Due to this feature the number of biocatalysts used in organic synthesis has rapidly increased during the last decades, especially for the production of chiral compounds. This review presents biotechnological processes for the production of chiral alcohols by reducing prochiral ketones with whole plant tissue. Chiral alcohols are important building blocks for the synthesis of pharmaceuticals, pesticides, pheromones, flavors, fragrances and advanced materials such as liquid crystals. Reductase-catalyzed reactions are dependent on cofactors, and therefore, one major task in process development is to provide an effective method for regeneration of the cofactors consumed. The need for expensive cofactors is eliminated by using the whole plant tissue since the plant automatically provides this requirement. Depending on the vegetable used, either enantiomer may be obtained in high yield and high enantiomeric excess (ee), which could be a critical factor for drug development/bioactivity evaluation perspective. In this paper, various processes carried out on laboratory scales are presented. Attention is turned to conversion, yield, enantiomeric excess (ee).

The use of biocatalysts for the industrial synthesis of chemicals has been attracting much attention as an environmental friendly synthetic method. Various plants, such as apple (Malus pumila), carrot (Daucus carota), cucumber (Cucumis sativus), onion (Allium cepa), potato (Solanum tuberosum), radish (Raphanus sativus) and sweet potato (Ipomoea batatas) were used as biocatalysts. Enzymes that plants produce are able to perform reactions under mild conditions (pH and temperature), with remarkable chemo-, regio-, and stereoselectivity. Due to this feature the number of biocatalysts used in organic synthesis has rapidly increased during the last decades, especially for the production of chiral compounds. This review presents biotechnological processes for the production of chiral alcohols by reducing prochiral ketones with whole plant tissue. Chiral alcohols are important building blocks for the synthesis of pharmaceuticals, pesticides, pheromones, flavors, fragrances and advanced mate...

Banana peel after chemical ant thermal modification was used as an alternative support to immobilize the commercial enzyme naringinase (Penicillum Decumbens); an immobilization yield greater than 70% was observed at pH 7. The morphology of the support was characterized by scanning electron microscopy with elemental analysis, showing the presence of pores and elements such as carbon, oxygen, sulfur, and zinc, while the immobilization of the enzyme was confirmed by infrared spectroscopy. For the free and immobilized enzyme, the KM and Vmax values were 0.0006 mg/mL and 2000 U, and 0.0003 mg/mL and 1666 U, respectively. The temperatures of greatest activity for the free and immobilized enzyme were 70°C and 50°C, respectively, and the optimum pH was 4.5 in both cases. It was found that, after the third use, the catalyst maintained 50% of the enzymatic activity. These results seem to suggest the potential of the synthesized material for its application in the food industry, specifically; ...

Banana peel after chemical ant thermal modification was used as an alternative support to immobilize the commercial enzyme naringinase (Penicillum Decumbens); an immobilization yields greater than 70% was observed at pH 7. The structural characteristics of the support were determined by scanning electron microscopy with elemental analysis, showing the presence of pores and elements such as carbon, oxygen, sulfur, and zinc, while the attachment of the enzyme was concluded by infrared spectroscopy. For the free and immobilized enzyme, the K M and V max values were 0.0006 molar and 2000 U, and 0.0003 molar and 1666 U, respectively. The temperatures of the greatest activity for the free was 70°C and for the immobilized enzyme was 50°C, respectively, and the best pH was 4.5 in both cases. It was found that, after the third use, the catalyst maintained 50% of the enzymatic activity. These results seem to suggest the potential of the synthesized material for its application in the debittering of citrus juices.

Antibiotics used in humans and farmed animals are an essential source of water
and soil contamination. Ampicillin is a micropollutant commonly found in water,
sludge, food, flora, and fauna. However, the methods used for its detection in
environmental samples are often complicated and expensive. Therefore,
developing more straightforward strategies to detect well-known target
antibiotics is necessary. In this context, enzyme-based detection methods
have been demonstrated to be selective, sensitive, rapid, and relatively
simple. In this study, a fluorescent byproduct from the ampicillin oxidation
using Chloroperoxidase (CPO) enzyme was used as a pointer compound to
determine ampicillin concentration in environmental water samples. We
oxidized 80% ampicillin for 1h, producing a fluorescent compound with m/z
274.2517. A response surface methodology (RSM) based on a central composite
design (CCD) was used to evaluate and optimize the effects of hydrogen
peroxide, enzyme concentration, and time as independent variables on the
maximum fluorescence signal as the response function. The methodology
proposes to build a calibration curve that relates the initial concentration of
ampicillin with fluorescence intensity after the reaction with CPO, which helps
detect ampicillin in the concentration range from 0.035 to 40 μM, with a limit of
detection of 0.026 μM. The application of the method to fortified
environmental water samples allowed percentages of recovery from 86 to
140%. The formation of the fluorescent compound was not affected by the
presence of salts commonly found in wastewater; however, it was affected by
other antibiotics. The proposed methodology was tested in the context of water
from water bodies, urban, and WWTP effluents.

