Spectrophotometric Assay for the Detection of 2,5-Diformylfuran and Its Validation through Laccase-Mediated Oxidation of 5-Hydroxymethylfurfural.

Modern biocatalysis requires fast, sensitive, and efficient high-throughput screening methods to screen enzyme libraries in order to seek out novel biocatalysts or enhanced variants for the production of chemicals. For instance, the synthesis of bio-based furan compounds like 2,5-diformylfuran (DFF) from 5-hydroxymethylfurfural (HMF) via aerobic oxidation is a crucial process in industrial chemistry. Laccases, known for their mild operating conditions, independence from cofactors, and versatility with various substrates, thanks to the use of chemical mediators, are appealing candidates for catalyzing HMF oxidation. Herein, Schiff-based polymers based on the coupling of DFF and 1,4-phenylenediamine (PPD) have been used in the set-up of a novel colorimetric assay for detecting the presence of DFF in different reaction mixtures. This method may be employed for the fast screening of enzymes (Z' values ranging from 0.68 to 0.72). The sensitivity of the method has been proved, and detection (8.4 µM) and quantification (25.5 µM) limits have been calculated. Notably, the assay displayed selectivity for DFF and enabled the measurement of kinetics in DFF production from HMF using three distinct laccase-mediator systems.

Laccases from Pleurotus ostreatus Applied to the Oxidation of Furfuryl Alcohol for the Synthesis of Key Compounds for Polymer Industry.

Laccases are oxidative enzymes with high synthetic potential. In this work, their value in biocatalysis is shown through the green and selective oxidation of furfuryl alcohol into furfural with the aid of mediators. The influence of different parameters, such as pH, enzyme/mediator composition, buffer type, cosolvent tolerance, and reaction times, is investigated. Under the optimal conditions, 20 mol % of TEMPO as mediator and 5.8 U mL-1 of laccases POXC and POXA1b from Pleurotus ostreatus, quantitative production of furfural is attained after 16 h. POXC laccase stands out for its ability to catalyze the reaction at pH 6.5, whereas POXA1b is notable for its high stability. Furfural conversions reach excellent values (95 %) after 72 h using only 5 mol % of TEMPO at 100 mM. Furthermore, furfuryl alcohol bioamination is achieved by employing the amine transaminase from Chromobacterium violaceum, providing furfuryl amine, a key compound for the polymer industry, through a one-pot sequential approach.

The power of two: An artificial microbial consortium for the conversion of inulin into Polyhydroxyalkanoates.

One of the major issues for the microbial production of polyhydroxyalkanoates (PHA) is to secure renewable, non-food biomass feedstocks to feed the fermentation process. Inulin, a polydisperse fructan that accumulates as reserve polysaccharide in the roots of several low-requirement crops, has the potential to face this challenge. In this work, a "substrate facilitator" microbial consortium was designed to address PHA production using inulin as feedstock. A microbial collection of Bacillus species was screened for efficient inulinase producer and the genome of the selected strain, RHF15, identified as Bacillus gibsonii, was analysed unravelling its wide catabolic potential. RHF15 was co-cultured with Cupriavidus necator, an established PHA producer, lacking the ability to metabolize inulin. A Central Composite Rotary Design (CCRD) was applied to optimise PHA synthesis from inulin by the designed artificial microbial consortium, assessing the impact of species inoculum ratio and inulin and N-source concentrations. In the optimized conditions, a maximum of 1.9 g L-1 of Polyhydroxybutyrate (PHB), corresponding to ~80% (gpolymer/gCDW) polymer content was achieved. The investigated approach represents an effective process optimization method, potentially applicable to the production of PHA from other complex C- sources.

Optimization of Inulin Hydrolysis by Penicillium lanosocoeruleum Inulinases and Efficient Conversion Into Polyhydroxyalkanoates.

