Surface Modification of Cellulose Acetate with Cutinase and ...

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Surface Modification of Cellulose Acetate with Cutinase and Cutinase Fused to Carbohydrate-binding Modules CBM of Endoglucanase C (CenC) from Cellulomonas fimi, which belongs to the family CBM4, is able to bind amorphous cellulose (Boraston et al., 2005). To our knowledge, this is the first report of the hydrolysis of surface acetyl groups from CDA and CTA with a cutinase. It constitutes a promising approach for the partial regeneration of cellulose reactivity and hydrophilicity in these fibres, here demonstrated by the enhanced reactive dye uptake of treated fabrics. The production of fusion cutinases with new functionalities is here described and a comparison with cutinase regarding its efficiency for CDA and CTA modification is presented. 2. Materials and methods 2.1. Reagents and enzymes The cellulose diacetate and triacetate plain woven fabrics used were kindly supplied by Mitsubishi Rayon Co. Ltd., Tokyo, Japan. The CDA fabric has 41/27 ends/picks per cm and 64 g m -2 . The CTA fabric has 45/31 ends/picks per cm and 98 g m -2 . All other reagents were laboratory grade reagents from Sigma-Aldrich, St. Louis, USA, unless stated otherwise. The cutinase (EC 3.1.1.74) from F. solani pisi used in this work was expressed and purified as previously reported by Araújo et al., 2007. Restriction enzymes were purchased from MBI Fermentas (Vilnius, Lithuania) and from Roche Diagnostics GmbH (Penzberg, Germany). Accuzyme TM DNA polymerase was purchased from Bioline GmbH (Luckenwalde, Germany) and recombinant Taq DNA polymerase was purchased from MBI Fermentas. T4 DNA ligase was purchased from Roche Diagnostics GmbH (Penzberg, Germany). 2.2. Esterase activity assay Esterase activity was determined following the product release (p-nitrophenol) continuously through the increase in the absorbance at 400 nm at 30º C. The assay conditions for the determination of cutinase activity were described previously (Matamá et al., 2006). All the assays were performed at least in triplicate. Standard solutions of 133
Subchapter 2.5 p-nitrophenol were used to obtain the calibration curve. One unit of esterase activity was defined as one μmol of p-nitrophenol released per minute. 2.3. Treatment of cellulose di- and triacetate fabric with cutinase All samples of cellulose acetate fabric were washed prior to use in order to remove possible impurities from manufacture and from human handling. Washing was performed at 35º C and 20 rpm, in stainless steel pots of 450 mL in capacity and housed in a laboratory scale machine, the Rotawash MKIII (vertical agitation simulating European washing machines, from SDL International Ltd.). The fabric was washed twice for 30 min in 40 mg L -1 Lutensol AT25 (non-ionic detergent, BASF, Ludwigshafen, Germany), then rinsed four times with distilled water for 30 min each and left to dry at room temperature. Several sets of experiments were carried out taking into account the amounts of enzyme, fabric and time of incubation. For all experiments, the treatment of cellulose acetate fabric was performed in 50 mM phosphate buffer pH 8 with vertical agitation, in the Rotawash machine operating at 30 ºC and 20 rpm. To evaluate the effect of enzyme concentration, samples of CDA and CTA fabric, with an average weight of 0.1 g, were incubated in duplicate for 8 hours with 0, 25, 50, 75 and 100 U mL -1 of cutinase, in a total volume of 5 mL. To obtain a progress curve, samples of CDA and CTA fabric, with an average weight of 0.1 g, were treated with 50 U mL -1 of cutinase, in a final volume of 10 mL for different periods of time. For each sample a control was run in parallel in which the buffer substituted the corresponding volume of enzyme. In another treatment, the average weight of both type of fabric was increased to 0.5 g and the incubation extended to 24 hours. The initial activity of cutinase was 25 U mL -1 in a final volume of 25 mL. For each sample a control was run in parallel without the enzyme. After enzymatic treatment, all fabric samples were washed at 35 ºC, in the Rotawash machine, to remove the adsorbed protein, according to the order: 250 mgL -1 Lutensol AT25 for 30 min, 70% ethanol for 20 min, 15% isopropanol for 15 min, three steps of increasing concentrations of NaCl for 10 min each, three steps in distilled water for 134
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Universidade do Minho Escola de Ci
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É AUTORIZADA A REPRODUÇÃO PARCIA
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Acknowledgments/ Agradecimentos
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Às 2 mosqueteiras ausentes, Joana
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Abstract The challenges facing the
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Resumo
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Resumo Os desafios que a indústria
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molecular ficou retida à superfíc
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Table of contents Acknowledgments /
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CATs- Catalases CBD- Carbohydrate b
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Chapter 1 General Introduction: App
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General Introduction: Application o
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1. Biotechnology in textile industr
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3. Role of enzymes in textile indus
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7. Serine proteases: subtilisins Ge
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12.1 Decolourization of dyes and te
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References General Introduction: Ap
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Chapter 2 Surface Modification of S
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Subchapter 2.1 52
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Subchapter 2.1 major material of co
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Subchapter 2.1 End uses include swe
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Subchapter 2.1 References Battistel
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Subchapter 2.1 60
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Subchapter 2.2 62
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Subchapter 2.2 Abstract Cutinase fr
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Subchapter 2.2 2. Materials and met
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Subchapter 2.2 Scheme 1. Thermodyna
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Subchapter 2.2 14000 rpm at 4 ºC.
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Subchapter 2.2 3. Results and discu
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Mutation Subchapter 2.2 Table III.
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Subchapter 2.2 Table IV. Hydrophobi
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Subchapter 2.2 References Ansaldi M
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Subchapter 2.2 80
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Page 135 and 136: Subchapter 2.4 Abstract The effect
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Page 145 and 146: Subchapter 2.4 122 K/S Increase (%)
Page 147 and 148: Subchapter 2.4 References Araújo R
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Page 184 and 185: SubChapter 3.1 Introduction
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Strategies Towards the Functionaliz
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4. Discussion Strategies Towards th
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References Strategies Towards the F
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Subchapter 3.3 Enzymatic Hydolysis
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Enzymatic Hydolysis of Wool with a
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1. Introduction Enzymatic Hydolysis
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References Enzymatic Hydolysis of W
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Chapter 4 General Discussion and Fu
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General Discussion and Future persp
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General Discussion and Future Persp
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References General Discussion and F
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Appendix
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SOB TE buffer Autoclave Tryptone 2%
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Dissolve the nucleic acids in 30 μ
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