This paper is dedicated to the synthesis of a copolymer with reducing properties obtained by functionalizing chitosan with quercetin and determining the antioxidant activity of the derivatives obtained depending on the molar mass of the polymer. For this purpose, low molecular weight chitosan was obtained by oxidizing commercial chitosan with hydrogen peroxide and further functionalization with quercetin by the covalent grafting method. The functionalization process was performed through the following steps: functionalization of chitosan with ethyl chloroformate to increase the reactivity of the amine group to the hydroxyl group of quercetin and grafting the quercetin molecule to the synthesized intermediate. The comparative antioxidant properties of the composite obtained by grafting technical chitosan with quercetin and by grafting low molecular weight chitosan were studied by the DPPH (2,2-diphenyl-1-picrylhydrazyl radical) method. The obtained results indicate that a decrease in the molecular weight of chitosan contributed to its grafting with quercetin. As a result, the functionalized polymer composite acquired a higher antioxidant activity and can be successfully used to inhibit the oxidation of various organic substrates in the cosmetic, food and pharmaceutical industries.

Background Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial c...

Peroxidases can be used in decolorization processes and the treatment of textiles effluents. This study evaluates the potential of the turnip peroxidase enzyme in the decolorization of the phthalocyanine textile dye Reactive Blue 21 (RB21). Some factors such as pH, the amount of H 2 O 2 and the enzyme were evaluated in order to determine the optimum conditions for the enzyme performance. The reaction products formed during the decolorization of the RB21 dye were analyzed by high-performance liquid chromatography-mass spectrometry coupling (LC-ESI/MS). LC-ESI/MS analysis showed that the decolorization of the dye RB21 by turnip peroxidase is due to the breaking up of the chromatogenous system. The tests for toxicity towards lettuce seeds showed an increase of the toxicity after enzymatic treatment of the dye. This study verifies the viability of the use of the turnip peroxidase enzyme in the biodegradation of textile dyes.

Two laccase based biocatalysts were produced by insolubilization (cross-linked enzyme aggregates-CLEAs) and by immobilization (covalent linking to silica support). Both biocatalysts were implemented parallely in a continuous stirred-tank membrane reactor to degrade 50 µM bisphenol A (BPA) in buffer pH 5. Kinetic models were derived based on various reaction mechanisms with BPA substrate molecules and enzyme activation-deactivation behaviors. The equation sets were solved for the different models and optimization was performed to fit the models to experimental data by adjustment of kinetic constants. Along with BPA measurement, laccase activities were monitored by the use of an external colorimetric substrate. It has been shown that a model with an apparent 2 nd order rate constant for reaction between BPA and insolubilized enzyme provides a good estimation of the continuous BPA removal.

Increasing awareness of the finite resources of the planet, including its environmental quality, constitutes a powerful driver for industries and governments not only to fight the effects of pollution but to avoid it at the source. Demands are mounting to replace conventional industrial processes by less-or non-polluting ones. During this gradual shift the treatment of wastes from current human activities and as a legacy of our industrial history remains a challenge. As these treatments merge with environmentally benign industrial processes a truly sustainable economy will become a reality. Enzymes or whole-cell biocatalysts in industrial processes are linked to lower consumption of energy and chemicals and are thus "green" agents. Enzymes catalyze specific reactions under moderate conditions hence they are instrumental for the selective removal of pollutants from waste streams. Enzyme specificity also precludes undesired sidereactions, while in some cases the pollutants treated could be transformed into added-value products, as illustrated in the waste biorefinery concept. The power of such biocatalysts is demonstrated in the case of emerging organic contaminants (EOCs) such as pharmaceuticals and personal care products (PPCPs), which affect treated and untreated water and associated matrices like sludges and sediments at very low but environmentally relevant concentrations ("micropollutants") through unwanted biological effects like endocrine disruption. Oxidative enzymes like fungal laccases and peroxidases are promising biocatalysts for the transformation and re...

