6 research outputs found
Single-batch, homogeneous phase depolymerization of cellulose catalyzed by a monocomponent endocellulase in ionic liquid [BMIM][Cl]
No full textThe stability and enzymatic activity of an industrial, recombinant cellulase dissolved either in 1-butyl-3-methylimidazolium chloride [BMIM][Cl] or in mixtures of [BMIM][Cl]/aqueous buffer have been investigated and the results are here reported. The preparation used was a recombinant monocomponent endocellulase from Trichoderma reesei or EGIII (now renamed Cel12A), commercially referred to as IndiAge® Super GX Plus. The key parameters studied were: the effect of temperature in the range between 75 and 90 °C, the enzyme stability at 75 °C and the ability of EGIII to hydrolyze cellulose in the presence of [BMIM][Cl]. This cellulase preparation turns out to be more stable and active in pure ionic liquid rather than in buffer. These results indicate that the recombinant, monocomponent endocellulase from T. reesei is a suitable biocatalyst for the depolymerization of cellulose in [BMIM][Cl] and it therefore opens the way to a possible one-pot process based on a homogeneous phase, enzyme-catalyzed depolymerization of cellulose
Absence of microsatellite instability in breast carcinomas with both p53 and c-erbB-2 alterations
No full text3D Structure of Sulfolobus solfataricus Carboxypeptidase Developed by Molecular Modeling is Confirmed by Site-Directed Mutagenesis and Small Angle X-Ray Scattering
No full textAbstractSulfolobus solfataricus carboxypeptidase (CPSso) is a thermostable zinc-metalloenzyme with a Mr of 43,000. Taking into account the experimentally determined zinc content of one ion per subunit, we developed two alternative 3D models, starting from the available structures of Thermoactinomyces vulgaris carboxypeptidase (Model A) and Pseudomonas carboxypeptidase G2 (Model B). The former enzyme is monomeric and has one metal ion in the active site, while the latter is dimeric and has two bound zinc ions. The two models were computed by exploiting the structural alignment of the one zinc- with the two zinc-containing active sites of the two templates, and with a threading procedure. Both computed structures resembled the respective template, with only one bound zinc with tetrahedric coordination in the active site. With these models, two different quaternary structures can be modeled: one using Model A with a hexameric symmetry, the other from Model B with a tetrameric symmetry. Mutagenesis experiments directed toward the residues putatively involved in metal chelation in either of the models disproved Model A and supported Model B, in which the metal-binding site comprises His108, Asp109, and His168. We also identified Glu142 as the acidic residue interacting with the water molecule occupying the fourth chelation site. Furthermore, the overall fold and the oligomeric structure of the molecule was validated by small angle x-ray scattering (SAXS). An ab initio original approach was used to reconstruct the shape of the CPSso in solution from the experimental curves. The results clearly support a tetrameric structure. The Monte Carlo method was then used to compare the crystallographic coordinates of the possible quaternary structures for CPSso with the SAXS profiles. The fitting procedure showed that only the model built using the Pseudomonas carboxypeptidase G2 structure as a template fitted the experimental data
Studies on One-Pot Enzymatic Depolymerization of Cellulose in Ionic Liquids
No full textLignocellulosic biomass is a renewable, low cost, non-food and abundant feedstock for biofuel production and a source of C atoms for chemistry alternative to fossil resources. The most important factor hampering massive exploitation of cellulose, the main component of plant biomass, is its well known low reactivity and recalcitrance towards chemical processing. The usual steps in producing biofuels from cellulosic sources are: pre-treatment followed by cellulose precipitation, enzymatic hydrolysis and fermentation. A possible greener alternative for the activation of cellulose fibrilles towards facile hydrolysis and/or derivatization passes through an emerging chemical pre-treatment step using ionic liquids (ILs) [1]. Unlikely, the more common cellulases have been reported to be inactivated by ILs [2].
In this work we describe a single-batch, homogeneous phase enzymatic depolymerization of cellulose catalyzed by a commercial cellulase (EC 3.2.1.4) in the presence of different ILs.
We used the industrial enzymatic preparation IndiAge® Super GX Plus (a monocomponent endoglucanase EGIII (Cel12A) from Trichoderma reesei produced by Genencor presently a subsidiary of Dupont). This enzyme already showed exceedingly good performance for the depolymerization of dissolved cellulose in [BMIM][Cl] without the usual pre-treatment, known as dissolution-regeneration of cellulose in its amorphous form [3].
After these promising results, we have extended our screening towards two more friendly ionic liquids such as 1-ethyl-3-methylimidazolium acetate ([EMIM][Ac]) and diethylphosphate ([EMIM][DEP]) and the present study presents a useful comparison of the effect on stability and activity of IndiAge® Super GX Plus in these media which could be amenable of scale-up and innovative industrial applications.
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[1] Dadi, A.P.; Varanasi, S.; Schall, C.A. Biotechnol. Bioeng., 2006, 95 (5), 904-910.
[2] Turner, M.B.; Spear, S.K.; Huddleston, J.G.; Holbrey, J.D.; Rogers, R.D. Green Chem., 2003, 5(4), 443-447.
