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10 pags., 2 figs., 1 tab., Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated β-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1-sacB that was able to produce 73.4 ± 1.6 g L-1 of FOS, with a productivity of 1.53 ± 0.03 g L-1 h-1 and a yield of 0.31 ± 0.03 gFOS gsucrose-1. This is the first report on the FOS production using a mutant Z. mobilis ZM4 strain in a one-step process. KEY POINTS: • Zymomonas mobilis was engineered to produce FOS in a one-step fermentation process. • Mutant strains produced FOS mixtures with high concentration of 6-kestose. • A new route to produce tailor-made FOS mixtures was presented., The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project FoSynBio—Synthetic biology approaches to design and construct microbial cell factories for the production of fructooligosaccharides (POCI-01–0145-FEDER-029549).

Synthetic colorants have gradually been replaced by natural pigments, mainly in the food industry. Among the several natural sources of pigments, filamentous fungi have gained special attention. However, the eventual biosynthesis of mycotoxins is a limitation for the practical use of this kind of microorganisms as pigment producers. In this work, Penicillium brevicompactum was studied for the first time as a potential pigment producer using submerged fermentation. The effect of several experimental parameters (culture medium composition, agitation speed, type of carbon source, temperature, concentration of supplements and carbon source, natural light and initial pH) in the production of pigments was evaluated. Under the optimal conditions (culture medium I containing lactose (20g/L) and peptone/yeast extract (8/8g/L), 23°C, initial pH 7.0, 150rpm, and natural light), a mixture of pigments (yellow, orange, and red) was obtained. This mixture is mycotoxin-free (ochratoxin A and mycophenolic acid) and presented promising antioxidant activity (FRAP: 58.58±4.58µmol Fe(II)/g; DPPH IC50: 18.48±0.26µmol TE/g and ABTS IC50: 28.38±3.79µmol TE/g). Furthermore, these pigments were proved to be very stable for a wide range of pH and temperatures. Overall, it was demonstrated that P. brevicompactum can be a safe source of natural pigments with interesting properties for the food industry., This work was financially supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit. Luís Abrunhosa acknowledges FCT for the assistant research contract CEECIND/00728/2017, obtained under CEEC Individual 2017., info:eu-repo/semantics/publishedVersion

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (>\thinspace99\\% of humic acids effectively removed) and sufficient quantity (10.5--13.8 \textmug g1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the Project LIG-NOZYMES—Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01–0145-FEDER-029773)., info:eu-repo/semantics/publishedVersion

" Available online 22 May 2022", This study explores the structural characterization, antioxidant and prebiotic activities of hydrolysates containing xylooligosaccharides (XOS) produced by different strategies: direct fermentation of beechwood xylan (FermBX) and enzymatic treatment of beechwood (EnzBX) and rice husk (EnzRH) xylans. EnzBX and EnzRH showed XOS with a backbone of (1 4)-linked-xylopyranosyl residues and branches of arabinose, galactose, and uronic acids. FermBX presented the highest content of total phenolic compounds (14 mg GAE/g) and flavonoids (0.6 mg QE/g), which may contribute to its antioxidant capacity 39.1 mol TE/g (DPPH), 45.7 mol TE/g (ABTS), and 79.9 mol Fe II/g (FRAP). The fermentation of hydrolysates decreased the abundance of microorganisms associated with intestinal diseases from Eubacteriales, Desulfovibrionales and Methanobacteriales orders, while stimulating the growth of organisms belonging to Bacteroides, Megamonas and Limosilactobacillus genera. The production of short-chain fatty acids, ammonia, and CO2 suggested the prebiotic potential. In conclusion, hydrolysates without previous purification and obtained from non-chemical approaches demonstrated promising biological activities for further food applications., This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) – Finance Code 001; by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) – Grant numbers 423285/2018-1 and 304857/2018-1; by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit; by the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte; by the COMPETE 2020 (POCI-01-0145- FEDER-006684); by the FoSynBio (POCI-01-0145-FEDER-029549); and LAQV/REQUIMTE (UIDB/50006/2020, UIDP/50006/2020) through national founds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement. GG acknowledges the Universidade Federal do Rio Grande for the financial support from Postgraduate Student Mobility Program (PROPESP/FURG). BC, CA, and SPS acknowledge their grants (SFRH/BD/132324/2017, 2020.00293 CEECIND, and SFRH/BD/136471/2018) from FCT. LB also acknowledge FCT for the junior research contract (CEECIND/03280/2020). EC thanks the research contract (CDL-CTTRI-88-ARH/2018 – REF. 049-88-ARH/2018) funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19., info:eu-repo/semantics/publishedVersion

Cellobiose 2-epimerases are interesting enzymes that can catalyze the production of lactose-based prebiotics, namely, epilactose and lactulose, through a single substrate reaction with noteworthy yields. In this work, the GRAS yeast Saccharomyces cerevisiae was for the first time used as a host for the heterologous production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus. The enzyme showed an optimal temperature and an optimal pH of 80 °C and 7.5, respectively. Under these conditions, the time-course production of epilactose from lactose (50 g/L) was evaluated. The maximum yield and productivity obtained were 27\% and 9 g L1 h1, respectively. This epilactose yield represents the highest value obtained from the several approaches reported using cellobiose 2-epimerase from C. saccharolyticus. In conclusion, it was shown that the enzyme produced by S. cerevisiae is suitable for the production of epilactose-enriched prebiotic mixtures with potential applications in the food industry., FCT -Fundação para a Ciência e a Tecnologia(Lisboa2020), info:eu-repo/semantics/publishedVersion

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality ( 99% of humic acids effectively removed) and sufficient quantity (10.5-13.8 µg g

Innovation of pastry products towards higher nutritional and commercial value remains a challenge to the growing field of healthy food. In this study, the prebiotic supplementation and sugar reduction were explored in a widely consumed pastry product with a low level of innovation. The prebiotic potential of commercial agave inulin and galacto-oligosaccharides (GOS) was evaluated and compared by an in vitro model using human fecal inocula. Rice muffins containing 100% of sugar or 75% of sugar supplemented with 0.8% GOS were produced and compared with commercial rice muffins regarding their physical and textural properties. GOS fermentation led to the highest production of lactate and short-chain fatty acids, besides the most significant reduction of the final pH value and of the ammonia and methane production. Inulin presented a higher selectivity towards Lactobacillaceae (51 ± 1% of all), while GOS are more efficient to stimulate Bifidobacteriaceae growth (65 ± 7% of all)., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte; COMPETE 2020 (POCI-01-0145-FEDER-006684), FoSynBio (POCI-01-0145-FEDER-029549) and NewFood (NORTE-01-0246-FEDER000043). CA and BBC acknowledge their grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from FCT. RM acknowledges the financial support to the Xunta de Galicia and FEDER by the project (EDD431B 2019/01)., info:eu-repo/semantics/publishedVersion