Peroxidases can be used in the decolorization process. There is a growing interest for new sources of this enzyme and for obtaining economically viable processes. In this work, a low-cost vegetable peroxidase extraction process is proposed; the resulting enzyme is characterized to determine its optimum pH, temperature, and stability conditions, and it is then applied in the decolorization of reactive dye Remazol Turquoise G 133%. The turnip peroxidase (TP) was utilized as an enzymatic source. This enzyme exhibited maximum activity at pH 7.0, and it was active in the temperature range of 30 to 50 °C, which favors its use in industrial processes. Acetone was the most efficient solvent to induce precipitation. The removal of Remazol Turquoise G 133% was 56.0% complete after 50 min, while 41.0% of the same dye was removed with the commercial horseradish peroxidase enzyme in 50 min. TP presents potential as a viable alternative in the decolorization of textile wastewaters.

Laccase (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are ubiquitous enzymes present in nature as they are known to originate from bacteria, fungi, plants, etc. Both laccase and tyrosinase are copper-containing phenoloxidases requiring readily available O2 without auxiliary cofactor for their catalytic transformation of numerous phenolic substrates. In the present study, laccase and tyrosinase have been insolubilized as combined crosslinked enzyme aggregates (combi-CLEA) using chitosan, a renewable and biodegradable polymer, as crosslinker. The combi-CLEA, with specific activity of 12.3 U/g for laccase and 167.4 U/g for tyrosinase, exhibited high enzymatic activity at pH5-8 and temperature at 5-30°C, significant resistance to denaturation and no diffusional restriction to its active site based upon the Michaelis-Menten kinetic parameters. Subsequently, the combi-CLEA was applied to the transformation of acetaminophen as a model phenolic compound in samples of real wastewaters in orde...

Antioxidant and cytocompatible chemically modified polysaccharides and their hydrogels were obtained by a biomimetic approach. For this purpose, carboxymethylpullulan grafted with ferulic acid (CMP-FA) was firstly synthesized with different substitution degrees (DS). Their hydrogels were secondly obtained by enzymatic cross-linking with laccase. Hydrogel swelling has been found dependent on both DS and media ionic strength. The CMP-FA antioxidant properties were evaluated by the DPPH method and ABTS assays. The DPPH radical scavenging effect was high for CMP-FA solutions (80% after 30 min) and lower for the corresponding hydrogels (70% after 7 h). The antibacterial properties of ferulic acid and CMP-FA derivatives were tested against Staphylococcus aureus but the minimal inhibitory concentration of CMP-FA was not reached in the range of concentrations studied. Finally the CMP-FA derivatives showed no cytotoxicity towards mouse fibroblast cells.

Magnetic iron dust, a byproduct by many chemical industries that performs the reduction of nitro compounds to amine, was used for laccase immobilization. The characterization of magnetic iron dust was done by X-ray diffraction, Fourier-transform infrared, and dynamic light scattering. Biodegradable polymer, chitosan, was coated on to the magnetic iron dust by reverse phase suspension method, which was confirmed by Fourier-transform infrared analysis. Immobilization of the laccase enzyme was done onto the chitosan-coated and non-coated magnetic iron dust. The immobilization was monitored by Fourier-transform infrared analysis. Binding efficiency, optimum pH, and optimum temperature for these immobilized laccases were investigated. X-ray diffraction pattern of magnetic iron dust confirmed presence of magnetite (Fe 3 O 4) and maghemite (g-Fe 2 O 3) with a particle size of 529.6 nm measured by dynamic light scattering. Laccase was immobilized on chitosan-coated and non-coated magnetic iron dust,

Chitosan and its derivatives functionalized by laccase-catalyzed oxidation of ferulic acid (FA) and ethyl ferulate (EF) were characterised for their physico-chemical, antioxidant and antibacterial properties. The enzymatic grafting of oxidised phenols led to FA-coloured and EF-colourless chitosan derivatives with good stability of colour and grafted phenols towards the chemical treatment by organic solvents. The efficiency of FA-products grafting onto chitosan was higher than that of EF-products. Moreover, the enzymatic grafting of phenols onto chitosan changed its morphological surface, increased its molecular weight and its viscosity. Furthermore, the chitosan derivatives presented improved antioxidant properties especially for FA-chitosan derivative when compared with chitosan with good antioxidant stability towards thermal treatment (100°C/1 h). Chitosan and its derivatives showed also similar antibacterial activities and more precisely bactericidal activities. This enzymatic procedure provided chitosan derivatives with improved properties such as antioxidant activity, thermal antioxidant stability as well as the preservation of initial antibacterial activity of chitosan.

