BBr3-assisted cleavage of most ethers does not follow the commonly assumed mechanism

Density‐functional computations were used to probe the reaction mechanism of BBr3‐assisted ether cleavage. After the initial formation of an ether–BBr3 adduct, secondary and tertiary alkyl ethers are cleaved through Br– transfer from the activated BBr3 to the alkyl moiety, as postulated in the literature. In contrast, all other ethers studied react through a novel pathway involving two ether–BBr3 adducts, one of which acts as Br– donor, and the second as the reaction substrate. The identification of the novel bimolecular mechanism for this classical reaction has further applications, because it implies that BBr3‐assisted ether cleavage may become impossible if the ether is surrounded by bulky portions of the molecule that prevent the approach of the attacking BBr3 adduct. Our data also allow the construction of an order of reactivity of alkyl ether deprotection: isopropyl, benzyl, tertiary alkyl, allyl, isobutyl and ethyl can be removed sequentially as their bromo derivatives; phenyl, cyanomethyl and chloromethyl groups can be sequentially removed as their corresponding alcohols.info:eu-repo/semantics/publishedVersio

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 24 full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL−1 of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor

Alkali and acid polysaccharides blend nanofibrous membranes prepared by electrospinning

Poster apresentado no "Smart and functional coatings conference", Torino, Italy, 2013Electrospinning allows the production of polymer fibres with diameters in the sub-micron size range, through the application of an external electric field, keeping intact the bulk properties of the polymers. Electrospun membranes possess some unique structural features, such as a high surface to volume ratio and very good mechanical performance, properties that are determinant to their use in several applications such as air and liquid filtration, tissue engineering, optical and chemical sensors [1]. In this work, alkali and acid biopolysaccharides blended with polyvinyl alcohol (PVA) were electrospinned into a polyvinylidene difluoride (PVDF) basal microfiltration membrane, with the goal of developing a mid-layer nanofibrous porous support for exploitable thin-film composite (TFC) membranes for water filtration. The alkali and acid biopolysaccharides chosen were, respectively, chitosan (CS), a cationic polyelectrolyte (in this case with deacetylation degree around 85), and cyanobacterial extracellular polymeric substances (EPS), an acidic polysaccharide isolated from Cyanothece sp.CCY 0110 [2]. The electrospun blended nanofibrous membranes were fully characterized in order to investigate their morphology, diameter, structure, mechanical and thermal properties. The results showed that these membranes have great potential for filtration purposes [3].This work was funded by FEDER funds through the Operational Competitiveness Programme – COMPETE and by National Funds through FCT – Fundação para a Ciência e a Tecnologia under the projects FCOMP-01-0124-FEDER-022718 (PEst-C/SAU/LA0002/2011), FCOMP-01-0124-FEDER-009389 (PTDC/CTM/100627/2008) and FCOMP-01-0124-FEDER-009697 (PTDC/EBB-EBI/099662/2008), and the grants SFRH/BPD/37045/2007 and SFRH/BPD/72400/2010. The authors also thank to the project INVISIBLE NETWORK nº. 13857 * SI I&DT Mobilizador

Immobilization of proteases with a water soluble–insoluble reversible polymer for treatment of wool

A commercial protease, Esperase, was covalently linked to Eudragit S-100, a reversible soluble–insoluble polymer by carbodiimide coupling. When compared to the native enzyme, the immobilized form presented a lower specific activity towards high molecular weight substrates but a higher thermal stability at all temperatures tested. The optimum pH of the immobilized protease was shifted towards the alkaline side by about one pH unit while there was no change in optimum temperature between the free and immobilized protease. The immobilized protease exhibited a good storage stability and re-usability. Enzymatic treatment of wool using proteases has been investigated for wool shrink-resist finishing. It was found that using the immobilized protease in the enzymatic treatment of wool there was a reduction of weight and fibre tensile strength loss because the proteolytic attack is only limited to the cuticle surfaces of wool fibres. This novel approach is a promising alternative for wool shrink-resist finishing to replace the conventional chlorine treatments. This environmentally friendly bioprocess needs to be further characterized to a complete understanding and optimization

Preparation and characterization of polysaccharides/PVA blend nanofibrous membranes prepared by electrospinning