Inulin, a polydisperse fructan found as a common storage polysaccharide in the roots of several plants, represents a renewable non-food biomass resource for the synthesis of bio-based products. Exploitation of inulin-containing feedstocks requires the integration of different processes, including inulinase production, saccharification of inulin, and microbial fermentation for the conversion of released sugars into added-value products. In this work paper, a new microbial source of inulinase, Penicillium lanosocoeruleum, was identified through the screening of a fungal library. Inulinase production using inulin as C-source was optimized, reaching up to 28 U mL-1 at the 4th day of growth. The fungal inulinase mixture (PlaI) was characterized for pH and temperature stability and activity profile, and its isoenzymes composition was investigated by proteomic strategies. Statistical optimization of inulin hydrolysis was performed using a central composite rotatable design (CCRD), by analyzing the effect of four factors. In the optimized conditions (T, 45.5°C; pH, 5.1; substrate concentration, 60 g L-1; enzyme loading, 50 U gsubstrate -1), up to 96% inulin is converted in fructose within 20 h. The integration of PlaI in a process for polyhydroxyalkanoate (PHA) production by Cupriavidus necator from inulin was tested in both separated hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). A maximum of 3.2 g L-1 of PHB accumulation, corresponding to 82% polymer content, was achieved in the SSF. The proved efficiency in inulin hydrolysis and its effective integration into a SSF process pave the way to a profitable exploitation of the PlaI enzymatic mixture in inulin-based biorefineries.

From untargeted metabolomics to the multiple reaction monitoring-based quantification of polyphenols in chocolates from different geographical areas.

Plants, including cocoa bean, are the main source of metabolites with multiple biological functions. Polyphenol extracts are widely used as a nutraceutical supplement for their well-known health-promoting role. In this paper, a preliminary untargeted metabolic screening was carried out by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)/TOF on a pool of chocolate samples made by cocoa beans of different geographical areas. Then, a targeted approach was developed for polyphenol quantification by an optimized Liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method multiple reaction monitoring (MRM) ion mode. Detection limit of polyphenol standard ranged between 1 and 25 pg/µl with variation coefficient lower than 15%. External calibration curves were used for quantification of polyphenols in 18 samples. Fifty polyphenols were detected in a single LC-MRM/MS run and quantified by monitoring almost 90 transitions in a 5-minute run. The polyphenols content of different cocoa beans from several countries was finally compared by principal component analysis (PCA) statistical analysis suggesting that the chocolate made by Ecuador cocoa beans showed the highest level of polyphenols.

Beyond natural laccases: extension of their potential applications by protein engineering.

Laccases bring exciting promises into the green industries, and the development of enzymes with improved properties is further raising their exploitation potential. Molecular engineering methods to build highly efficient catalysts both through rational and random mutagenesis were extensively applied. Moreover, computational approaches are becoming always more reliable in aiding proper design of efficient and tailored catalyst for specific applications. In this review, the results of the last 10 years about industrial application of engineered laccases in different fields are analyzed. Tailoring laccase towards a target substrate and defining a proper screening strategy for the selection of the "jackpot mutant" represent the keys of a winning mutagenesis pathway. Likewise, laccase chimerae, built by the fusion of laccases with relevant proteins, emerged as an added value in the designing of flexible and well-rounded biocatalysts. Despite being promising in most of the reported examples, evolved laccases are currently tested at a laboratory scale and a feedback from the industry world is continuously required to strengthen the biotechnological exploitation of these improved enzymes.

Conversion of no/low value waste frying oils into biodiesel and polyhydroxyalkanoates.

A sustainable bioprocess was developed for the valorization of a no/low value substrate, i.e. waste frying oils (WFOs) with high content of free fatty acids (FFAs), otherwise unsuitable for biodiesel production. The bioprocess was verified using both recombinant (Escherichia coli) and native (Pseudomonas resinovorans) polyhydroxyalkanoates (PHAs) producing cell factories. Microbial fermentation of WFOs provided a 2-fold advantage: i) the reduction of FFAs content resulting into an upgrading of the "exhausted waste oils" and ii) the production of a bio-based microbial polymer. Proper strain designing and process optimization allowed to achieve up to 1.5 g L-1 of medium chain length, mcl-PHAs, together with an efficient conversion (80% yield) of the treated WFO into biodiesel.

Butanol production from laccase-pretreated brewer's spent grain.