Chitosan particles were functionalized with ferulic acid (FA) and ethyl ferulate (EF) as substrates using laccase from Myceliophtora thermophyla as biocatalyst. The reactions were performed with chitosan particles under an eco-friendly procedure, in a heterogeneous system at 30 • C, in phosphate buffer (50 mM, pH 7.5). The FA-chitosan derivative presented an intense yellow-orange color stable while the EF-chitosan derivative was colorless. The spectroscopic analyses indicated that the reaction products bound covalently to the free amino groups of chitosan exhibiting a novel absorbance band in the UV/Vis spectra between 300 and 350 nm, at C-2 region by the duplication of C-2 signal in the 13 C NMR spectrum, via Schiff base bond (N C) exhibiting novel bands in the FT-IR spectrum at 1640 and 1620 cm −1. Additionally, antioxidant capacities of chitosan derivatives showed that the chitosan derivatives presented improved antioxidant properties, especially for FA-chitosan derivative (EC 50 were 0.52 ± 0.04, 0.20 ± 0.02 mg/ml for DPPH • and ABTS • + scavenging, respectively).

Antioxidant and cytocompatible chemically modified polysaccharides and their hydrogels were obtained by a biomimetic approach. For this purpose, carboxymethylpullulan grafted with ferulic acid (CMP-FA) was firstly synthesized with different substitution degrees (DS). Their hydrogels were secondly obtained by enzymatic cross-linking with laccase. Hydrogel swelling has been found dependent on both DS and media ionic strength. The CMP-FA antioxidant properties were evaluated by the DPPH method and ABTS assays. The DPPH radical scavenging effect was high for CMP-FA solutions (80% after 30 min) and lower for the corresponding hydrogels (70% after 7 h). The antibacterial properties of ferulic acid and CMP-FA derivatives were tested against Staphylococcus aureus but the minimal inhibitory concentration of CMP-FA was not reached in the range of concentrations studied. Finally the CMP-FA derivatives showed no cytotoxicity towards mouse fibroblast cells.

A simple new spectrophotometric method has been developed for estimation of Atovaquone in bulk and tablet dosage form. Atovaquone is estimated to be 251 nm in methanol. The Beer's law is obeyed in the concentration range of 1-10 μg/mL of the drug. The slope and intercept values are 0.111 and 0.012, respectively. Results of analysis of this method have been validated statically and by recovery studies. The method is applied to the marketed tablet formulation. A result of the analysis of tablet formulation, given as a percentage of label claim ± standard deviation, is 99.14 ± 0.66. The precision and accuracy has been examined by performing recovery studies and found to be 100.09 ± 1.14. The developed method is simple, sensitive, and reproducible, and can be used for the routine analysis of Atovaquone in bulk and tablet dosage form.

Peroxidases can be used in the decolorization process. There is a growing interest for new sources of this enzyme and for obtaining economically viable processes. In this work, a low-cost vegetable peroxidase extraction process is proposed; the resulting enzyme is characterized to determine its optimum pH, temperature, and stability conditions, and it is then applied in the decolorization of reactive dye Remazol Turquoise G 133%. The turnip peroxidase (TP) was utilized as an enzymatic source. This enzyme exhibited maximum activity at pH 7.0, and it was active in the temperature range of 30 to 50 °C, which favors its use in industrial processes. Acetone was the most efficient solvent to induce precipitation. The removal of Remazol Turquoise G 133% was 56.0% complete after 50 min, while 41.0% of the same dye was removed with the commercial horseradish peroxidase enzyme in 50 min. TP presents potential as a viable alternative in the decolorization of textile wastewaters.

FeFe]-hydrogenases are the enzymes responsible for high yield H2 production during dark fermentation in bio-hydrogen production plants. The culturable bacterial population present in a pilot-scale plant efficiently producing H2 from waste materials was isolated, classified and identified by means of 16S rDNA gene analysis. The culturable part of the mixed population consists of nine bacterial species that include non-hydrogen producers (Lactobacillus, Enterococcus and Staphylococcus) and several Clostridium that are directly responsible for H2 production. An extensive analysis of the expression of [FeFe]-hydrogenases in the three best producer strains was achieved by RT-PCR, covering the complete set of known genes for each species. This revealed that during H2 production there are several different [FeFe]-hydrogenases simultaneously expressed, with genes belonging to the same phylogenetic and structural classification sharing similar transcriptional profiles.

