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Richard A. Gross - One of the best experts on this subject based on the ideXlab platform.
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immobilized Cutinases preparation solvent tolerance and thermal stability
Enzyme and Microbial Technology, 2018Co-Authors: Abhijit N. Shirke, Joshua Baik, Yi Zou, Richard A. GrossAbstract:Abstract Developing active immobilized enzymes and characterization of their use conditions is critically important prior to initiating studies of enzyme selectivity and substrate specificity in organic media. To this end, physical immobilization by hydrophobic interactions was performed with three well-characterized Cutinases (Aspergillus oryzae Cutinase (AoC), Humicola insolens Cutinase (HiC), and Thielavia terrestris Cutinase (TtC)) using Lewatit VP OC 1600 as the macroporous support. We found that immobilization yields >98% were achieved for all three Cutinases under the following immobilization conditions: 100 mg/g loading ratio, immobilization buffers of 100 mM phosphate pH 8 (AoC and HiC) and 100 mM acetate pH 5 (TtC), mixing at 150 rpm and 30 °C for 24 h. Among the three Cutinases, HiC has the highest tolerance towards solvents of increased polarity while TtC has the highest thermal stability (up to 80 °C) in a bulk reaction system that consists of the reactants butanol and lauric acid. In nonane, these Cutinases retain >64% of their activity at 90 °C. Furthermore, kinetic stability (residual activity as a function of time) analysis reveals that the Cutinases retain >75% residual activity at 70 °C in 3 h. Moreover, at 80 °C, the kinetic stability of TtC is higher than that of HiC and AoC. Collectively, the results herein set the stage for the in-depth evaluation of these catalysts for selective transformations in organic media.
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Stabilizing Leaf and Branch Compost Cutinase (LCC) with Glycosylation: Mechanism and Effect on PET Hydrolysis
2018Co-Authors: Abhijit N. Shirke, Glenn L. Butterfoss, Christine White, Jacob A. Englaender, Allison Zwarycz, Robert J. Linhardt, Richard A. GrossAbstract:Cutinases are polyester hydrolases that show a remarkable capability to hydrolyze polyethylene terephthalate (PET) to its monomeric units. This revelation has stimulated research aimed at developing sustainable and green Cutinase-catalyzed PET recycling methods. Leaf and branch compost Cutinase (LCC) is particularly suited toward these ends given its relatively high PET hydrolysis activity and thermostability. Any practical enzymatic PET recycling application will require that the protein have kinetic stability at or above the PET glass transition temperature (Tg, i.e., 70 °C). This paper elucidates the thermodynamics and kinetics of LCC conformational and colloidal stability. Aggregation emerged as a major contributor that reduces LCC kinetic stability. In its native state, LCC is prone to aggregation owing to electrostatic interactions. Further, with increasing temperature, perturbation of LCC’s tertiary structure and corresponding exposure of hydrophobic domains leads to rapid aggregation. Glycosylation was employed in an attempt to impede LCC aggregation. Owing to the presence of three putative N-glycosylation sites, expression of native LCC in Pichia pastoris resulted in the production of glycosylated LCC (LCC-G). LCC-G showed improved stability to native state aggregation while increasing the temperature for thermal induced aggregation by 10 °C. Furthermore, stabilization against thermal aggregation resulted in improved catalytic PET hydrolysis both at its optimum temperature and concentration
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Comparative thermal inactivation analysis of Aspergillus oryzae and Thiellavia terrestris Cutinase: Role of glycosylation.
Biotechnology and bioengineering, 2016Co-Authors: Abhijit N. Shirke, J. Andrew Jones, Glenn L. Butterfoss, Mattheos A. G. Koffas, Jin Ryoun Kim, Richard A. GrossAbstract:Cutinase thermostability is important so that the enzymes can function above the glass transition of what are often rigid polymer substrates. A detailed thermal inactivation analysis was performed for two well-characterized Cutinases, Aspergillus oryzae Cutinase (AoC) and Thiellavia terrestris Cutinase (TtC). Both AoC and TtC are prone to thermal aggregation upon unfolding at high temperature, which was found to be a major reason for irreversible loss of enzyme activity. Our study demonstrates that glycosylation stabilizes TtC expressed in Pichia pastoris by inhibiting its thermal aggregation. Based on the comparative thermal inactivation analyses of non-glycosylated AoC, glycosylated (TtC-G), and non-glycosylated TtC (TtC-NG), a unified model for thermal inactivation is proposed that accounts for thermal aggregation and may be applicable to other Cutinase homologues. Inspired by glycosylated TtC, we successfully employed glycosylation site engineering to inhibit AoC thermal aggregation. Indeed, the inhibition of thermal aggregation by AoC glycosylation was greater than that achieved by conventional use of trehalose under a typical condition. Collectively, this study demonstrates the excellent potential of implementing glycosylation site engineering for thermal aggregation inhibition, which is one of the most common reasons for the irreversible thermal inactivation of Cutinases and many proteins. Biotechnol. Bioeng. 2017;114: 63-73. © 2016 Wiley Periodicals, Inc.