Consumers are conscientiously changing their eating preferences toward healthier options, such as functional foods enriched with pre- and probiotics. Prebiotics are attractive bioactive compounds with multidimensional beneficial action on both human and animal health, namely on the gastrointestinal tract, cardiometabolism, bones or mental health. Conventionally, prebiotics are non-digestible carbohydrates which generally present favorable organoleptic properties, temperature and acidic stability, and are considered interesting food ingredients. However, according to the current definition of prebiotics, application categories other than food are accepted, as well as non-carbohydrate substrates and bioactivity at extra-intestinal sites. Regulatory issues are considered a major concern for prebiotics since a clear understanding and application of these compounds among the consumers, regulators, scientists, suppliers or manufacturers, health-care providers and standards or recommendation-setting organizations are of utmost importance. Prebiotics can be divided in several categories according to their development and regulatory status. Inulin, galactooligosaccharides, fructooligosaccharides and lactulose are generally classified as well established prebiotics. Xylooligosaccharides, isomaltooligosaccharides, chitooligosaccharides and lactosucrose are classified as "emerging" prebiotics, while raffinose, neoagaro-oligosaccharides and epilactose are "under development." Other substances, such as human milk oligosaccharides, polyphenols, polyunsaturated fatty acids, proteins, protein hydrolysates and peptides are considered "new candidates." This chapter will encompass actual information about the non-established prebiotics, mainly their physicochemical properties, market, legislation, biological activity and possible applications. Generally, there is a lack of clear demonstrations about the effective health benefits associated with all the non-established prebiotics. Overcoming this limitation will undoubtedly increase the demand for these compounds and their market size will follow the consumer's trend.

Consumers are conscientiously changing their eating preferences toward healthier options, such as functional foods enriched with pre- and probiotics. Prebiotics are attractive bioactive compounds with multidimensional beneficial action on both human and animal health, namely on the gastrointestinal tract, cardiometabolism, bones or mental health. Conventionally, prebiotics are non-digestible carbohydrates which generally present favorable organoleptic properties, temperature and acidic stability, and are considered interesting food ingredients. However, according to the current definition of prebiotics, application categories other than food are accepted, as well as non-carbohydrate substrates and bioactivity at extra-intestinal sites. Regulatory issues are considered a major concern for prebiotics since a clear understanding and application of these compounds among the consumers, regulators, scientists, suppliers or manufacturers, health-care providers and standards or recommendation-setting organizations are of utmost importance. Prebiotics can be divided in several categories according to their development and regulatory status. Inulin, galactooligosaccharides, fructooligosaccharides and lactulose are generally classified as well established prebiotics. Xylooligosaccharides, isomaltooligosaccharides, chitooligosaccharides and lactosucrose are classified as "emerging" prebiotics, while raffinose, neoagaro-oligosaccharides and epilactose are "under development." Other substances, such as human milk oligosaccharides, polyphenols, polyunsaturated fatty acids, proteins, protein hydrolysates and peptides are considered "new candidates." This chapter will encompass actual information about the non-established prebiotics, mainly their physicochemical properties, market, legislation, biological activity and possible applications. Generally, there is a lack of clear demonstrations about the effective health benefits associated with all the non-established prebiotics. Overcoming this limitation will undoubtedly increase the demand for these compounds and their market size will follow the consumer's trend.

The potential use of alternative culture media towards the development of a sustainable bioprocess to produce lipases by Diutina rugosa is clearly demonstrated. First, a synthetic medium containing glucose, peptone, yeast extract, oleic acid, and ammonium sulfate was proposed, with lipase activity of 143 U/L. Then, alternative culture media formulated with agro-industrial residues, such as molasses, corn steep liquor (CSL), and olive mill waste (OMW), were investigated. An experimental design was conducted, and only CSL concentration was found to have a positive effect in lipase production. The highest lipase activity (561 U/L) was produced on a mixture of molasses (5 g/L), CSL (6 g/L), OMW (0.5\\% v/v), 0.5 g/L of ammonium sulfate, and 3 g/L of peptone at 24 h of cultivation. Lipase production was also carried out in a 1-L bioreactor leading to a slightly higher lipase activity at 24 h of cultivation. The semi-purified enzyme exhibits an optimum temperature and pH of 40 \textdegreeC and 7.0, respectively. Finally, the media cost per unit of lipase produced (UPC) was influenced by the medium components, specially by the inducer used. The lowest UPC was obtained when the agro-industrial residues were combined and used at the improved concentrations., The study is funded by CAPES, CNPq, and FUNCAP (from Brazil) for the financial support that made this work possible. In addition, the study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte, and the Project LIGNOZYMES (POCI-01-0145- FEDER-029773)., info:eu-repo/semantics/publishedVersion

The potential use of alternative culture media towards the development of a sustainable bioprocess to produce lipases by Diutina rugosa is clearly demonstrated. First, a synthetic medium containing glucose, peptone, yeast extract, oleic acid, and ammonium sulfate was proposed, with lipase activity of 143 U/L. Then, alternative culture media formulated with agro-industrial residues, such as molasses, corn steep liquor (CSL), and olive mill waste (OMW), were investigated. An experimental design was conducted, and only CSL concentration was found to have a positive effect in lipase production. The highest lipase activity (561 U/L) was produced on a mixture of molasses (5 g/L), CSL (6 g/L), OMW (0.5% v/v), 0.5 g/L of ammonium sulfate, and 3 g/L of peptone at 24 h of cultivation. Lipase production was also carried out in a 1-L bioreactor leading to a slightly higher lipase activity at 24 h of cultivation. The semi-purified enzyme exhibits an optimum temperature and pH of 40 °C and 7.0, respectively. Finally, the media cost per unit of lipase produced (UPC) was influenced by the medium components, specially by the inducer used. The lowest UPC was obtained when the agro-industrial residues were combined and used at the improved concentrations.