A mesophilic alkaline tolerant fermentative microbe was isolated from estuarine sediment samples and designated as Clostridium butyricum TM-9A, based on 16S rRNA gene sequence. Batch experiments were conducted for investigation of TM-9A strain for its growth and hydrogen productivity from glucose, in an iron containing basal solution supplemented with yeast extract as organic nitrogen source. Hydrogen production started to evolve when cell growth entered exponential phase and reached maximum production rate at late exponential phase. Maximum hydrogen production was observed at 37 C, initial pH of 8.0 in the presence of 1% glucose. Optimization of process parameters resulted in increase in hydrogen yield from 1.64 to 2.67 mol of H 2 /mol glucose. Molar yield of H 2 increased further from 2.67 to 3.1 mol of H 2 /mol of glucose with the decrease in hydrogen partial pressure, obtained by lowering the total pressure in the head space of the batch reactor. Acetate and butyrate were the measure volatile fatty acids generated during hydrogen fermentation. TM-9A strain produced hydrogen efficiently from a range of pentose and hexose sugars including di-, tri and poly-saccharides like;

In this work, a novel type of functionalized magnetic graphene oxide (MGO) nano-sheets which have the ability to capture His-tagged CotA laccase was synthesized. N␣,N␣-Bis(carboxymethyl)-l-lysine hydrate (NTA-NH 2) was attached on MGO then chelated with Ni 2+ and Cu 2+ separately. The Cu 2+-chelated MGO (MGO-NTA-Cu 2+) exhibited the highest adsorption capacity of 177 mg/g-support when compared with both MGO and Ni 2+-chelated MGO (MGO-NTA-Ni 2+) which exhibited an adsorption capacity of 49.9 and 145 mg/g-support, respectively. CotA laccase immobilized on MGO-NTA-Cu 2+ nano-sheets showed the highest activity recovery when compared with that immobilized on MGO-NTA-Ni 2+ nano-sheets. The catalytic properties of MGO-NTA-Cu-CotA laccase were significantly improved in comparison with those of the free laccase. MGO-NTA-Cu-CotA laccase showed efficient decolorization for Congo Red (CR), and the decolorization rate reached 100% after 5 h reaction at 60 • C and pH 8. Furthermore, MGO-NTA-Cu-CotA laccase retained 89.4% of its initial activity after 10 consecutive cycles. Together these results indicated that the Cu 2+ chelated MGO nano-sheets are promising support for efficient CotA laccase immobilization for environmental and industrial applications under high temperature and alkaline condition.

The residual effluent from the corn tortilla industry, referred to as Nejayote in Mexico, causes serious pollution problems because of its high chemical oxygen demand (COD). However, as this effluent contains valuable corn phytochemicals, such as ferulic acid and its derivatives, it also represents an opportunity to produce high-added-value biocompounds. In this work, we addressed environmental and biosynthetic approaches by applying laccase oxidative treatment in the presence of chitosan to reduce the environmental impact of the effluents from the tortilla industry. A central composite design pattern was used to understand the relationship between several variables (pH, temperature, reaction time, and enzyme quantity) and two responses (removal of ferulic acid derivatives and COD decrease). The optimum conditions for maximizing both responses were pH 6, 35 • C, 24 h, and 0.3025 nmoles laccase, which resulted in 70 and 78% decreases in phenolics and COD, respectively. UV-vis, fluorescence and FTIR analyses revealed enzymatic grafting of chitosan with phenolics from the effluent. Grafted chitosan presented a higher capacity to scavenge DPPH radicals (EC 50 1.9 mg/mL) than neat chitosan (EC 50 2.5 mg/mL) and higher viscosity values. The obtained results indicate that a process involving a single enzymatic step could be adequate to decrease the effluent COD and to generate polymers with potential applications in the food and pharmaceutical industries.

Background Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial c...