BACKGROUND: Beer is the most popular alcoholic beverage worldwide. In the manufacture of beer, various by-products and residues are generated, and the most abundant (85% of total by-products) are spent grains. Thanks to its high (hemi)cellulose content (about 50% w/w dry weight), this secondary raw material is attractive for the production of second-generation biofuels as butanol through fermentation processes. RESULTS: This study reports the ability of two laccase preparations from Pleurotus ostreatus to delignify and detoxify milled brewer's spent grains (BSG). Up to 94% of phenols reduction was achieved. Moreover, thanks to the mild conditions of enzymatic pretreatment, the formation of other inhibitory compounds was avoided allowing to apply the sequential enzymatic pretreatment and hydrolysis process (no filtration and washing steps between the two phases). As expected, the high detoxification and delignification yields achieved by laccase pretreatment resulted in great saccharification. As a fact, no loss of carbohydrates was observed thanks to the novel sequential strategy, and thus the totality of polysaccharides was hydrolysed into fermentable sugars. The enzymatic hydrolysate was fermented to acetone-butanol-ethanol (ABE) by Clostridium acetobutilycum obtaining about 12.6 g/L ABE and 7.83 g/L butanol within 190 h. CONCLUSIONS: The applied sequential pretreatment and hydrolysis process resulted to be very effective for the milled BSG, allowing reduction of inhibitory compounds and lignin content with a consequent efficient saccharification. C. acetobutilycum was able to ferment the BSG hydrolysate with ABE yields similar to those obtained by using synthetic media. The proposed strategy reduces the amount of wastewater and the cost of the overall process. Based on the reported results, the potential production of butanol from the fermentation of BSG hydrolysate can be envisaged.

Green synthesis of conductive polyaniline by Trametes versicolor laccase using a DNA template.

The green synthesis of highly conductive polyaniline by using two biological macromolecules, i.e laccase as biocatalyst, and DNA as template/dopant, was achieved in this work. Trametes versicolor laccase B (TvB) was found effective in oxidizing both aniline and its less toxic/mutagenic dimer N-phenyl-p-phenylenediamine (DANI) to conductive polyaniline. Reaction conditions for synthesis of conductive polyanilines were set-up, and structural and electrochemical properties of the two polymers were extensively investigated. When the less toxic aniline dimer was used as substrate, the polymerization reaction was faster and gave less-branched polymer. DNA was proven to work as hard template for both enzymatically synthesized polymers, conferring them a semi-ordered morphology. Moreover, DNA also acts as dopant leading to polymers with extraordinary conductive properties (∼6 S/cm). It can be envisaged that polymer properties are magnified by the concomitant action of DNA as template and dopant. Herein, the developed combination of laccase and DNA represents a breakthrough in the green synthesis of conductive materials.

Laccase pretreatment for agrofood wastes valorization.

Apple pomace, potato peels, and coffee silverskin are attractive agrofood wastes for the production of biofuels and chemicals, due to their abundance and carbohydrate content. As lignocellulosic biomasses, their conversion is challenged by the presence of lignin that prevents hydrolysis of polysaccharides, hence demanding a pretreatment step. In this work, the effectiveness of Pleurotus ostreatus laccases (with and without mediator) to remove lignin, improving the subsequent saccharification, was assessed. Optimized conditions for sequential protocol were set up for all agrofood wastes reaching delignification and detoxification yields correlated with high saccharification. Especially noteworthy were results for apple pomace and coffee silverskin for which 83% of and 73% saccharification yields were observed, by using laccase and laccase mediator system, respectively. The herein developed sequential protocol, saving soluble sugars and reducing the amount of wastewater, can improve the overall process for obtaining chemicals or fuels from agrofood wastes.

New lipases by mining of Pleurotus ostreatus genome.

The analysis of Pleurotus ostreatus genome reveals the presence of automatically annotated 53 lipase and 34 carboxylesterase putative coding-genes. Since no biochemical or physiological data are available so far, a functional approach was applied to identify lipases from P. ostreatus. In the tested growth conditions, four lipases were found expressed, with different patterns depending on the used C source. Two of the four identified proteins (PleoLip241 and PleoLip369), expressed in both analysed conditions, were chosen for further studies, such as an in silico analysis and their molecular characterization. To overcome limits linked to native production, a recombinant expression approach in the yeast Pichia pastoris was applied. Different expression levels were obtained: PleoLip241 reached a maximum activity of 4000 U/L, whereas PleoLip369 reached a maximum activity of 700 U/L. Despite their sequence similarity, these enzymes exhibited different substrate specificity and diverse stability at pH, temperature, and presence of metals, detergents and organic solvents. The obtained data allowed classifying PleoLip241 as belonging to the "true lipase" family. Indeed, by phylogenetic analysis the two proteins fall in different clusters. PleoLip241 was used to remove the hydrophobic layer from wool surface in order to improve its dyeability. The encouraging results obtained with lipase treated wool led to forecast PleoLip241 applicability in this field.