Supplementary data to this article can be found online athttps://doi.org/10.1016/j.lwt.2020.109322., Until now the prebiotic potential of pure trisaccharide raffinose on human health assessed through high-throughput sequencing remains poorly investigated. In this work, an in vitro model using human fecal inocula of two healthy volunteers (D1 and D2) was used to study the prebiotic potential of raffinose and compare it with the well-stablished and commercial prebiotic lactulose. The intestinal microbiota showed preference for raffinose as substrate showing the highest consumption value at 48h (96.0±0.9% D1 and 95.3±0.7% D2). The fermentation of raffinose decreased the medium pH, the ammonia concentration and the relative amount of Proteobacteria, while increasing the total production of lactate and short chain fatty acids (129.9±2.6mmol/L D1 and 179.6±0.6mmol/L D2), CO2 (10.8±0.8 mmol/Lmedium D1 and 5.2±0.3 mmol/Lmedium D2) and the relative amount of Bifidobacterium and Lactobacillus. This study suggests that raffinose holds potential functional properties for human health., CA an BBC acknowledge her grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from Portuguese Foundation for Science andTechnology (FCT). The study receivedfinancial support from FCT underthe scope of the strategic funding of UID/BIO/04469/2020 unit;COMPETE 2020 (POCI-01-0145-FEDER-006684), through nationalfunds and where applicable co-financed by the FEDER, within thePT2020 Partnership Agreement; the Projects FoSynBio (POCI-01-0145-FEDER-029549) and NewFood–Food Technologies Valorization(NORTE-324 01-0246-FEDER-000043). The authors also acknowledgeBioTecNorte operation (NORTE-01-0145-FEDER-000004) funded bythe European Regional Development Fund under the scope ofNorte2020 -Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.seppur.2018.07.062., Esterases catalyze the cleavage and formation of ester bonds of a broad range of substrates presenting a widespread spectrum of industrial applications. This work aimed to partially purify and characterize an esterase from Aureobasidium pullulans LABIOTEC 01 produced in a culture medium containing olive mill wastewater. Esterase purification was evaluated using different strategies, namely the enzyme recovery by PEG-salt aqueous two-phase systems (ATPSs); and the enzyme precipitation with ammonium sulfate, acetone, and ethanol. The best purification factor (18±2) was obtained when the ATPS composed of 20% (w/w) polyethylene glycol (PEG) 6000 and 5.8% (w/w) potassium phosphate buffer (PPB) pH 8.0 was combined with acetone precipitation. The partially purified enzyme presented an optimum pH of 5.0, although it remained active in the pH range of 4.5 to 7.5 ( 50% relative activity). The optimum temperature was found to be 60°C. Furthermore, the addition of salts such as FeCl3, CuSO4 and MnCl2 promoted an increase in the enzymatic activity (above 100%). The enzyme was found to be stable and showed high activity when exposed to polar solvents such as dimethyl sulfoxide, dimethylformamide, and methanol. The use of an integrated strategy of purification combining simple purification methods such as ATPS and precipitation was herein reported for the first time for esterase. This strategy proved to be an interesting approach to partially purify the esterase produced under submerged fermentation by A. pullulans. Furthermore, the enzyme showed potential to be applied in industrial biocatalytic processes using high temperature and different salts or solvents. Also, the production of esterase using olive mill wastewater as substrate demonstrated to be a suitable alternative to reduce and valorize agro-industrial residues., ACL acknowledges his post-doctoral grant and research funds from CAPES and CNPq (Brazilian funding agencies). SCS and LRR acknowledge their grants SFRH/BPD/88584/2012 and SFRH/BSAB/142873/ 2018, respectively, from the Portuguese Foundation for Science and Technology (FCT). The study also received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the Project MultiBiorefinery - Multi‐purpose strategies for broadband agro‐forest and fisheries by‐products valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403) and the Project Lignozymes - Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-010145-FEDER-029773). The authors also acknowledge financial support from the BioTecNorte operation (NORTE-01-0145-FEDER-000004) fundedbytheEuropeanRegionalDevelopment Fundunderthescopeof Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Brewers spent grain (BSG) is an inexpensive and abundant brewery by-product that can be used to produce prebiotic arabino-xylooligosaccharides (AXOS). In this study, Bacillus subtilis 3610 was used, for the first time, to produce AXOS through direct fermentation of BSG. Additionally, the microorganism was genetically modified to improve the AXOS production. The xylanase gene xyn2 from Trichoderma reesei coupled with a secretion tag endogenous to B. subtilis was cloned in pDR111 and integrated into its chromosome. After optimization by experimental design, AXOS with a degree of polymerization ranging from 2 to 6 were obtained. The maximum production yield expressed in xylose equivalents per amount of BSG (54.2 ± 1.1 mg/g) represents an increase of 33% comparing to the wild type. When compared with the enzymatic hydrolysis process, single-step fermentation with B. subtilis proved to be a very promising low-cost strategy for the simultaneous production of AXOS and valorization of BSG., CA, SCS, EC and LRR acknowledge their grants (PD/BD/105870/ 2014, SFRH/BPD/88584/2012, SFRH/BPD/70589/2010 and SFRH/ BSAB/142873/2018) from Portuguese Foundation for Science and Technology(FCT).Thestudyreceived financialsupportfromFCTunder the scope of the strategic funding of UID/BIO/04469/2013 unit; COMPETE2020 (POCI-01-0145-FEDER-006684); QOPNAresearch Unit (FCT UID/QUI/00062/2013), through national founds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the projects MultiBiorefinery (POCI-01-0145-FEDER016403); FoSynBio (POCI-01-0145-FEDER-029549) and Lignozymes (POCI-01-0145-FEDER-029773). The authors also acknowledge financial support from the BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Collagenases are proteolytic enzymes capable of degrading both native and denatured collagen, reported to be applied in industrial, medical and biotechnological sectors. Liquid-liquid extraction using aqueous two-phase system (ATPS) is one of the most promising bioseparation techniques, which can substitute difficult solid-liquid separation processes, offering many advantages over conventional methods including low-processing time, low-cost material and low-energy consumption. The collagenase produced by Penicillium sp. UCP 1286 showed a stronger affinity for the bottom salt-rich phase, where the highest levels of collagenolytic activity were observed at the center point runs, using 15.0% (w/w) PEG 3350 g/mol and 12.5% (w/w) phosphate salt at pH 7.0 and concentration. The enzyme was characterized by thermal stability, pH tolerance and effect of inhibitors, showing optimal collagenolytic activity at 37 °C and pH 9.0 and proved to be a serine protease. ATPS showed high efficiency in the collagenase purification, confirmed by a single band in SDS/PAGE, and can in fact be applied as a quick and inexpensive alternative method., This work was supported by the National Council of Technological and Scientific Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). Sara Silvério also acknowledges her post-doc grant (SFRH/BPD/88584/2012) from FCT (SFRH/BPD/88584/2012) (Portuguese Science and Technology Foundation), Portugal.

A sustainable and rational use of the existing resources with limited generation of residues and using environmentally friendly processes is a current concern that is being addressed under the concepts of green chemistry and circular economy. Biotechnological processes, namely, the use of microorganisms and/or enzymes to cost-efficiently produce and convert renewable biological resources and waste streams into valuable products across several economic sectors (such as food, feed, bio-based products, and bioenergy), comprise a powerful alternative. The so-called circular bioeconomy is currently taking advantage of the recent developments in omics techniques, gene editing, systems, and synthetic biology, as well as of the novel technological developments of bioreactors and downstream processes. In addition, the potential of many existing residues from different sources, which remain to be mapped and thoroughly characterized, is largely unexplored and represents a great opportunity to design new products and processes, hence a great opportunity for the economy, for the environment, and ultimately for the society.