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Influence of surface charge, binding site residues and glycosylation on Thielavia terrestris Cutinase biochemical characteristics
Applied Microbiology and Biotechnology, 2016Co-Authors: Abhijit N. Shirke, Glenn L. Butterfoss, Danielle Basore, Christopher Bystroff, Samantha Holton, Evan Baugh, George Makhatadze, Richard A. GrossAbstract:Cutinases are esterases of industrial importance for applications in recycling and surface modification of polyesters. The Cutinase from Thielavia terrestris (TtC) is distinct in terms of its ability to retain its stability and activity in acidic pH. Stability and activity in acidic pHs are desirable for esterases as the pH of the reaction tends to go down with the generation of acid. The pH stability and activity are governed by the charged state of the residues involved in catalysis or in substrate binding. In this study, we performed the detailed structural and biochemical characterization of TtC coupled with surface charge analysis to understand its acidic tolerance. The stability of TtC in acidic pH was rationalized by evaluating the contribution of charge interactions to the Gibbs free energy of unfolding at varying pHs. The activity of TtC was found to be limited by substrate binding affinity, which is a function of the surface charge. Additionally, the presence of glycosylation affects the biochemical characteristics of TtC owing to steric interactions with residues involved in substrate binding.
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toward rational thermostabilization of aspergillus oryzae Cutinase insights into catalytic and structural stability
Proteins, 2016Co-Authors: Abhijit N. Shirke, Glenn L. Butterfoss, Danielle Basore, Richard Bonneau, Christopher Bystroff, Richard A. GrossAbstract:Cutinases are powerful hydrolases that can cleave ester bonds of polyesters such as poly(ethylene terephthalate) (PET), opening up new options for enzymatic routes for polymer recycling and surface modification reactions. Cutinase from Aspergillus oryzae (AoC) is promising owing to the presence of an extended groove near the catalytic triad which is important for the orientation of polymeric chains. However, the catalytic efficiency of AoC on rigid polymers like PET is limited by its low thermostability; as it is essential to work at or over the glass transition temperature (Tg) of PET, that is, 70 °C. Consequently, in this study we worked toward the thermostabilization of AoC. Use of Rosetta computational protein design software in conjunction with rational design led to a 6 °C improvement in the thermal unfolding temperature (Tm) and a 10-fold increase in the half-life of the enzyme activity at 60 °C. Surprisingly, thermostabilization did not improve the rate or temperature optimum of enzyme activity. Three notable findings are presented as steps toward designing more thermophilic Cutinase: (a) surface salt bridge optimization produced enthalpic stabilization, (b) mutations to proline reduced the entropy loss upon folding, and (c) the lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the Tm of the global structure.
Christian Cambillau - One of the best experts on this subject based on the ideXlab platform.
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a Cutinase from trichoderma reesei with a lid covered active site and kinetic properties of true lipases
Journal of Molecular Biology, 2014Co-Authors: Alain Roussel, Christian Cambillau, Antti Nyyssölä, Frederic Carriere, Sawsan Amara, Eduardo Mateosdiaz, Stephanie Blangy, Hanna Kontkanen, Ann WesterholmparvinenAbstract:Cutinases belong to the α/β-hydrolase fold family of enzymes and degrade cutin and various esters, including triglycerides, phospholipids and galactolipids. Cutinases are able to degrade aggregated and soluble substrates because, in contrast with true lipases, they do not have a lid covering their catalytic machinery. We report here the structure of a Cutinase from the fungus Trichoderma reesei (Tr) in native and inhibitor-bound conformations, along with its enzymatic characterization. A rare characteristic of Tr Cutinase is its optimal activity at acidic pH. Furthermore, Tr Cutinase, in contrast with classical Cutinases, possesses a lid covering its active site and requires the presence of detergents for activity. In addition to the presence of the lid, the core of the Tr enzyme is very similar to other Cutinase cores, with a central five-stranded β-sheet covered by helices on either side. The catalytic residues form a catalytic triad involving Ser164, His229 and Asp216 that is covered by the two N-terminal helices, which form the lid. This lid opens in the presence of surfactants, such as β-octylglucoside, and uncovers the catalytic crevice, allowing a C11Y4 phosphonate inhibitor to bind to the catalytic serine. Taken together, these results reveal Tr Cutinase to be a member of a new group of lipolytic enzymes resembling Cutinases but with kinetic and structural features of true lipases and a heightened specificity for long-chain triglycerides.