Apresentação efetuada no Smart & Funcional Coatings–European Conference Turin, 26th September 2013A polyvinyl alcohol (PVA) / cyanobacterial EPS blend nanofibrous membranes were fabricated by electrospinning using polyvinylidene fluoride (PVDF) as a basal membrane, in order to obtain thin-layer composite nanofiltration membranes. The production of the nanofibers using EPS and PVA as plasticizer in different ratios was produced in a NF-103 MECC Nanon electrospinning equipment with an applied electric field between 15 and 25 kV and a flow of 0,2 mL/h. Morphological, mechanical, chemical and thermal characterization of the electrospun fibers deposited on the basal membranes, were evaluated by atomic force microscopy (AFM), scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), dynamical and mechanical analysis (DMA), thermogravimetry (TGA) and differential scanning calorimetry (DSC). The AFM and SEM results show the presence of fibers with dimensions between 54 and 121 nm with low bead formation. In the EDS analysis presence of sulfur elements was observed confirming the inclusion of EPS in the nanofibers. The morphology and diameter of the nanofibers were mainly affected by the concentration of the blend solution and the weight ratio of the blend, respectively. The best PVA/EPS nanofibers were achieved in a ratio of 12 % PVA and 0.4 % EPS. The solution conductivity was ranging 1500 to 3500 μS/cm with a viscosity of about 100 to 500 cP. The DMA results confirmed the miscibility of PVA/EPS blends. The elastic modulus of the nanocomposite mats increased significantly as a consequence of the reinforcing effect of EPS. Thermal and mechanical analysis demonstrated that there were strong intermolecular hydrogen bonds between the molecules EPS-PVA in the blends. The heat-treated electrospun blended membranes showed better tensile mechanical properties when compared with PVA alone, and resisted more against disintegration. Dead-­end filtration of a standard solutionof hexavalent chromium (2mg/mL) prepared using potassium dichromate (K2Cr2O7). Increase in chromium binding capacity of ~5% in PVA/polysaccharide blended membranes