Sugar fatty acid esters (SFAEs) are composed of one or more fatty acid chains linked to a carbohydrate molecule. They are commonly used as non-ionic and biodegradable surfactants and emulsifiers. However, SFAEs also present other interesting features such as tasteless, odorless, non-toxicity, and bioactivity which make them suitable for industrial applications in the food, pharmaceutical, and cosmetics industries. Traditionally, SFAEs are obtained from chemical synthesis despite the recognized disadvantages associated to this process. More recently, the synthesis of SFAEs using enzymes as biocatalysts proved to be a suitable and greener alternative. However, the use of organic solvents as reaction medium has been pointed out as one of the greatest limitations of the enzymatic process. To overcome this issue, ionic liquids (ILs) have been proposed as alternative solvents. ILs are frequently described as “tunable” solvents due to the possibility of combining different cations and anions to obtain the desired physicochemical properties. Based on their unique characteristics, ILs have been recognized as suitable solvents for biocatalytic processes involving different enzymes and reactions. Nevertheless, the selection of the suitable IL for the enzymatic synthesis of SFAEs is considered a key aspect in the bioprocess since compounds chemically different (sugars, fatty acids, and enzymes) are simultaneously present in the reaction medium. Ideally, the IL used as solvent should facilitate the solubilization of the substrates and not negatively affect the enzymatic activity and stability. The enzymatic synthesis of SFAEs can occur though two different approaches, namely esterification and transesterification. Several ILs and biocatalysts have been successfully reported for the synthesis of different types of SFAEs. Considering the enormous versatility of combining different sugars with fatty acids, it is expected that novel and promising SFAEs could be synthesized in the future using ILs as reaction medium.

The updated definition of prebiotic expands the range of potential applications in which emerging xylooligosaccharides (XOS) can be used. It has been demonstrated that XOS exhibit prebiotic effects at lower amounts compared to others, making them competitively priced prebiotics. As a result, the industry is focused on developing alternative approaches to improve processes efficiency that can meet the increasing demand while reducing costs. Recent advances have been made towards greener and more efficient processes, by applying process integration strategies to produce XOS from costless lignocellulosic residues and using genetic engineering to create microorganisms that convert these residues to XOS. In addition, collecting more in vivo data on their performance will be key to achieve regulatory claims, greatly increasing XOS commercial value., CA, SCS and LRR acknowledge their grants (UMINHO/BPD/4/2019, SFRH/BPD/88584/2012 and SFRH/BSAB/142873/2018) from University of Minho and Portuguese Foundation for Science and Technology(FCT).The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2019 unit; COMPETE 2020 (POCI-01-0145-FEDER-006684), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Project FoSynBio (POCI-01-0145FEDER-029549) and Lignozymes (POCI-01-0145-FEDER-029773). The authors also acknowledge BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Lactulose is a lactose-based carbohydrate with well-known prebiotic effect and recognized medical applications. Currently, the commercially available lactulose is chemically synthesized. Nevertheless, the process leads to low yields and high levels of by-products. Alternatively, lactulose can be produced by enzymatic synthesis, which provides a cleaner production under mild conditions. Two different enzymatic routes were reported for lactulose production. Lactulose can be obtained through hydrolysis and transfer reactions catalyzed by a glycosidase. Alternatively, lactulose can be produced by direct isomerization of lactose to lactulose catalyzed by cellobiose-2-epimerase. An interesting characteristic of lactulose is also its capacity to act as substrate in additional enzymatic synthesis which leads to the formation of attractive compounds, such as lactulose-based oligosaccharides and lactulose esters. Besides increasing the interest and potential of lactulose, these lactulose-based compounds can also offer new and promising functionalities and applications. Herein, we review the enzymes involved in the synthesis of lactulose, as well as the reaction conditions and yields. The potential of different enzymes is discussed and it is shown that reaction conditions and composition of products depend on the type of enzyme and its microbial source. The conversion of lactulose into lactulose-based compounds is also covered, describing in detail the biocatalysts involved, the reaction conditions used, and the potential of the final products obtained.

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.carbpol.2018.09.088., The global demand of prebiotics such as xylooligosaccharides (XOS) has been growing over the years, motivating the search for different production processes with increased efficiency. In this study, a cloned Bacillus subtilis 3610, containing the xylanase gene xyn2 of Trichoderma reesei coupled with an endogenous secretion tag, was selected for XOS production through direct fermentation of beechwood xylan. A mixture of XOS with a degree of polymerization ranging from 4-6 was obtained, presenting high stability after a static in vitro digestion (98.5±0.2%). The maximum production yield expressed as total XOS per amount of xylan (306±4mg/g) was achieved after 8h of fermentation operating under one-time impulse fed-batch. The optimal conditions found were pH 6.0 and 42.5°C, using 2.5g/L of initial concentration of xylan increased up to 5.0g/L at 3h. Xylopentaose was the major oligosaccharide produced, representing 47% of the total production yield., CA, SCS, ACP, EC and LRR acknowledge their grants (PD/BD/ 105870/2014, SFRH/BPD/88584/2012, SFRH/BPD/ 101181/2014, SFRH/BPD/70589/2010 and SFRH/BSAB/142873/2018) from Portuguese Foundation for Science and Technology (FCT). The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit; COMPETE 2020 (POCI-010145-FEDER-006684); QOPNA research Unit (FCT UID/QUI/00062/ 2013), through national founds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Project MultiBiorefinery (POCI-01-0145-FEDER-016403); the Project FoSynBio (POCI-01-0145-FEDER-029549) and Lignozymes (POCI-01-0145FEDER-029773). The authors also acknowledge BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.foodchem.2018.07.080., Xylooligosaccharides (XOS) are prebiotic nutraceuticals that can be sourced from lignocellulosic biomass, such as agro-residues. This study reports for the first time an optimization study of XOS production from agro-residues by direct fermentation using two Trichoderma species. A total of 13 residues were evaluated as potential substrates for single-step production. The best results were found for Trichoderma reesei using brewers spent grain (BSG) as substrate. Under optimal conditions (3 days, pH 7.0, 30°C and 20 g/L of BSG), a production yield of 38.3 ± 1.8 mg/g (xylose equivalents/g of BSG) was achieved. The obtained oligosaccharides were identified as arabino-xylooligosacharides (AXOS) with degree of polymerization from 2 to 5. One-step fermentation proved to be a promising strategy for AXOS production from BSG, presenting a performance comparable with the use of commercial enzymes. This study provides new insights towards the bioprocess integration, enabling further developments of low-cost bioprocesses for the production of these valuable compounds., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684) and under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the Project MultiBiorefinery – Multi purpose strategies for broadband agro forest and fisheries by products valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403) the Project Lignozymes (POCI-010145-FEDER-029773) and the Project FoSynBio (POCI-01-0145FEDER-029549). The authors also acknowledge financial support from BioTecNorte (NORTE-01-0145-FEDER-000004) Project BioInd – Biotechnology and Bioengineering for improved Industrial and AgroFood processes(NORTE-07–0124-FEDER-000028) funded by the European Regional Development Fund under the scope of Norte2020 Programa Operacional Regional do Norte. CA and SCS acknowledge their grants (PD/BD/105870/2014 SFRH/BPD/88584/2012) from FCT., info:eu-repo/semantics/publishedVersion