A step forward in laccase exploitation: Recombinant production and evaluation of techno-economic feasibility of the process.

Protein heterologous production offers viable opportunities to tailor laccase properties to specific industrial needs. The high redox potential laccase POXA1b from Pleurotus ostreatus was chosen as case study of marketable enzyme, due to its desirable properties in terms of activity/stability profile, and already assessed applicability. POXA1b was heterologously produced in Pichia pastoris by investigating the effect of inducible and constitutive expression systems on both the yield and the cost of its production. System performances were first assessed in shaken-flasks and then scaled-up in bioreactor. The production level obtained in the inducible system is 42U/mL, while the activity value achieved with the constitutive one is 60U/mL, the highest obtained in constitutive systems so far. The economic feasibility of recombinant laccase production was simulated, describing the case of an Italian small-medium enterprise. Two scenarios were evaluated: Scenario (I) production based on methanol inducible system; Scenario (II) production based on the constitutive system, fed with glycerol. At all the scales the glycerol-based fermentation is more economic than the methanol-based one. The price forecast for rPOXA1b production is 0.34€kU-1 for glycerol-based process, and is very competitive with the current price of commercial laccase.

Oxidoreductases on their way to industrial biotransformations.

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.

Exploitation of Trametes versicolor for bioremediation of endocrine disrupting chemicals in bioreactors.

Endocrine disrupting chemicals (EDCs) are environmental contaminants causing increasing concerns due to their toxicity, persistence and ubiquity. In the present study, degradative capabilities of Trametes versicolor, Pleurotus ostreatus and Phanerochaete chrysosporium to act on five EDCs, which represent different classes of chemicals (phenols, parabens and phthalate) and were first applied as single compounds, were assessed. T. versicolor was selected due to its efficiency against target EDCs and its potentialities were exploited against a mixture of EDCs in a cost-effective bioremediation process. A fed-batch approach as well as a starvation strategy were applied in order to reduce the need for input of 'fresh' biomass, and avoid the requirement for external nutrients. The fungus was successfully operated in two different bioreactors over one week. Semi-batch cultures were carried out by daily adding a mixture of EDCs to the bioreactors in a total of five consecutive degradation cycles. T. versicolor was able to efficiently remove all compounds during each cycle converting up to 21 mg L-1 day-1 of the tested EDCs. The maintained ability of T. versicolor to remove EDCs without any additional nutrients represents the main outcome of this study, which enables to forecast its application in a water treatment process.

Efficient immobilization of a fungal laccase and its exploitation in fruit juice clarification.

The clarification step represents, in fruit juices industries, a bottleneck process because residual phenols cause severe haze formation affecting juice quality and impairing customers acceptance. An enzymatic step can be efficiently integrated in the process, and use of immobilized enzymes entails an economical advantage. In this work, covalent immobilization of recombinant POXA1b laccase from Pleurotus ostreatus on epoxy activated poly(methacrylate) beads was optimized thanks to a Response Surface Methodologies approach. Through regression analysis the process was well fitted by a quadratic polynomial equation (R(2)=0.9367, adjusted R(2)=0.8226) under which laccase activity reached 2000 ± 100 Ug(-1) of beads, with an immobilization efficiency of 98%. The immobilized biocatalyst was characterized and then tested in fruit juice clarification reaching up to 45% phenol reduction, without affecting health-effective flavanones content. Furthermore, laccase treated juice displays an improved sensory profile, due to the reduction of vinyl guaiacol, a potent off-flavor possessing a peppery/spicy aroma.

Immobilization of a Pleurotus ostreatus laccase mixture on perlite and its application to dye decolourisation.