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Cutinase a lipolytic enzyme with a preformed oxyanion hole
Biochemistry, 1994Co-Authors: Chrislaine Martinez, Aurelie Nicolas, H Van Tilbeurgh, Mariepierre Egloff, C Cudrey, Robert Verger, Christian CambillauAbstract:: Cutinases, a group of cutin degrading enzymes with molecular masses of around 22-25 kDa (Kolattukudy, 1984), are also able to efficiently hydrolyse triglycerides (De Geus et al., 1989; Lauwereys et al., 1991), but without exhibiting the interfacial activation phenomenom (Sarda et al., 1958). They belong to a class of proteins with a common structural framework, called the alpha/beta hydrolase fold (Martinez et al., 1992; Ollis et al., 1992). We describe herein the structure of Cutinase covalently inhibited by diethyl-p-nitrophenyl phosphate (E600) and refined at 1.9-A resolution. Contrary to what has previously been reported with lipases (Brzozowski et al., 1991; Derewenda et al., 1992; Van Tilbeurgh et al., 1993), no significant structural rearrangement was observed here in Cutinase upon the inhibitor binding. Moreover, the structure of the active site machinery, consisting of a catalytic triad (S120, H188, D175) and an oxyanion hole (Q121 and S42), was found to be identical to that of the native enzyme, whereas the oxyanion hole of Rhizomucor lipase (Brzozowski et al., 1991; Derewenda et al., 1992), like that of pancreatic lipase (van Tilbeurgh et al., 1993), is formed only upon enzyme-ligand complex formation. The fact that Cutinase does not display interfacial activation cannot therefore only be due to the absence of a lid but might also be attributable to the presence of a preformed oxyanion hole.
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fusarium solani Cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent
Nature, 1992Co-Authors: Chrislaine Martinez, Pieter De Geus, Marc Lauwereys, G Matthyssens, Christian CambillauAbstract:LIPASES belong to a class of esterases whose activity on trigly-cerides is greatly enhanced at lipid–water interfaces1. This phenomenon, called interfacial activation2, has a structural explanation: a hydrophobic lid, which at rest covers the catalytic site, is displaced on substrate or inhibitor binding, and probably interacts with the lipid matrix3–6. Fusarium solani pisi Cutinase belongs to a group of homologous enzymes of relative molecular mass 22–25K (ref. 7) capable of degrading cutin, the insoluble lipid-polyester matrix covering the surface of plants7, and hydrolysing triglycerides7,8. Cutinases differ from classical Upases in that they do not exhibit interfacial activation; they are active on soluble as well as on emulsified triglycerides. Cutinases therefore establish a bridge between esterases and lipases. We report here the three-dimensional structure of a recombinant Cutinase from F. solani pisi, expressed in Escherichia coli9,10. Cutinase is an α–β protein; the active site is composed of the triad Ser 120, His 188 and Asp 175. Unlike other lipases, the catalytic serine is not buried under surface loops, but is accessible to solvent. This could explain why Cutinase does not display interfacial activation.