Lactosucrose is a synthetic trisaccharide composed of galactose, glucose and fructose. This compound is obtained through enzymatic synthesis using lactose and sucrose as substrates. The enzymes involved in the process are able to catalyse both hydrolysis and transfer reactions. The yield and productivity of the process are usually affected by the occurrence of parallel hydrolysis of the newly formed product (lactosucrose). Therefore, it is important to find efficient strategies to avoid or minimize product degradation. Furthermore, in the last decades the demand for lactosucrose has significantly increased. This compound is considered a potential prebiotic and several beneficial effects associated to its consumption have been described. As a result, it has been included in the formulations of functional foods. This review covers the most relevant information about lactosucrose, including its synthesis and purification, beneficial effects at physiological level, and also its potential applications., Fundação para a Ciência e a Tecnologia (FCT) Strategic Project of UID/BIO/04469/2013 unit, the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462) and the project “BioInd – Biotechnology and Bioengineering for im- proved Industrial and Agro-Food processes”, REF. NORTE-07- 0124-FEDER-000028 co-funded by the Programa Operacional Regional do Norte (ON. 2–ONovo Norte), QREN, FEDER.

ß-galactosidases (EC 3.2.1.23) are interesting enzymes with potential application in the pharmaceutical and food industry. In this work, a screening study was carried out to identify new fungal sources of -galactosidase. A total of 50 fungi were evaluated using a chromogenic test performed in agar plates. The most promising microorganisms were validated as effective -galactosidase producers under submerged fermentation conditions. The crude -galactosidases were characterized regarding their optimal pH (3.0-5.5) and temperature (45-65 °C). All enzymes showed ability to synthesize lactose-based prebiotics, namely lactulose (maximal yield 3.3%) and a galacto-oligosaccharide (GOS) (maximal yield 20%). Additionally, some enzymatic extracts with fructosyltransferase activity allowed to produce other type of prebiotics, namely fructo-oligosaccharides (FOS). This work, reports for the first time the simultaneous synthesis of different mixtures of GOS (2-15% yield and 0.07-0.5 g/L·h-1 productivity) and FOS (4-30% yield and 0.1-1 g/L·h-1 productivity) by crude extracts exhibiting dual enzymatic activity., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684),and also by the Project BioTecNorte(NORTE-01-0145-FEDER000004) and the project MultiBiorefinery (POCI-01-0145-FEDER016403) funded by the European Regional Development Fund under thescope ofNorte2020 –Programa OperacionalRegional doNorte.SCS acknowledges her post doc grant (SFRH/BPD/88584/2012) from FCT. EA Macedo is a member of the Associate Laboratory LSRE-LCM, Project POCI-01-0145-FEDER-006984, funded by FEDER through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) – and by national funds through FCT – Fundação para a Ciência e a Tecnologia., info:eu-repo/semantics/publishedVersion

This work presents an inexpensive, simple and fast procedure to purify trypsin based on affinity binding with ferromagnetic particles of azocasein composite (mAzo). Crude extract was obtained from intestines of fish Nile tilapia (Oreochromis niloticus) homogenized in buffer (01 g tissue/ml). This extract was exposed to 100mg of mAzo and washed to remove unbound proteins by magnetic field. Trypsin was leached off under high ionic strength (3 M NaCl). Preparation was achieved containing specific activity about 60 times higher than that of the crude extract. SDS-PAGE showed that the purified protein had molecular weight (24 kDa) in concordance with the literature for the Nile tilapia trypsin. The mAzo composite can be reused and applied to purify trypsin from other sources., Authors are thankful to CNPq (grant 307273/2013-0) and CAPES (Scholarship to MHMEA) Brazil agencies as well as FCT (Scholarship to SICS and CN) Portugal Agencie.

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.carbpol.2019.115460., Xylooligosaccharides (XOS) are emergent prebiotics exhibiting high potential as food ingredients. In this work, in vitro studies were performed using human fecal inocula from two healthy donors (D 1 and D2) to evaluate the prebiotic effect of commercial lactulose and XOS produced in a single-step by recombinant Bacillus subtilis 3610. The fermentation of lactulose led to the highest production of lactate (D1: 33.7±0.5mM; D2:19.7±0.3mM) and acetate (D1: 77.5±0.6mM; D2: 81.0±0.7mM), while XOS led to the highest production of butyrate (D1: 9.0±0.6mM; D2: 10.5±0.8mM) and CO2 (D1: 8.92±0.02mM; D2: 11.4±0.3mM). Microbiota analysis showed a significant decrease in the relative abundance of Proteobacteria for both substrates and an increase in Bifidobacterium and Lactobacillus for lactulose, and Bacteroides for XOS., CA and BBC acknowledge their grants (UMINHO/BPD/4/2019 and SFRH/BD/132324/2017) from the Portuguese Foundation for Science and Technology (FCT). The study received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2019 unit; COMPETE 2020 (POCI-01-0145-FEDER-006684), through national funds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the Project FoSynBio (POCI-01-0145FEDER-029549), and NewFood (NORTE-01-0246-FEDER-000043). The authors also acknowledge BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

ß-galactosidase (EC 3.2.1.23) are interesting enzymes able to catalyze lactose hydrolysis and transfer reactions to produce lactose-based prebiotics with potential application in the pharmaceutical and food industry. In this work, Aspergillus lacticoffeatus is described, for the first time, as an effective -galactosidase producer. The extracellular enzyme production was evaluated in synthetic and alternative media containing cheese whey and corn steep liquor. Although -galactosidase production occurred in all media (expect for the one composed solely by cheese whey), the highest enzymatic activity values (460 U/mL) were obtained for the synthetic medium. Ochratoxin A production in synthetic medium was also evaluated and 9 days of fermentation was identified as a suitable fermentation time to obtain a crude extract enzyme with mycotoxin concentration below the legal comparable value established for wine and grape juices (2 ng/mL). The optimal pH and temperature for the crude extract enzyme was found in the range of 3.54.5 and 5060 °C, respectively. The -galactosidase activity was reduced in the presence of Ba2 +, Fe2 +, Li+, K+ and galactose, while additives (except for ascorbic acid) and detergents exhibited a positive effect on enzymatic activity. This enzyme was able to catalyze the synthesis of prebiotics, namely lactulose (2.5 g/L) and a galacto-oligosaccharide (trisaccharide, 6.3 g/L), either when whole cells or crude enzyme was used as biocatalyst. The lactulose production using fungal whole cells is herein reported for the first time. Additionally, A. lacticoffeatus was also found to produce an enzyme with fructosyltransferase activity and other prebiotics, namely fructo-oligosaccharide 1-kestose (2.4 g/L)., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/ BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER006684),and also by the Project BioTecNorte (NORTE-01-0145-FEDER000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. SCS acknowledges her post doc grant (SFRH/BPD/88584/2012) from FCT. LA acknowledges his post doc grant (UMINHO/BPD/51/2015) from UID/BIO/04469/2013., info:eu-repo/semantics/publishedVersion