Enzymatic treatment of wool with modified proteases

Tese de doutoramento em Engenharia Têxtil.The tendency of wool to felt and shrink is mainly due to its scaly structure. The chlorine-Hercosett is the most widespread process used to modify the scales of wool fibres with the purpose of providing resistance to felting and shrinkage. There have been many attempts to replace this chlorine process by an environmental friendly enzymatic process that wouid similarly degrade the scales. However, although proteases are large molecuies, their attack is not oniy Iimited to the scales; they penetrate inside the fibre causing unacceptabie weight and strength ioss. lt is believed that if the proteases are chemicaliy modified in order to increase their molecular weight, then they will act just on the surface of the fibres, thus providing wool with anti-shrinking behaviour, which is the main idea of this research. In this work the screening of the attack of the proteoiytic enzymes inside the fibre was made by means of several techniques. Among them, special attention was paid to the study of the protein adsorption inside the wool fibres. It was demonstrated that the penetration of protein (measured as the maximum adsorption capacity in g protein/g wool) was higher when the wool was previously subjected to a surfactant washing and bleaching. Furthermore, it was observed that the diffusion of the proteases into wool was dependent on their size. The free enzyme penetrated into wool fibre cortex while the modified enzyme, with a bigger size, was retained at the surface, in the cuticle layer. lt was also confirmed that the diffusion of proteases was facilitated by the hydrolytic action. Scanning electron microphotographs were also used to observe the intensity of the proteolytic attack. Some techniques of increasing the proteases molecular weight were attempted, namely the covalent crosslinking method using the bifunctional reagent glutaraldehyde. It was observed that the Iow amount of free Iysine residues available in the protease for crosslinking was affecting the process A more successful technique was attained by covalently coupling the enzyme to a soluble-insoluble polymer of high molecular weight. An enzyme conjugated to such a carrier may be used as a catalyst in its soluble form and then be recovered via the insoluble state. Moreover, this system overcomes the problem of the non accessibility of the enzyme to the macromolecular substrate, wool, whilst in the soluble state. When comparing to the native enzyme, the immobilized form presented a lower specific activity towards high molecular weight substrates but a higher thermal stability at ali temperatures tested. lt also exhibited a good storage stability and reusability, which makes this enzyme conjugate quite interesting from an industrial point of view. Wool fabrics were treated with the immobilized serine protease using harsh conditions and subjected subsequently to several machine washings, after which they presented a significant Iower weight loss than wool treated with the native enzyme, in the same conditions. Using a moderate enzymatic treatment, a reduction to about half of the initial area shrinkage was attained, both for free and immobilized enzymes. However, the immobilized Esperase presented 92% of the original tensile strength resistance while native Esperase kept only 75% of its initial resistance. The coupling of the protease to the polymer, Eudragit S-100, was optimized by using experimental design techniques. This optimization strategy allowed for an enzyme conjugate wherein the enzyme was covalently crosslinked to the polymer, with high activity yield and high operational stability at 6000. Ali these results prove that modified proteases attained by this immobilization method, using a soluble-insoluble polymer of high molecular weight, can be a promising alternative for wool bio-finishing processes at an industrial levei, since it is an effective way of removing wool scales and can be an environmental friendly option to the conventional chiorine treatments. This process needs to be further characterized for its complete understanding and optimization.A tendência da lã para feltrar e encolher é devida, principalmente, à sua estrutura em escamas. O tratamento anti-feltragem normalmente utilizado para modificar as escamas das fibras de lã utiliza cloro, pelo que, várias tentativas têm sido levadas a cabo para substituir este processo por um processo enzimático amigo do ambiente. Estes processos recorrem à utilização de proteases que, apesar do seu tamanho, atacam não só a cutícula mas também penetram rapidamente dentro da fibra, provocando perdas de peso e resistência nas fibras, inaceitáveis do ponto de vista comercial. Assim sendo, este trabalho teve como principal objectivo o desenvolvimento de técnicas que permitissem modificar as proteases, aumentando o seu peso molecular e restringindo, deste modo, o seu ataque à superfície da fibra, removendo apenas a cutícula. Várias técnicas foram utilizadas para a monitorização do ataque das enzimas proteolíticas ao interior da fibra, como por exemplo a adsorção de proteína nas fibras. Verificou-se que a penetração de proteína (medida como a máxima capacidade de adsorção em g proteina/g lã) foi superior quando a lã foi sujeita a um pré-tratamento de lavagem alcalina com surfactante e posterior branqueamento. Foi ainda constatado que a difusão das proteases na lã depende do seu tamanho. A enzima livre penetrou no córtex da fibra de lã enquanto que a enzima modificada, de maior tamanho, ficou retida à superfície, na cutícula. Foi também confirmado que a difusão das proteases no interior da fibra foi facilitada pela sua acção hidrolítica. A técnica de microscopia electrónica foi também utilizada para observar a intensidade do ataque proteolitico. Para aumentar o peso molecular das proteases foram tentadas algumas estratégias, entre as quais o método de ligação covalente ao glutaraldeldo, um reagente bifuncional. Demonstrou-se que o baixo teor em resíduos usina da protease, disponíveis para ligação, afecta o processo. Uma técnica mais eficaz foi a ligação covalente da enzima a um polímero solúvel-insolúvel de elevado peso molecular. De facto, as enzimas ligadas a este tipo de suportes podem ser utilizadas como catalisadores na sua forma solúvel sendo posteriormente recuperadas no seu estado insolúvel. Para além disso, este tipo de imobilização contorna os problemas da não acessibilidade da enzima ao seu substrato macromolecular, a lã. A enzima imobilizada, quando comparada com a enzima nativa, apresentou uma actividade específica menor para substratos de elevado peso molecular, mas uma estabilidade térmica superior, a todas as temperaturas testadas. A protease imobilizada no polímero entérico exibiu ainda uma boa estabilidade de armazenamento e boa reutilização, o que torna este conjugado enzimático muito interessante do ponto de vista industrial. Foram tratados tecidos de pura lã em condições severas com a protease imobilizada, que foram depois sujeitos a várias lavagens domésticas à máquina. Os tecidos tratados com a enzima modificada apresentaram uma menor perda de peso do que a lã tratada com a enzima nativa, nas mesmas condições. Usando um tratamento enzimático moderado, verificou-se uma redução para cerca de metade no encolhimento da lã, para ambas as enzimas. No entanto, a Esperase imobilizada conservou cerca de 92% da sua resistência à tracção enquanto a Esperase nativa apresentou apenas 75% da sua resistência original. O processo de acoplamento da protease ao polímero, Eudragit S-100, foi optimizado usando técnicas de desenho experimental. Esta estratégia de optimização permitiu obter um conjugado no qual a enzima se encontra covalentemente ligada ao polímero, com elevado rendimento em actividade e uma alta estabilidade operacional a 60°C. Os resultados obtidos mostram que as proteases modificadas pelo método de imobilização descrito neste trabalho, (usando um polimero solúvel-insolúvel de elevada massa molecular), podem ser uma alternativa promissora para os processos de bio-acabamento da lã, uma vez que, constituem um modo eficaz de remover as escamas da lã, podendo ser uma opção ambientalmente aceite para substituir os tratamentos convencionais com cloro. Este processo necessita, no entanto, de ser melhor caracterizado para a sua completa compreensão e optimização.Fundação para a Ciência e Tecnologia (FCT)

Impact of brewery wastewater inhibitors in pure and mixed cultures of the yeast Rhodosporidium toruloides NCYC 921 and the microalga Tetradesmus obliquus ACOI 204/07