Kemičari već desetljećima razvijaju razne tehnike pripravljanja enantiomerno čistih spojeva. Njihova se primjena može pronaći svugdje u svakodnevici, ali daleko su najbitniji u proizvodnji lijekova. Kiralni produkt nije moguće dobiti polazeći iz akiralnog polaznog spoja. Stoga, nastanu li novi kiralni centri u molekuli, produkti će se nalaziti u obliku enantiomernog para u omjeru 50:50 koji se naziva racemična smjesa. Takvu smjesu je teško razdvojiti jer enantiomeri imaju ista fizikalna svojstva. Asimetrična sinteza uključuje reakcije u kojima je omjer nastalih stereoizomera pomaknut prema jednom od njih te se za takve reakcije kaže da su stereoselektivne. Asimetrična sinteza može se podijeliti na tri glavna pristupa – rezoluciju, strategiju kiralnog bazena te asimetričnu indukciju. U većini se slučajeva sva tri pristupa kombiniraju prilikom pripravljanja enantiomerno čistog produkta. Rezolucija je metoda razdvajanja smjese enantiomera fizikalnim metodama na način da se u smjesu dod...

Problem zastite prirodnog okružja pred kemicare stavlja važan posao pronalaženja kemijskih reakcija koje su manje stetne za okolis. U tu svrhu osmisljavaju se manje toksicni industrijski procesi, u kojima sudjeluju ili prilikom reakcije nastaju manje kolicine ekoloski stetnih tvari i nusprodukata. Zamjena organskih otapala vodom kao medijem velik je korak naprijed. Konacno, potpuno izuzimanje otapala u kemijskim reakcijama za ljude i okolis je najpoželjnije. Mehanokemijske organske reakcije bez uporabe otapala nov su nacin priprave organskih spojeva tzv. "Highspeed vibratonal milling" (brzi vibracijski mlin - u daljem tekstu HSVM) tehnikom. U ovom tekstu dan je kratak opis te metode i pregled dosadasnjih znanstvenih rezultata dobivenih uporabom te tehnike.

Invertase was immobilized using the cross-linked enzyme aggregate (CLEA) methodology using soy protein as co-feeder. The immobilized invertase retained around 30% of the initial activity after enzyme leaching assay. Maximum activity for the immobilized invertase was at 50.8 • C (5.8 • C higher than the soluble invertase) and at pH 5.0 (similar to the soluble invertase). The catalytic efficiency (k cat /K m) of invertase did reduce approximately tenfold after immobilization, probably due to diffusional delays. The immobilized invertase could be recycled tenfold in 4 h-batches of hydrolysis of sucrose at 40 • C and pH 6.0, maintaining the reaction conversion above 75%. The hydrolysis of sucrose catalyzed by immobilized invertase showed to be economically feasible in an operational window built based on economic metrics for a fed-batch process, with three intermittent feeds of sucrose to restore the substrate concentration at 100 g L −1 when the conversion reaches 95%. This work represents an advance in the field, because using a carrier-free and recyclable biocatalyst the specific productivity (gram of products per gram of biocatalyst per hour) of invert sugar was as high as those previously reported for invertase immobilized on solid carriers, which may dilute its volumetric activity and increase the cost of the biocatalyst.

Solids from wastewater of the tortilla industry, known as nejayote, are a potential source of chemopreventive phenolic compounds. Phenolic compounds were identified and quantified in nejayote solids obtained by filtration, as well as its antioxidant activity and chemopreventive effect. Bound phenolics and antioxidant capacity of nejayote were concentrated in the nejayote solution obtained after filtration (FSS) through 280 mm. Additionally, FSS contained 84, 79, 72 and 97% more bound ferulic, coumaric, 8-O-4-diferulic and 5-5 0-diferulic acids, respectively, compared to total nejayote solids. Feruloyl putrescines, especially coumaroyl-feruloyl putrescine, isolated from bound FSS exerted 105% and 12.45% higher chemopreventive effect than nejayote and bound FSS, respectively. Feruloyl putrescines are potent phase II inducers due to their double nitrogen linked Michael acceptors structure. Feruloyl putrescines in nejayote are a source of natural chemopreventive agents capable of inducing phase II enzymes.

