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 extracted and purified from agarose and cloned into the SalI/NotI restricted and dephosphorilated pCWT::CBMN1, resulting in the final pCWT::PTbox::CBMN1 vector. 2.11.3. Expression and purification of cutinase fusion proteins The constructs pCWT::wtCBMCBHI, pCWT::sCBMCBHI, pCWT::CBMN1 and pCWT::PTbox::CBMN1 were first established in E. coli strain XL1-Blue. Medium-scale purifications of plasmid DNA were made and used to transform the E. coli strain BL21(DE3). Clones harbouring the constructs were grown, at 15º C and 200 rpm, in 2.5 L Luria-Broth medium supplemented with 100 µg mL -1 ampicillin until an absorbance A600 nm of 0.3-0.5 was reached. Cells were induced with 0.7 mM isopropyl- 1-thio-β-D-galactopyranoside, and further incubated for 16 hours at 15º C. The cells were harvested by centrifugation at 4º C (7500 xg, 10 min), washed with PBS pH 7.4 and frozen at -80º C. The ultrasonic disruption of the bacterial cells was accomplished on ice with a 25.4 mm probe in an Ultrasonic Processor VCX-400 watt (Cole-Parmer Instrument Company, Illinois, USA). The lysate was centrifuged for 30 min at 16000 xg and 4 ºC. The supernatant was collected, pH was adjusted to 7.6 and imidazole was added to a final concentration of 25 mM. Protein purification was performed with the affinity chromatography system HiTrap Chelating HP (GE Healthcare Bio-Sciences Europe GmbH, Munich, Germany) coupled to a peristaltic pump. The 5 mL column was loaded with 100 mM Ni 2+ and equilibrated with the binding buffer (20 mM phosphate buffer pH 7.6, 500 mM NaCl, 25 mM imidazole). The samples were loaded and washed with 10 column volumes of binding buffer followed by buffers with 50 and 100 mM imidazole. The fusion proteins (figure. 1) were eluted with 550 mM imidazole buffer. The fractions obtained were monitored by SDS-PAGE with Coomassie Brilliant Blue staining. The elution buffer was changed to 50 mM phosphate buffer pH 8 with HiTrap Desalting 5 mL columns (GE Healthcare Bio-Sciences Europe GmbH, Munich, Germany). Prior to the 2.5 L culture scale up, Western blotting was performed with monoclonal Anti-polyHistidine-Peroxidase Conjugate from mouse to confirm the expression of the fusion proteins. The detection was made with ECL Western blotting 139
Subchapter 2.5 reagents and analysis system (Amersham Biosciences Europe GmbH, Freiburg, Germany). Figure 1. Schematic representation of the recombinant wild-type cutinase from F. solani pisi (Araújo et al., 2007) and its new fusion proteins with the fungal carbohydrate-binding module of CBH I, from T. reesei, and the bacterial carbohydrate- binding module N1 of CenC, from C. fimi. The primary sequences of the linkers used are specified in the figure. 2.12. Treatment of cellulose di- and triacetate fabric with cutinase fused to carbohydrate-binding modules All samples of cellulose acetate fabric used were previously washed as already described in 2.3. Cellulose acetate fabric samples with an average weight of 0.1 g were incubated with 100 U mL -1 of cutinase and cutinase-CBMN1, 50 U mL -1 of cutinase-PTboxCBMN1 and cutinase-wtCBMT.reesei, 25 U mL -1 of cutinase-sCBMT.reesei in 10 mL of 50 mM phosphate buffer pH 8 with 0.01% sodium azide, under continuous vertical agitation at 30º C and 20 rpm, for 18 hours. A control for both types of fabric was run in parallel in 140
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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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Subchapter 2.3 82
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Subchapter 2.3 Abstract Two polyami
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Subchapter 2.3 aliphatic polyester.
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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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