In the present study, a crude laccase preparation from Pleurotus ostreatus was successfully immobilized on perlite, a cheap porous silica material, and tested for Remazol Brilliant Blue R (RBBR) decolourisation in a fluidized bed recycle reactor. Results showed that RBBR decolourisation is mainly due to enzyme action despite the occurrence of dye adsorption-related enzyme inhibition. Fine tuning of immobilization conditions allowed balancing the immobilization yield and the resulting rate of decolourisation, with the adsorption capacity of the solid biocatalyst. In the continuous lab scale reactor, a maximum conversion degree of 56.1% was achieved at reactor space-time of 4.2 h. Stability and catalytic parameters of the immobilized laccases were also assessed in comparison with the soluble counterparts, revealing an increase in stability, despite a reduction of the catalytic performances. Both effects are most likely ascribable to the occurrence of multipoint attachment phenomena.

Fungal laccases degradation of endocrine disrupting compounds.

Over the past decades, water pollution by trace organic compounds (ng/L) has become one of the key environmental issues in developed countries. This is the case of the emerging contaminants called endocrine disrupting compounds (EDCs). EDCs are a new class of environmental pollutants able to mimic or antagonize the effects of endogenous hormones, and are recently drawing scientific and public attention. Their widespread presence in the environment solicits the need of their removal from the contaminated sites. One promising approach to face this challenge consists in the use of enzymatic systems able to react with these molecules. Among the possible enzymes, oxidative enzymes are attracting increasing attention because of their versatility, the possibility to produce them on large scale, and to modify their properties. In this study five different EDCs were treated with four different fungal laccases, also in the presence of both synthetic and natural mediators. Mediators significantly increased the efficiency of the enzymatic treatment, promoting the degradation of substrates recalcitrant to laccase oxidation. The laccase showing the best performances was chosen to further investigate its oxidative capabilities against micropollutant mixtures. Improvement of enzyme performances in nonylphenol degradation rate was achieved through immobilization on glass beads.

Effective mutations in a high redox potential laccase from Pleurotus ostreatus.

Since the first report on a laccase, there has been a notable development in the interest towards this class of enzymes, highlighted from the number of scientific papers and patents about them. At the same time, interest in exploiting laccases-mainly high redox potential-for various functions has been growing exponentially over the last 10 years. Despite decades of work, the molecular determinants of the redox potential are far to be fully understood. For this reason, interest in tuning laccase redox potential to provide more efficient catalysts has been growing since the last years. The work herein described takes advantage of the filamentous fungus Aspergillus niger as host for the heterologous production of the high redox potential laccase POXA1b from Pleurotus ostreatus and of one of its in vitro selected variants (1H6C). The system herein developed allowed to obtain a production level of 35,000 U/L (583.3 µkat/L) for POXA1b and 60,000 U/L (1,000 µkat/L) for 1H6C, corresponding to 13 and 20 mg/L for POXA1b and 1H6C, respectively. The characterised proteins exhibit very similar characteristics, with some exceptions regarding catalytic behaviour, stability and spectro-electrochemical properties. Remarkably, the 1H6C variant shows a higher redox potential with respect to POXA1b. Furthermore, the spectro-electrochemical results obtained for 1H6C make it tempting to claim that we spectro-electrochemically determined the redox potential of the 1H6C T2 site, which has not been studied in any detail by spectro-electrochemistry yet.

Crystallization and preliminary X-ray crystallographic analysis of the small subunit of the heterodimeric laccase POXA3b from Pleurotus ostreatus.

Laccases are multicopper oxidases of great biotechnological potential. While laccases are generally monomeric glycoproteins, the white-rot fungus Pleurotus ostreatus produces two closely related heterodimeric isoenzymes composed of a large subunit, homologous to the other fungal laccases, and a small subunit. The sequence of the small subunit does not show significant homology to any other protein or domain of known function and consequently its function is unknown. The highest similarity to proteins of known structure is to a putative enoyl-CoA hydratase/isomerase from Acinetobacter baumannii, which shows an identity of 27.8%. Diffraction-quality crystals of the small subunit of the heterodimeric laccase POXA3b (sPOXA3b) from P. ostreatus were obtained using the sitting-drop vapour-diffusion method at 294 K from a solution consisting of 1.8 M sodium formate, 0.1 M Tris-HCl pH 8.5. The crystals belonged to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = 126.6, c = 53.9 Å. The asymmetric unit contains two molecules related by a noncrystallographic twofold axis. A complete data set extending to a maximum resolution of 2.5 Šwas collected at 100 K using a wavelength of 1.140 Å.