Wastewater from petroleum refineries contains toxic, carcinogenic and recalcitrant chlorophenols. This comes with the mandatory environmental aspect of the recycling of the organic wastes. This work aimed to prepare; fluorapatite (FAP) by a simple calcination process of waste animal bones (ABs), ZnO nanoparticles by a wet chemical method and ZnO/FAP binary oxide nano-bio-composite by a simple mechano-mixing technique and study their photo-catalytic degradation properties on chlorophenols. The obtained catalysts were characterized by high-resolution X-ray diffractometer, Fourier transform infrared spectroscopy, scanning electron microscope, transmission electron microscope, Brunauer Emmett-Teller and Barret-Joyner-Halenda methods and N 2 adsorption-desorption isotherms. The three prepared catalysts showed excellent photo-catalytic degradation activity under UV-irradiation towards 3-chlorphenol and 2,3-dichlorphenol. The photo-degradation followed the Langmuir-Hinshelwood L-H kinetic model with R 2 ≥ 0.962; however, the rate constant was strongly affected by the type of the catalyst and chlorophenols. New proposed mathematical correlations were elucidated to assess the relationship between the cumulative removal of the studied chlorophenols and the production of their photocatalytic oxidation intermediates. In the view of the ubiquity, environmental compatibility, and cost-effectiveness; the prepared green FAP from ABs as a readily available, sustainable and cheap waste byproduct of food industry proved to be a favorable photo-catalyst for remediating petroleum refineries wastewater. In an attempt to enhance the photo-catalytic degradation activities of ZnO towards the chlorophenols, the mechano-mixing process suggested a new approach to produce commercial amounts of nano-biocomposites of ZnO/FAP nanostructures with high quality and suitable structural and morphological features that would revolutionize the field of biomaterials.

Invertase was immobilized using the cross-linked enzyme aggregate (CLEA) methodology using soy protein as co-feeder. The immobilized invertase retained around 30% of the initial activity after enzyme leaching assay. Maximum activity for the immobilized invertase was at 50.8 • C (5.8 • C higher than the soluble invertase) and at pH 5.0 (similar to the soluble invertase). The catalytic efficiency (k cat /K m) of invertase did reduce approximately tenfold after immobilization, probably due to diffusional delays. The immobilized invertase could be recycled tenfold in 4 h-batches of hydrolysis of sucrose at 40 • C and pH 6.0, maintaining the reaction conversion above 75%. The hydrolysis of sucrose catalyzed by immobilized invertase showed to be economically feasible in an operational window built based on economic metrics for a fed-batch process, with three intermittent feeds of sucrose to restore the substrate concentration at 100 g L −1 when the conversion reaches 95%. This work represents an advance in the field, because using a carrier-free and recyclable biocatalyst the specific productivity (gram of products per gram of biocatalyst per hour) of invert sugar was as high as those previously reported for invertase immobilized on solid carriers, which may dilute its volumetric activity and increase the cost of the biocatalyst.

Hydrogen has emerged as a promising alternative because it can be derived from a renewable energy sources and used in fuel cells with high efficiency and thus appears as the most promising alternative to fossil fuels. Among the various biological processes known to produce biohydrogen, dark fermentation offers an excellent potential for practical application such as treatment of organic wastes. However, commercialization of the process depends on advances in bioprocess design and optimization along with an understanding of the structure of biohydrogen producing communities and their improvement. The present paper highlights the major factors affecting biohydrogen production as well as the importance of consortium development and molecular understanding of the microorganisms involved in the process toward the realization of a stable hydrogen economy. Further, technological advancements based on improved bioreactor designs and integrated systems based on process economy have been discussed. Based on the recent research on dark fermentative hydrogen production, several new findings and achievements in the field have been highlighted.

Background Bio-hydrogen production via dark fermentation of low-value waste is a potent and simple mean of recovering energy, maximising the harvesting of reducing equivalents to produce the cleanest fuel amongst renewables. Following several position papers from companies and public bodies, the hydrogen economy is regaining interest, especially in combination with circular economy and the environmental benefits of short local supply chains, aiming at zero net emission of greenhouse gases (GHG). The biomasses attracting the largest interest are agricultural and urban green wastes (pruning of trees, collected leaves, grass clippings from public parks and boulevards), which are usually employed in compost production, with some concerns over the GHG emission during the process. Here, an alternative application of green wastes, low-value compost and intermediate products (partially composted but unsuitable for completing the process) is studied, pointing at the autochthonous microbial c...

Hydrogen production has been one of the most studied problematics nowadays because of the worldwide needs of change in energy sources for daily use, industrial use and laboratory use. In addition, we can also mention the importance of this energy sources to be clean, self-sustainable and of course profitable for the industry, that is the reason why in this work we studied one of the most common hydrogen-producer microorganisms, Clostridium butyricum. This bacterial specie was chosen because it's metabolic pathways have been widely studied and it's an easy-obtainable microorganism present in almost any kind of anaerobic environment such as some zones of soil, human intestines and others, in addition to that, we can also mention some other species for hydrogen production: Kleibsella pneumoniae and Citrobacter freundii which can also be used for the modelation process and the industrial process of production. In this work we developed a process model about the metabolic pathways of C. butyricum in order to understand the hydrogen production process and reveal the points where it can be optimized. Mainly, the model was designed so it could predict the comportment of the process in the exponential phase of growth of the bacteria. Stoichiometric analysis was performed in order to create theoretical curves of the production of hydrogen and other metabolites. Part of the study was performed based in experimental results exposed by other authors, the model was developed in JMCAD (an opensource software for complex dynamic system modeling based in Java. The predicted results are comparable with the experimental ones as it can be seen in the results section, the R of all the curves was (near) to 0,97 so we could ensure a close relation with the reality. Some suggestions in order to maintain the exponential phase of growth in the cultures of C. butyricum are exposed and explained further in the article.