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fusarium solani Cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent
Nature, 1992Co-Authors: Chrislaine Martinez, Pieter De Geus, Marc Lauwereys, G Matthyssens, Christian CambillauAbstract:Lipases belong to a class of esterases whose activity on triglycerides is greatly enhanced at lipid-water interfaces. This phenomenon, called interfacial activation, has a structural explanation: a hydrophobic lid, which at rest covers the catalytic site, is displaced on substrate or inhibitor binding and probably interacts with the lipid matrix. Fusarium solani pisi Cutinase belongs to a group of homologous enzymes of relative molecular mass 22-25K (ref. 7) capable of degrading cutin, the insoluble lipid-polyester matrix covering the surface of plants, and hydrolysing triglycerides. Cutinases differ from classical lipases in that they do not exhibit interfacial activation; they are active on soluble as well as on emulsified triglycerides. Cutinases therefore establish a bridge between esterases and lipases. We report here the three-dimensional structure of a recombinant Cutinase from F. solani pisi, expressed in Escherichia coli. Cutinase is an alpha-beta protein; the active site is composed of the triad Ser 120, His 188 and Asp 175. Unlike other lipases, the catalytic serine is not buried under surface loops, but is accessible to solvent. This could explain why Cutinase does not display interfacial activation.
Joaquim M. S. Cabral - One of the best experts on this subject based on the ideXlab platform.
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REVIEW ARTICLE Cutinase structure, function and biocatalytic applications
2016Co-Authors: Cristina M. L. Carvalho, Maria Raquel Aires-barros, Joaquim M. S. CabralAbstract:This review analyses the role of Cutinases in nature and their potential biotechnological applications. The cloning and expression of a fungal Cutinase from Fusarium solani f. pisi, in Escherichia coli and Saccharomyces cerevisiae hosts are described. The three dimensional structure of this Cutinase is also analysed and its function as a lipase discussed and compared with other lipases. The biocatalytic applications of Cutinase are described taking into account the preparation of different Cutinase forms and the media where the different types of enzymatic reactions have been performed, namely hydrolysis, esterification, transesterification and resolution of racemic mixtures. The stability of Cutinase preparations is discussed, particularly in anionic reversed micelles considering th
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effect of tween 80 on stability and secretion of hydrophobic tagged Cutinases
Chemical and Biochemical Engineering Quarterly, 2009Co-Authors: Cecília R.c. Calado, M Brandao, J Biscaia, Joaquim M. S. Cabral, Luís P. FonsecaAbstract:To significantly enhance the downstream processing, two Cutinase variants were constructed by genetic fusion small hydrophobic peptides (WP) 2 and (WP) 4 , respectively. However, the fusion of these peptides impairs Cutinase secretion by the host cell Saccharomyces cerevisiae and increases Cutinase inactivation in the culture broth due to Cutinase aggregation, resulting in Cutinase activities per biomass of 56 % of Cutinase-(WP) 2 and of 7 % of Cutinase-(WP) 4 , in relation to Cutinase without the hydrophobic tags. It was observed that the addition of non-ionic surfactant Tween-80 into the culture broth could minimise the Cutinase inactivation. The addition of Tween-80 also results in the enhancement of Cutinase secretion by the yeast cell, leading to 1.25 and 2.51 fold-higher extracellular Cutinase-(WP) 2 and Cutinase-(WP) 4 , respectively, in relation to cultivations performed in the absence of surfactant. Therefore, the addition of Tween-80 on the culture broth partly minimises the effect of fusion of the hydrophobic tags on the inactivation of the enzymatic activity and on the reduction of the protein secretion. By this way, the use of Tween-80 on the S. cerevisiae cultivation may contribute to the efficiency enhancement of the downstream processing of tagged Cutinases.
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prediction of retention time of Cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatography
Journal of Chromatography A, 2007Co-Authors: M E Lienqueo, O Salazar, K Henriquez, Cecília R.c. Calado, Luís P. Fonseca, Joaquim M. S. CabralAbstract:Hydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of Cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan-proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Phitagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE<0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE<0.022) for the two tagged Cutinases at three different stationary phases, except for the case of cut_(wp)2 in octyl sepharose-2 M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of Cutinases tagged with hydrophobic peptides in HIC.
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Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi Cutinase fusion proteins.
Journal of Biotechnology, 2003Co-Authors: Maria Teresa Cunha, Maria João Costa, Cecília R.c. Calado, Luís P. Fonseca, Maria Raquel Aires-barros, Joaquim M. S. CabralAbstract:Genetic engineering was integrated with the production and purification of Fusarium solani pisi Cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular Cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding Cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were Cutinase wild type and two fusion proteins of Cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)2 and (WP)4. The (WP)4 fusion protein enabled a 300-fold increase of the Cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type Cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the Cutinase-(WP)2 proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)4 fusion proteins, respectively.