[3] D’Arrigo. P.; Allegretti, C.; Tamborini, S.; Formantici, C.; Galante, Y.; Pollegioni, L.; Mele, A. J. Mol. Cat. B, 2014, 106, 76-80
Enzyme resistance and biostability of hydroxyalkylated cellulose and galactomannan as thickeners in waterborne paints
No full textAvaliação dos efeitos da aplicação da enzima celulase nas propriedades de sustratos têxteis de algodão
No full textTese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em Engenharia Quimica.Na indústria têxtil as operações convencionais de preparação, tingimento e acabamento são responsáveis pela geração de uma grande quantidade de carga poluidora, especialmente pelo elevado consumo de substâncias químicas como álcalis e agentes tensoativos. Para minimizar estes problemas tem-se aplicado tratamentos enzimáticos, os quais são seletivos à ação sobre componentes químicos específicos e ocorrem a baixas temperaturas, na faixa de 60°C. A celulase é uma das enzimas mais utilizadas na indústria têxtil, principalmente nos processos de preparação e acabamento, em especial no biopolimento. No presente trabalho foram investigadas diferentes condições de empacotamento das fibras de algodão, assim como determinada a influência de diferentes pré-tratamentos sobre a ação da celulase. Verificou-se que a escolha adequada do pré-tratamento pode ser uma possível rota para promover o ataque da enzima em situações onde o mesmo é desfavorável, devido ao aumento da resistência por efeitos de empacotamento. Foram investigadas diferentes meadas de fios (OE 12/1, OE 14/1, OE 16/1, penteado 13/1 e cardado 16/1), verificando-se um aumento da ação enzimática após o tratamento com vapor, onde o fio penteado 13/1 (0,98 µmol.mL-1 de Açúcares Redutores Totais (ART) liberados) apresentou uma maior ação da enzima, seguido pelo fio cardado 16/1 (0,94 µmol.mL-1 de ART liberados). Para os fios OE 12/1 e OE 14/1, a condição de maior atuação enzimática, medida pela formação de ART, foi na ausência de pré-tratamento, onde se observou uma liberação de ART após 60 min. de reação de 0,86 e 0,95 µmol.mL-1, respectivamente. A avaliação do biopolimento em bobinas mostrou que a condição que apresentou menor pilosidade, alongamento, força máxima de ruptura, torção, grau de polimerização e a maior concentração de ART liberados foi para o fio OE 16/1 pré-tratado com vapor por 15 minutos e biopolido com celulase (Quimilase BP). Observou-se encolhimento em todas as amostras de fios analisadas, sendo o maior encolhimento observado no branco do fio penteado 13/1 pré-tratado com Inchamento 24 horas, seguido pelas amostras de branco sem pré-tratamento dos fios OE 14/1 e penteado 13/1. Nas análises dos cordéis observou-se que os tratamentos enzimáticos precedidos por pré-tratamentos de vapor para os fios 24/1 auxiliam na minimização dos efeitos de variação dimensional em comparação aos não tratados. Na avaliação do DP dos cordéis (cardado e penteado) observou-se que cordéis de título 24/1 apresentaram maior grau de polimerização que os 30/1. A análise estatística dos resultados dos cordéis mostrou que o maior ataque enzimático foi obtido quando se tratou com Quimilase BP o cordel de fio cardado 30/1 pré-tratado com vapor 15 minutos Foi observada uma influência do grau de empacotamento da fibra, sendo que de acordo com as condições de pré-tratamento dos substratos podem-se obter diferentes acessibilidades da enzima à fibra, mostrando assim a importância em se adaptar as condições de biopolimento de acordo com os substratos e processos.In the textile industry, conventional operations such as cleaning, dyeing and finishing are responsible for generating a large amount of pollutant, especially due to a high use of chemical substances such as alkalis and surfactant. To minimize these problems has been applied enzymatic treatments, which are selective for action on specific chemical components and occur at low temperatures in the range of 60 ° C. One of the most widely used enzymes in the textile industry is cellulase which acts mainly in the preparation and finishing, especially in the biopolishing. In the present work we investigated different packing conditions of cotton fibers, and determined the influence of different pretreatments on the action of cellulase. It was found that choosing the appropriate pretreatment may be in a possible route to promote the attack of the enzyme in situations where it is disadvantageous due to increased resistance to the effects of packaging. We investigated different yarn, OE 12/1, OE 14/1, OE 16/1, Combed 13/1 and Carded 16/1, and found an increase in the enzymatic activity after treatment with steam, where the Combed yarn 13/1 (0.98 µmol.mL-1 glucose released) presented an increase in the enzyme action, followed by Carded yarn 16/1 (approximately 0.94 µmol.mL-1 glucose released). For the OE 12/1 and OE 14/1 yarn, the condition of higher enzyme activity, measured by the formation of glucose, was in the absence of pre-treatment, where there was a release of glucose in the reaction medium after 60 min. of 0.86 and 0.95 µmol.mL-1, respectively. The evaluation of the biopolishing in bobbin showed wich the condition with the lowest hairiness, elongation, force of rupture, torsion, degree of polymerization and the largest concentration of glucose was released to the yarn OE 16 / 1 pre-treated with steam for 15 minutes and biopolish with cellulase (Quimilase BP). Shrinkage was observed in all yarn samples analyzed, and the greater shrinkage observed in white combed yarn 13/1 pre-treated with swell 24 hours, with the blank samples without pre-treatment of OE yarn 14/1 and combed yarn 13/1. In the analysis of the twine was observed that the enzymatic treatments preceded by pre-treatments of steam to yarns 24/1 was helped in minimizing the effects of dimensional variation compared to the untreated. In the analysis of the degree of polymerization (DP) of the twine (carded and combed) was observed that twines with title 24/1 had a higher DP that the 30/1. The statistical analysis of the twines results showed that the greater enzymatic attack was obtained when the twine of carded yarn 30/1 was treated with Quimilase BP and pre-treated with steam 15 minutes. We observed an influence of the fiber degree of packaging, and according to the conditions of substrates pre-treatment, can obtain different enzyme accessibility of the fiber thus showing the importance to adapt the biopolishing conditions according with the substrates and processes