A new Penicillium sp. strain isolated from the soil of Caatinga, a Brazilian Biome (UCP 1286) was selected for collagenase production. Fermentation system allowing obtention of collagenolytic activity about 2.7 times higher than existing data, with the highest values of collagenolytic and specific activity (379.80 U/mL, 1460.77 U/mg, respectively), after 126 hours. Applying a factorial design, enzyme production was increased by about 65% compared to the preliminary results. The factorial design demonstrated the existence of two factors with statistical significance on the production of the enzyme: pH and temperature, both with negative effects. Enzyme was found to be more active at pH 9.0 and 37 °C, and also to be very stable in comparison with the collagenase produced by other microorganisms. The enzyme seems to belong to collagenolytic serine proteases family. Concerning the substrate specificity, it was observed that the highest enzyme activity corresponds to azocoll, there was no relevant activity on azocasein and the enzyme showed to be more specific to type V collagen and gelatin than the commercial colagenase produced by Clostridium histolyticum. Major band observed at electrophoresis was approximately 37 kDa. Zymogram analysis confirmed the collagenolytic activity. All data indicates this enzyme as promising biotechnology product., This work was supported by Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) (IBPG-0137-2.08/12) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Sara Silvério also acknowledges her post-doc grant (SFRH/BPD/88584/2012) from FCT (Fundação para a Ciência e a Tecnologia), Portugal.

Lactulose is a lactose-based carbohydrate with well-known prebiotic effect and recognized medical applications. Currently, the commercially available lactulose is chemically synthesized. Nevertheless, the process leads to low yields and high levels of by-products. Alternatively, lactulose can be produced by enzymatic synthesis, which provides a cleaner production under mild conditions. Two different enzymatic routes were reported for lactulose production. Lactulose can be obtained through hydrolysis and transfer reactions catalyzed by a glycosidase. Alternatively, lactulose can be produced by direct isomerization of lactose to lactulose catalyzed by cellobiose-2-epimerase. An interesting characteristic of lactulose is also its capacity to act as substrate in additional enzymatic synthesis which leads to the formation of attractive compounds, such as lactulose-based oligosaccharides and lactulose esters. Besides increasing the interest and potential of lactulose, these lactulose-based compounds can also offer new and promising functionalities and applications. Herein, we review the enzymes involved in the synthesis of lactulose, as well as the reaction conditions and yields. The potential of different enzymes is discussed and it is shown that reaction conditions and composition of products depend on the type of enzyme and its microbial source. The conversion of lactulose into lactulose-based compounds is also covered, describing in detail the biocatalysts involved, the reaction conditions used, and the potential of the final products obtained.

The Gibbs free energy of transfer of a methylene group, G*(CH2), is reported as a measure of the relative hydrophobicity of the equilibrium phases. Furthermore, G*(CH2) is a characteristic parameter of each tie-line, and for that reason can be used for comparing different tie-lines of a given aqueous two-phase system (ATPS) or even to establish comparisons among different ATPSs. In this work, the partition coefficients of a series of four dinitrophenylated-amino acids were experimentally determined, at 23 °C, in five different tie-lines of PEG8000(sodium or potassium) citrate ATPSs. G*(CH2) values were calculated from the partition coefficients and used to evaluate the relative hydrophobicity of the equilibrium phases. PEG8000potassium citrate ATPSs presented larger relative hydrophobicity than PEG8000sodium citrate ATPSs. Furthermore, the results obtained indicated that the PEG-rich phase (top phase) has higher affinity to participate in hydrophobic hydration interactions than the salt-rich phase (bottom phase)., This work was partially supported by FCT/MEC and FEDER under Programe PT2020 (Project UID/EQU/50020/2013), and co-financed by QREN, ON2 and FEDER (Projects NORTE-07-0162FEDER-000050 and NORTE-07-0124-FEDER-0000011), for which the authors are thankful. SCS alsoa cknowledges her post-doc grant (SFRH/BPD/88584/2012) from FCT.

Lactulose is a lactose-based carbohydrate with well-known prebiotic effect and recognized medical applications. Currently, the commercially available lactulose is chemically synthesized. Nevertheless, the process leads to low yields and high levels of by-products. Alternatively, lactulose can be produced by enzymatic synthesis, which provides a cleaner production under mild conditions. Two different enzymatic routes were reported for lactulose production. Lactulose can be obtained through hydrolysis and transfer reactions catalyzed by a glycosidase. Alternatively, lactulose can be produced by direct isomerization of lactose to lactulose catalyzed by cellobiose-2-epimerase. An interesting characteristic of lactulose is also its capacity to act as substrate in additional enzymatic synthesis which leads to the formation of attractive compounds, such as lactulose-based oligosaccharides and lactulose esters. Besides increasing the interest and potential of lactulose, these lactulose-based compounds can also offer new and promising functionalities and applications. Herein, we review the enzymes involved in the synthesis of lactulose, as well as the reaction conditions and yields. The potential of different enzymes is discussed and it is shown that reaction conditions and composition of products depend on the type of enzyme and its microbial source. The conversion of lactulose into lactulose-based compounds is also covered, describing in detail the biocatalysts involved, the reaction conditions used, and the potential of the final products obtained. (c) 2016 Institute of Food Technologists®