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optimisation of culture conditions and characterisation of Cutinase produced by recombinant saccharomyces cerevisiae
Enzyme and Microbial Technology, 2002Co-Authors: Cecília R.c. Calado, Joaquim M. S. Cabral, ângela M Taipa, Luís P. FonsecaAbstract:Abstract The optimum culture dissolved oxygen concentration and culture pH for the production of Cutinase from Fusarium solani pisi by the recombinant Saccharomyces cerevisiae SU50 strain was investigated. Dissolved oxygen concentrations in the fermentation culture of 5, 30 and 60% of air saturation were evaluated. A high Cutinase production, specific Cutinase activity and the highest Cutinase yield on biomass and the highest specific Cutinase production rate were obtained with a 5% of air saturation, which could have impact on process economics. Furthermore, at a low dissolved oxygen concentration, the specific growth rate, specific Cutinase production rate, Cutinase yield on biomass, Cutinase activity and specific Cutinase activity were increased when the pH control was changed from 5.25 to 6.25. The Cutinase, accumulated in the yeast culture presents two glycosilated isoforms with molecular weights of 22.8 and 24.9 kDa measured by SDS-PAGE. Furthermore, Cutinase in clarified culture samples presents a linear relationship between estereolytic and lypolitic activity and a high stability at room temperature.
Luís P. Fonseca - One of the best experts on this subject based on the ideXlab platform.
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effect of tween 80 on stability and secretion of hydrophobic tagged Cutinases
Chemical and Biochemical Engineering Quarterly, 2009Co-Authors: Cecília R.c. Calado, M Brandao, J Biscaia, Joaquim M. S. Cabral, Luís P. FonsecaAbstract:To significantly enhance the downstream processing, two Cutinase variants were constructed by genetic fusion small hydrophobic peptides (WP) 2 and (WP) 4 , respectively. However, the fusion of these peptides impairs Cutinase secretion by the host cell Saccharomyces cerevisiae and increases Cutinase inactivation in the culture broth due to Cutinase aggregation, resulting in Cutinase activities per biomass of 56 % of Cutinase-(WP) 2 and of 7 % of Cutinase-(WP) 4 , in relation to Cutinase without the hydrophobic tags. It was observed that the addition of non-ionic surfactant Tween-80 into the culture broth could minimise the Cutinase inactivation. The addition of Tween-80 also results in the enhancement of Cutinase secretion by the yeast cell, leading to 1.25 and 2.51 fold-higher extracellular Cutinase-(WP) 2 and Cutinase-(WP) 4 , respectively, in relation to cultivations performed in the absence of surfactant. Therefore, the addition of Tween-80 on the culture broth partly minimises the effect of fusion of the hydrophobic tags on the inactivation of the enzymatic activity and on the reduction of the protein secretion. By this way, the use of Tween-80 on the S. cerevisiae cultivation may contribute to the efficiency enhancement of the downstream processing of tagged Cutinases.
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prediction of retention time of Cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatography
Journal of Chromatography A, 2007Co-Authors: M E Lienqueo, O Salazar, K Henriquez, Cecília R.c. Calado, Luís P. Fonseca, Joaquim M. S. CabralAbstract:Hydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of Cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan-proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Phitagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE<0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE<0.022) for the two tagged Cutinases at three different stationary phases, except for the case of cut_(wp)2 in octyl sepharose-2 M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of Cutinases tagged with hydrophobic peptides in HIC.
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Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi Cutinase fusion proteins.
Journal of Biotechnology, 2003Co-Authors: Maria Teresa Cunha, Maria João Costa, Cecília R.c. Calado, Luís P. Fonseca, Maria Raquel Aires-barros, Joaquim M. S. CabralAbstract:Genetic engineering was integrated with the production and purification of Fusarium solani pisi Cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular Cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding Cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were Cutinase wild type and two fusion proteins of Cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)2 and (WP)4. The (WP)4 fusion protein enabled a 300-fold increase of the Cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type Cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the Cutinase-(WP)2 proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)4 fusion proteins, respectively.