A simple new spectrophotometric method has been developed for estimation of Atovaquone in bulk and tablet dosage form. Atovaquone is estimated to be 251 nm in methanol. The Beer's law is obeyed in the concentration range of 1-10 μg/mL of the drug. The slope and intercept values are 0.111 and 0.012, respectively. Results of analysis of this method have been validated statically and by recovery studies. The method is applied to the marketed tablet formulation. A result of the analysis of tablet formulation, given as a percentage of label claim ± standard deviation, is 99.14 ± 0.66. The precision and accuracy has been examined by performing recovery studies and found to be 100.09 ± 1.14. The developed method is simple, sensitive, and reproducible, and can be used for the routine analysis of Atovaquone in bulk and tablet dosage form.

U organskoj kemiji sve se više pažnje posvećuje sintezi u mikro- i mezo- protočnim sustavima (engl. flow chemistry), koja ima brojne prednosti nad šaržnom sintezom. Glavne prednosti provedbe organske sinteze u takvim protočnim reaktorima su veća učinkovitost, ekološka prihvatljivost i sigurnost. Unatoč tome sinteza u protočnim sustavima ne može se primjenjivati kao univerzalni pristup za sve probleme koji mogu zateći organske sintetske kemičare te prije provedbe odabranih reakcija treba razmotriti isplativost s obzirom na šaržnu sintezu. Sigurnosti i ekološkoj prihvatljivosti sinteza u protočnim reaktorima značajno doprinosi upotreba malog volumena kemikalija i otapala budući da se reakcije provode u mikro- ili mezo- reaktorima napravljenima u pravilu od inertnih materijala. Zbog brojnih prednosti, organske reakcije u protočnim sustavima predmet su kontinuiranog istraživanja, pri čemu se uvjeti provedbe reakcija optimiraju u svrhu povećanja učinkovitosti i sigurnosti procesa te njeg...

Background: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H 2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways. Results: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H 2 production. The growth, H 2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H 2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H 2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase. Conclusions: This work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H 2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.

Background: Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H 2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways. Results: In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H 2 production. The growth, H 2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H 2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H 2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase. Conclusions: This work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H 2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.

Laccase (EC 1.10.3.2) and tyrosinases (EC 1.14.18.1) are ubiquitous enzymes present in nature as they are known to originate from bacteria, fungi, plants, etc. Both laccase and tyrosinase are copper-containing phenoloxidases requiring readily available O2 without auxiliary cofactor for their catalytic transformation of numerous phenolic substrates. In the present study, laccase and tyrosinase have been insolubilized as combined crosslinked enzyme aggregates (combi-CLEA) using chitosan, a renewable and biodegradable polymer, as crosslinker. The combi-CLEA, with specific activity of 12.3 U/g for laccase and 167.4 U/g for tyrosinase, exhibited high enzymatic activity at pH5-8 and temperature at 5-30°C, significant resistance to denaturation and no diffusional restriction to its active site based upon the Michaelis-Menten kinetic parameters. Subsequently, the combi-CLEA was applied to the transformation of acetaminophen as a model phenolic compound in samples of real wastewaters in orde...

The main purpose of this research was to qualitatively and quantitatively identify chitosan-based viscose fibre functional group surfaces as modified by two chemically-similar phenolics, namely fisetin and quercetin. Potentiometric titration was used to determine fibre-dissociable weak acids as a consequence of the presence of chitosan and deposited flavonoids. In addition, a conventional spectrophotometric method using C.I. Acid Orange 7 dye was used for determining the amino groups only. Finally, the antioxidant and antimicrobial properties of the fibres were evaluated, respectively. It has been clearly shown that the introduction of flavonoid onto the fibres introduces a significant amount of anionic phenolic hydroxyl groups, leading to fibre antioxidant activity and a decrease in fibre antimicrobial efficiency.