Collagenolytic proteases can hydrolyze both denatured and native collagen and are becoming increasingly important commercially. The aim of this work was to select a new strain of Penicillium sp. isolated from the soil of Caatinga for collagenase production. A factorial design 24 was performed to determine the best conditions of enzyme production. Collagenolytic activity reported on this work is about 2 times larger than existing data. According to the growth kinetics, after 126 hours of production, were obtained the highest values of collagenolytic activity and specific activity. The highest values of collagenolytic activity and specific activity were obtained on a culture medium containing 0.25% (w/v) gelatin, 200 rpm, pH 8.0 and 24 °C. Only two factors were statistically significant: pH and temperature, both with negative effects. The experimental design made possible to define fermentation culture conditions able to increase by 66% the value of the initial enzyme activity., Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Aqueous two-phase systems (ATPSs) composed by UCON (ethylene oxide/propylene oxide copolymer) and potassium phosphate salts were for the first time evaluated in the recovery of Peniophora cinerea laccase from complex fermented medium. The ATPSs were obtained by combining the random copolymer UCON with KH2PO4, potassium phosphate buffer pH 7 or K2HPO4. According to the results, protein partition occurred predominantly toward the saline phase (bottom phase) of the ATPSs, while some contaminants such as pigments partitioned mainly to the top phase. In preliminary tests, it was found that the salt with the lowest pH value (KH2PO4, pH 4.6) stimulated the enzyme activity, while the other salts (pH between 7.0 and 9.5) caused a strong inhibition. However, the salt inhibition was not observed in the equilibrium phases of the UCON-Potassium phosphate ATPSs. The laccase recovery was high for all the biphasic systems, but the highest value (134%) was obtained when using UCON combined with KH2PO4. When compared to conventional concentration and purification methods (lyophilization, ammonium sulfate precipitation, ultrafiltration, and ion exchange chromatography), ATPS was demonstrated to be an efficient alternative for P. cinerea laccase recovery from fermented medium., Sergio Moreira gratefully acknowledges the financial support from Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil), and Sara C. Silverio acknowledges her Ph.D. grant (SFRH/BD/43439/2008) from Fundacao para a Ciencia e a Tecnologia (FCT, Portugal).

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production. © 2012 Springer-Verlag.

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production., S.C. Silverio acknowledges the financial support from FCT (Fundacao para a Ciencia e para a Tecnologia, Ph.D. grant SFRH/BD/43439/2008), Portugal; and S. Moreira acknowledges CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), Brazil.

Polyethylene glycol (PEG) with average molecular weight of 8000 was used as a model polymer and modified by introducing an amino acid (phenylalanine) in its hydroxyl terminals. The effect of different percentages of the conjugate polymer in relation to the total polymer on binodal curve of PEG-8000–potassium phosphate buffer (KPB), pH 7.2 aqueous two-phase systems and solute partitioning was explored. The partition of lysozyme (Lyz) and bovine serum albumin (BSA) and also of the conjugated polymer was evaluated in ATPS composed of 17% (w/w) total polymer and 12.5% (w/w) KPB, pH 7.2. Lyz partition profile follows the expected trend decreasing with the increase of modified polymer concentration. However BSA shows a partition profile that is approximately symmetrical to the one observed for the partition of the conjugate polymer, which depends on PEG–phenylalanine concentration in the system. Determination of the relative hydrophobicity of the equilibrium phases and of the protein surface hydrophobicity (So) delivered information that shed some light in the mechanisms controlling the partitioning behavior. Therefore, it becomes clear that the hydrophobic character of the protein taken together with the molecular weight explain the different behavior of these two proteins with the increase of PEG–phenylalanine substitution in the systems., L.A. Ferreira and S.C. Silverio acknowledge the financial support (Grants SFRH/BPD/47607/2008 and SFRH/BD/43439/2008, respectively) from Fundacao para a Ciencia e a Tecnologia (Lisboa, Portugal).

Eight different poly(ethylene glycol) (PEG)/salt aqueous two-phase systems (ATPS's) were characterized at 23 °C. PEGs with different molecular weights (1500 and 8000) were combined with different salts (sodium or potassium citrates and formates) to study the effect of each ATPS constituent in the phase diagram. The binodal curve and several tie-lines were determined for each system using common analytical techniques described in the literature for ATPS's (cloud point method, conductivity, and gravimetric analysis after lyophilization). According to the results, it was found that each ATPS constituent produced a particular effect in the phase diagram. For the salts, changes in the anion produced larger variations in the heterogeneous region size than changes of the cation. Thus, the effect of salt type seems to be largely dependent on the anion, which makes the dominant contribution. The ability to form ATPS's is connected with the Gibbs energy of hydration, ΔG hyd, of the anions: more negative ΔG hyd values facilitate ATPS formation. Changes in the PEG molecular weight affected also the phase diagram: the size of the heterogeneous region increases with the increase in the polymer molecular weight., This work is supported by project PEst-C/EQB/LA0020/2011, financed by FEDER through COMPETE-Programa Operacional Factores de Competitividade and by FCT-Fundacao para a Ciencia e a Tecnologia, Portugal. S.C.S. and O.R acknowledge financial support of FCT (Ph.D. grant SFRH/BD/43439/2008) and Programme Ciencia 2007 (FCT), respectively. A.W. and E.L. acknowledge financial support of IAESTE trainee programme and Ministerio de Educacion y Ciencia of Spain (postdoctoral fellowship EX2009-0404), respectively.

Commercial laccase formulation was immobilized on modified green coconut fiber silanized with 3-glycidoxypropyltrimethoxysilane, aiming to achieve a cheap and effective biocatalyst. Two different strategies were followed: one point (pH 7.0) and multipoint (pH 10.0) covalent attachment. The influence of immobilization time on enzymatic activity and the final reduction with sodium borohydride were evaluated. The highest activities were achieved after 2 h of contact time in all situations. Commercial laccase immobilized at pH 7.0 was found to have higher activity and higher affinity to the substrate. However, the immobilization by multipoint covalent attachment improved the biocatalyst thermal stability at 50 A degrees C, when compared to soluble enzyme and to the immobilized enzyme at pH 7.0. The Schiff's bases reduction by sodium borohydride, in spite of causing a decrease in enzyme activity, showed to contribute to the increase of operational stability through bonds stabilization. Finally, these immobilized enzymes showed high efficiency in the continuous decolourization of reactive textile dyes. In the first cycle, the decolourization is mainly due to dyes adsorption on the support. However, when working in successive cycles, the adsorption capacity of the support decreases (saturation) and the enzymatic action increases, indicating the applicability of this biocatalyst for textile wastewater treatment.

Aqueous two-phase systems (ATPS) are recognized as very suitable techniques for the recovery of target solutes in biological applications. Three new phase diagrams of (UCON 50-HB-5100 + potassium citrate + water), (UCON 50-HB-5100 + sodium formate + water), and (UCON 50-HB-5100 + potassium formate + water) systems were measured at 23 C. The binodal curves were successfully described using the empirical equation suggested by Merchuk and co-workers. The reliability of the tie-line data experimentally determined was evaluated using the equations reported by OthmerTobias and Bancroft and satisfactory linearity was obtained for all ATPS. Among the salts studied, potassium citrate proved to be the most effective in ATPS formation, providing the largest heterogeneous region. Besides, the effect of both anions and cations in the size of the heterogeneous region and in the slope of the tie-lines has been compared. For the same salts and conditions, the heterogeneous region using UCON as the phase-forming polymer is larger than using polyethylene glycol. Furthermore, laccase partition in the UCON-salt ATPS was studied and it was found that in all cases enzyme partition occurred preferably to the bottom phase (salt-rich phase). Laccase concentration in the salt-rich phase was approximately 2-fold that in the top phase, thus UCON-salt ATPS can be a suitable biphasic system for laccase extraction.

