Surface Modification of Cellulose Acetate with Cutinase and ...

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Strategies Towards the Functionalization of Bacillus subtilis Subtilisin E for Wool Finishing Applications Strategies Towards the Functionalization of Bacillus subtilis Subtilisin E for Wool Finishing Applications RITA ARAÚJO 1 , ARTUR CAVACO-PAULO 2 *, MARGARIDA CASAL a 1 Center of Environmental and Molecular Biology, Department of Biology, University of Minho, Campus of Gualtar, 4710-057 Braga, Portugal 2 Center of Textile Engineering, University of Minho, Campus of Azurém, 4800-058 Guimarães, Portugal Keywords: Subtilisin E, protein engineering, in vitro refolding, wool hydrolysis *Correspondence to: Artur Cavaco-Paulo University of Minho, Textile Engineering Department 4800-058 Guimarães, Portugal Tel: +351 253 510271 Fax: +351 253 510293 E-mail: artur@det.uminho.pt 175
Subchapter 3.2 Abstract Subtilisin E is an alkaline serine protease secreted by the Gram positive bacteria Bacillus subtilis and widely used in industry as biocatalyst for various processes. The most common application of subtilisins is in laundry detergents but, due to environmental concerns, the application of subtilisins to treat wool is under study. There are some reports regarding the attempts to substitute the conventional chlorine treatment by an enzymatic process capable of providing the same characteristics to the fabric, like anti-shrinking and better uptake and fixation of the dyestuff. However, the uncontrolled hydrolysis degree due to diffusion of the enzyme inside the wool fibre causes unacceptable losses of strength. To overcome this fact, and taking advantage of the xray crystallographic structure, we have genetically modified subtilisin E, increasing its molecular weight, to restrict the hydrolysis to the surface of the wool fibres. Therefore, three genetically modified enzymes with a molecular weight 2-fold to 4-fold higher than the native subtilisin E were produced and assessed for activity. The prokaryotic expression systems, pET25b (+), pET11b and pBAD C were explored for the production of recombinant enzymes. The results demonstrated that regardless the expression system or strain used, chimeric subtilisins were not expressed with the correct folding. No active and soluble recombinant protein was recovered under the testing conditions. Despite this drawback, we have described here a novel approach to increase subtilisin molecular weight. The reported results are noteworthy and can indicate good guidelines for future work aiming the solubilization of recombinant chimeric subtilisins. 176
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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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Subchapter 2.3 2.3. Determination o
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Subchapter 2.3 2.7. Enzymatic hydro
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Subchapter 2.3 The crystallinity va
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Subchapter 2.3 Table I. Protein ads
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Subchapter 2.3 In the same set of e
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Subchapter 2.3 98 Protein in soluti
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Subchapter 2.3 100 Protein in solut
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Subchapter 2.3 Table II. Time of wa
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Subchapter 2.3 104 Abs 60 50 40 30
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Subchapter 2.3 Acknowledgments The
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Subchapter 2.3 Lau Y, Bruice C. 199
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Subchapter 2.4 110
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Subchapter 2.4 Abstract The effect
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Subchapter 2.4 A balance between en
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Subchapter 2.4 flask with stirring
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Subchapter 2.4 118 TPA (mM) TPA (mM
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Subchapter 2.4 of native cutinase a
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Subchapter 2.4 122 K/S Increase (%)
Page 147 and 148: Subchapter 2.4 References Araújo R
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Page 152 and 153: Surface Modification of Cellulose A
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Page 188 and 189: Enzymatic Modification of Wool Surf
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Page 224 and 225: Subchapter 3.3 Enzymatic Hydolysis
Page 226 and 227: Enzymatic Hydolysis of Wool with a
Page 228 and 229: 1. Introduction Enzymatic Hydolysis
Page 238 and 239: References Enzymatic Hydolysis of W
Page 240 and 241: Chapter 4 General Discussion and Fu
Page 242 and 243: General Discussion and Future persp
Page 244 and 245: General Discussion and Future Persp
Page 246 and 247: 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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