ABSTRACT: Brewery wastewater (BWW) is an appealing low-cost substrate for the production of single cell oils by oleaginous microorganisms. However, it may contain inhibitor compounds that may affect the microbial metabolism. This work investigated, for the first time, the presence of potential inhibitor compounds in primary brewery wastewater (PBWW) and secondary brewery wastewater (SBWW) for the pure and mixed cultivation of the yeast Rhodosporidium toruloides NCYC 921 and the microalga Tetradesmus obliquus ACOI 204/07. Three organic acids (OrgAc) were identified in the brewery effluents (acetic, propionic and butyric acids). Yeast and microalga pure and mixed cultivations were performed in PBWW and SBWW in order to understand the behaviour of the microorganisms, individually and together. Flow cytometry (FC) was used to monitor each microbial population during the mixed cultivations, and to study the yeast and microalga cell viability throughout all cultivations. The yeast cells in pure cultures grown in both effluents were severely affected by the OrgAc presence confirmed by the cell stress results obtained by FC. However, in the mixed cultures, the yeast cells were able to develop, and the levels of stress conditions were considerably lower. Only in microalga pure and mixed cultures efficient OrgAc removal was observed.info:eu-repo/semantics/publishedVersio

Effect of some pocess parameters in enzymatic dyeing of wool

This article reports on the dyeing of wool using an enzymatic system comprising laccase; dye precursor, 2,5- iaminobenzenesulfonic acid; and dye modifiers, catechol and resorcinol. Enzymatic dyeing was performed as a batchwise process at the temperature and pH of maximum enzyme activity. The effects of the process variables reaction time, enzyme, and modifier concentration on fabric color were studied, according to an appropriate experimental design. Different hues and depths of shades could be achieved by varying the concentration of the modifiers and the time of laccase treatment. The duration of the enzymatic reaction appeared to be the most important factor in the dyeing process. Thus, the dyeing process, performed at low temperature and mild pH, was advantageous in terms of reduced enzyme and chemical dosage

Chemical Modifications on Proteins Using Glutaraldehyde

In this work the effect of crosslinking the enzyme esperase (E.C. 3.4.21.62) and the proteins bovine serum albumin and casein with the bifunctional compound glutaraldehyde on molecular mass increase was studied. Two common techniques for measuring molecular mass of proteins were used: SEC and SDS-PAGE. These techniques revealed that the proteins bovine albumin and casein, when subjected to chemical crosslinking with glutaraldehyde, volume fraction 0.25 %, increased their molecular mass by 20- and 40-fold, respectively. It was also observed that Mr increased proportionally to the increase of glutaraldehyde concentration in the solution, and that the addition of glutaraldehyde should be done slowly, in small amounts, in order to attain bigger protein aggregates. When the proteolytic enzyme esperase was subjected to glutaraldehyde, no increase in its Mr was achieved. Several assumptions can be made to explain these results, the most reasonable being the low amount of free lysine groups available for crosslinking. This study confirms that glutaraldehyde is not an adequate crosslinker for esperase

Concomitant wastewater treatment with lipid and carotenoid production by the oleaginous yeast Rhodosporidium toruloides grown on brewery effluent enriched with sugarcane molasses and urea

ABSTRACT: In this study, secondary brewery wastewater (SBWW) supplemented with sugarcane molasses (SCM) was used for SBWW treatment with concomitant lipid and carotenoid production by the yeast Rhodosporidium toruloides NCYC 921. In order to improve the biomass production, ammonium sulfate, yeast extract and urea were tested as nitrogen sources. Urea was chosen as the best low-cost nitrogen source. A fed-batch cultivation was carried out with SBWW supplemented with 10 g L−1 of sugarcane molasses as carbon source, and 2 g L−1 of urea as nitrogen source. A maximum biomass concentration of 42.5 g L−1 was obtained at t=126.5 h and the maximum biomass productivity was 0.55 g L−1 h−1 at t=48.25 h. The maximum lipid content was 29.9 % w/w (DCW) at t=94 h of cultivation and the maximum carotenoid content was 0.23 mg g−1 at 120 h of cultivation. Relatively to the SBWW treatment, after the batch phase, 45.8 % of total Kjeldahl nitrogen removal, 81.7 % of COD removal and 100 % of sugar consumption were observed. Flow cytometry analysis revealed that 27.27 % of the cells had injured membrane after the inoculation. This proportion was reduced to 10.37 % at the end of the cultivation, indicating that cells adapted to the growth conditions.info:eu-repo/semantics/publishedVersio