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optimisation of culture conditions and characterisation of Cutinase produced by recombinant saccharomyces cerevisiae
Enzyme and Microbial Technology, 2002Co-Authors: Cecília R.c. Calado, Joaquim M. S. Cabral, ângela M Taipa, Luís P. FonsecaAbstract:Abstract The optimum culture dissolved oxygen concentration and culture pH for the production of Cutinase from Fusarium solani pisi by the recombinant Saccharomyces cerevisiae SU50 strain was investigated. Dissolved oxygen concentrations in the fermentation culture of 5, 30 and 60% of air saturation were evaluated. A high Cutinase production, specific Cutinase activity and the highest Cutinase yield on biomass and the highest specific Cutinase production rate were obtained with a 5% of air saturation, which could have impact on process economics. Furthermore, at a low dissolved oxygen concentration, the specific growth rate, specific Cutinase production rate, Cutinase yield on biomass, Cutinase activity and specific Cutinase activity were increased when the pH control was changed from 5.25 to 6.25. The Cutinase, accumulated in the yeast culture presents two glycosilated isoforms with molecular weights of 22.8 and 24.9 kDa measured by SDS-PAGE. Furthermore, Cutinase in clarified culture samples presents a linear relationship between estereolytic and lypolitic activity and a high stability at room temperature.
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production of wild type and peptide fusion Cutinases by recombinant saccharomyces cerevisiae mm01 strains
Biotechnology and Bioengineering, 2002Co-Authors: Cecília R.c. Calado, Maurice Mannesse, Maarten R Egmond, Joaquim M. S. Cabral, Luís P. FonsecaAbstract:This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous Cutinases: a wild-type Cutinase and two Cutinases in which the primary structure was fused with the peptides (WP)2 and (WP)4, respectively. Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and Cutinase production were analyzed and compared for the three yeast strains. The highest Cutinase productions and productivities were obtained in the fed-batch culture, where wild-type Cutinase was secreted up to a level of Cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg−1 with activity per protein broth (specific activity) of 266 Umg−1, whereas Cutinase-(WP)2 was secreted with a specific cell activity of 2.1 Umg−1 with a specific activity of 200 Umg−1, and Cutinase-(WP)4 with a specific cell activity of 0.7 Umg−1 with a specific activity of 15 Umg−1. The results indicate that the fusion of hydrophobic peptides to Cutinase that changes the physical properties of the fused protein limits Cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture). © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 692–698, 2002.
Cecília R.c. Calado - One of the best experts on this subject based on the ideXlab platform.
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effect of tween 80 on stability and secretion of hydrophobic tagged Cutinases
Chemical and Biochemical Engineering Quarterly, 2009Co-Authors: Cecília R.c. Calado, M Brandao, J Biscaia, Joaquim M. S. Cabral, Luís P. FonsecaAbstract:To significantly enhance the downstream processing, two Cutinase variants were constructed by genetic fusion small hydrophobic peptides (WP) 2 and (WP) 4 , respectively. However, the fusion of these peptides impairs Cutinase secretion by the host cell Saccharomyces cerevisiae and increases Cutinase inactivation in the culture broth due to Cutinase aggregation, resulting in Cutinase activities per biomass of 56 % of Cutinase-(WP) 2 and of 7 % of Cutinase-(WP) 4 , in relation to Cutinase without the hydrophobic tags. It was observed that the addition of non-ionic surfactant Tween-80 into the culture broth could minimise the Cutinase inactivation. The addition of Tween-80 also results in the enhancement of Cutinase secretion by the yeast cell, leading to 1.25 and 2.51 fold-higher extracellular Cutinase-(WP) 2 and Cutinase-(WP) 4 , respectively, in relation to cultivations performed in the absence of surfactant. Therefore, the addition of Tween-80 on the culture broth partly minimises the effect of fusion of the hydrophobic tags on the inactivation of the enzymatic activity and on the reduction of the protein secretion. By this way, the use of Tween-80 on the S. cerevisiae cultivation may contribute to the efficiency enhancement of the downstream processing of tagged Cutinases.
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prediction of retention time of Cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatography
Journal of Chromatography A, 2007Co-Authors: M E Lienqueo, O Salazar, K Henriquez, Cecília R.c. Calado, Luís P. Fonseca, Joaquim M. S. CabralAbstract:Hydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of Cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan-proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Phitagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE<0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE<0.022) for the two tagged Cutinases at three different stationary phases, except for the case of cut_(wp)2 in octyl sepharose-2 M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of Cutinases tagged with hydrophobic peptides in HIC.
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Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi Cutinase fusion proteins.