Aqueous two-phase systems (ATPS) are recognized as very suitable techniques for the recovery of target solutes in biological applications. Three new phase diagrams of (UCON 50-HB-5100 + potassium citrate + water), (UCON 50-HB-5100 + sodium formate + water), and (UCON 50-HB-5100 + potassium formate + water) systems were measured at 23 C. The binodal curves were successfully described using the empirical equation suggested by Merchuk and co-workers. The reliability of the tie-line data experimentally determined was evaluated using the equations reported by Othmer–Tobias and Bancroft and satisfactory linearity was obtained for all ATPS. Among the salts studied, potassium citrate proved to be the most effective in ATPS formation, providing the largest heterogeneous region. Besides, the effect of both anions and cations in the size of the heterogeneous region and in the slope of the tie-lines has been compared. For the same salts and conditions, the heterogeneous region using UCON as the phase-forming polymer is larger than using polyethylene glycol. Furthermore, laccase partition in the UCON-salt ATPS was studied and it was found that in all cases enzyme partition occurred preferably to the bottom phase (salt-rich phase). Laccase concentration in the salt-rich phase was approximately 2-fold that in the top phase, thus UCON-salt ATPS can be a suitable biphasic system for laccase extraction., This work is supported by project PEst-C/EQB/LA0020/2011, financed by FEDER through COMPETE - Programa Operacional Factores de Competitividade and by FCT - Fundacao para a Ciencia e a Tecnologia, Portugal. SCS and OR acknowledge financial support of FCT (Ph.D. grant SFRH/BD/43439/2008) and Programme Ciencia 2007 (FCT), respectively. EL acknowledges financial support of Ministerio de Educacion y Ciencia of Spain (doctoral fellowship EX2009-0404)

*Aqueous Two-Phase Systems (ATPS) are obtained by mixing two aqueous solutions of different constituents that become immiscible under certain critical conditions, like temperature, concentration, etc. Both phases are composed mainly by water (>80%) and each one is enriched in a different component [1]. ATPS formed by two polymers or a polymer and a salt represent the traditional systems. Nevertheless, other alternative biphasic systems can be obtained using surfactants, micellar compounds or ionic liquids. Due to the high percentage of water present in their composition, ATPS can provide a gentle environment for the extraction and recovery of sensitive biological materials, such as proteins. Different methodologies can be used for protein quantification, such as UV absorbance spectroscopy (280 nm), chemical methods (like Kjeldahl) or colorimetric determination based on dye binding assays (like Bradford, BCA or Lowry). Among them, the Bradford assay is the most currently used for protein quantification in ATPS due to its simplicity, sensitivity and fast response. This assay is based on the absorbance shift observed when the dye Coomassie® Brilliant Blue G-250 binds to protein. The peak absorbance of the acidic solution of Coomassie® changes from 465 to 595 nm when binding occurs. The binding process is fast (5 minutes) and the protein-dye complex formed is stable for about 1 hour [2]. The use of a single reactive and the sensitivity of the dye to small amounts of protein make the Bradford method widely used for protein determination. However, the method can suffer significant interference from some compounds, like detergents and alkaline agents. It is also known that the presence of some salts under certain concentration can compromise the method response. Nevertheless, poor information is found in the literature about interference of specific phase-forming components of ATPS. In this work, the interference in the Bradford method caused by several salts and polymers usually used in ATPS formation was investigated. For that purpose, calibration curves were obtained for bovine serum albumin (BSA) in the presence of different concentrations of sulfates (Na2SO4, (NH4)2SO4, MgSO4 and LiSO4), phosphates (Na2HPO4, NaH2PO4, K2HPO4 and KH2PO4), citrates (citric acid and trisodium citrate), acetates (acetic acid and sodium acetate) and polymers (PES Mw=100000, UCON Mw=3900, Ficoll Mw=70000 and PVP Mw=40000). Comparison of these curves with that obtained for BSA in distilled water, allow us to conclude that salts produce a more significant effect in BSA calibration curve than polymers. In all cases, a reduction in the absorbance at 595 nm (A595) was observed. For most of the salts tested, reduction ≥20% was obtained in A595 when a concentration ≥1% (w/w) was present. The only exceptions were observed for the salts NaH2PO4, KH2PO4, citric acid and acetic acid that, probably due to their acidic pH, presented reductions in the A595 lower than 12% for 1% (w/w). For sulfates, the change of the cation also provided a change in the calibration curve and (NH4)2SO4 showed the higher interference (A595 reduction of 25% for a concentration of 1% (w/w)). Fig. 1 shows the reduction in A595 in the presence of different (NH4)2SO4 concentrations.

Six phase diagrams for UCON−phosphate salt aqueous two-phase systems (ATPSs), at 23 °C, are presented. The ATPSs were obtained combining UCON (a random copolymer of 50 % ethylene oxide and 50 % propylene oxide) and a phosphate salt (KH2PO4, K2HPO4, potassium phosphate buffer pH = 7, NaH2PO4, Na2HPO4, and sodium phosphate buffer pH = 7). Among the salts used, Na2HPO4 proved to be the most effective in ATPS formation, providing the greatest heterogeneous region in the phase diagram. The previous established relation between the anion valence and the concentration needed to form a biphasic system was confirmed: higher valence requires lower concentration. Na+ salts require, in all cases, lower concentration to form ATPSs than the corresponding K+ salts. The size of the heterogeneous region in the phase diagrams increases with an increase of the pH value of the salt used in ATPS preparation., Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/43439/2008, Fundo Europeu de Desenvolvimento Regional (FEDER) - FEDER/POCI/2010

The partition coefficients for several solutes (five nitrophenylated-monosaccharides and four proteins) were experimentally determined, at 23 ◦C, in three different tie-lines of two polymer–salt aqueous twophase systems (ATPS): UCON-K2HPO4 and UCON-NaH2PO4. These partition coefficients together with others obtained from the literature for five dinitrophenylated-amino acids were used to investigate the suitability of the Collander equation to correlate partition coefficients in polymer–salt ATPS. This equation was first proposed to describe the linear correlation between partition coefficients of solutes in different water–organic solvent systems. More recently, it was proved that partition coefficients for several biomolecules in polymer–polymer ATPS can also be correlated with this equation. In this work, several correlations were tested: partition coefficients obtained for different tie-lines within the same system and also partition coefficients obtained from different systems. In both cases, a linear relation was observed, despite a less satisfactory correlation was found when different ATPS were compared. Overall, it was demonstrated that the Collander equation can be used to satisfactorily correlate solute partitioning in the studied polymer–salt ATPS., FEDER., Fundação para a Ciência e a Tecnologia (FCT)