Journal of Biotechnology, 2003Co-Authors: Maria Teresa Cunha, Maria João Costa, Cecília R.c. Calado, Luís P. Fonseca, Maria Raquel Aires-barros, Joaquim M. S. CabralAbstract:Genetic engineering was integrated with the production and purification of Fusarium solani pisi Cutinases, in order to obtain the highest amount of enzyme activity units, after purification. An aqueous two-phase system (ATPS) of polyethylene glycol 3350, dipotassium phosphate and whole broth was used for the extraction of three extracellular Cutinases expressed in Saccharomyces cerevisiae. The production/extraction process was evaluated regarding Cutinases secretion in the medium, partition behaviour and extraction yields in the ATPS. The proteins studied were Cutinase wild type and two fusion proteins of Cutinase with the tryptophane-proline (WP) fusion tags, namely (WP)2 and (WP)4. The (WP)4 fusion protein enabled a 300-fold increase of the Cutinase partition coefficient when comparing to the wild type. However, the secretion of the fusion proteins was lower than of the wild type Cutinase secretion. A batch extraction strategy was compared with a continuous extraction in a perforated rotating disc contactor (PRDC). The batch and continuous systems were loaded with as much as 60% (w/w) whole cultivation broth. The continuous extraction strategy provided a 2.5 higher separation capacity than the batch extraction strategy. Considering the integrated process, the Cutinase-(WP)2 proved to lead to the highest product activity, enabling five and six times more product activity than the wild type and the (WP)4 fusion proteins, respectively.
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optimisation of culture conditions and characterisation of Cutinase produced by recombinant saccharomyces cerevisiae
Enzyme and Microbial Technology, 2002Co-Authors: Cecília R.c. Calado, Joaquim M. S. Cabral, ângela M Taipa, Luís P. FonsecaAbstract:Abstract The optimum culture dissolved oxygen concentration and culture pH for the production of Cutinase from Fusarium solani pisi by the recombinant Saccharomyces cerevisiae SU50 strain was investigated. Dissolved oxygen concentrations in the fermentation culture of 5, 30 and 60% of air saturation were evaluated. A high Cutinase production, specific Cutinase activity and the highest Cutinase yield on biomass and the highest specific Cutinase production rate were obtained with a 5% of air saturation, which could have impact on process economics. Furthermore, at a low dissolved oxygen concentration, the specific growth rate, specific Cutinase production rate, Cutinase yield on biomass, Cutinase activity and specific Cutinase activity were increased when the pH control was changed from 5.25 to 6.25. The Cutinase, accumulated in the yeast culture presents two glycosilated isoforms with molecular weights of 22.8 and 24.9 kDa measured by SDS-PAGE. Furthermore, Cutinase in clarified culture samples presents a linear relationship between estereolytic and lypolitic activity and a high stability at room temperature.
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production of wild type and peptide fusion Cutinases by recombinant saccharomyces cerevisiae mm01 strains
Biotechnology and Bioengineering, 2002Co-Authors: Cecília R.c. Calado, Maurice Mannesse, Maarten R Egmond, Joaquim M. S. Cabral, Luís P. FonsecaAbstract:This study focused on the growth of Saccha-romyces cerevisiae MM01 recombinant strains and the respective production of three extracellular heterologous Cutinases: a wild-type Cutinase and two Cutinases in which the primary structure was fused with the peptides (WP)2 and (WP)4, respectively. Different cultivation and strategies were tested in a 2-L shake flask and a 5-L bioreactor, and the respective cell growth and Cutinase production were analyzed and compared for the three yeast strains. The highest Cutinase productions and productivities were obtained in the fed-batch culture, where wild-type Cutinase was secreted up to a level of Cutinase activity per dry cell weight (specific cell activity) of 4.1 Umg−1 with activity per protein broth (specific activity) of 266 Umg−1, whereas Cutinase-(WP)2 was secreted with a specific cell activity of 2.1 Umg−1 with a specific activity of 200 Umg−1, and Cutinase-(WP)4 with a specific cell activity of 0.7 Umg−1 with a specific activity of 15 Umg−1. The results indicate that the fusion of hydrophobic peptides to Cutinase that changes the physical properties of the fused protein limits Cutinase secretion and subsequently leads to a lower plasmid stability and lower yeast cell growth. These effects were observed under different cultivation conditions (shake flask and bioreactor) and cultivation strategies (batch culture versus fed-batch culture). © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 692–698, 2002.
