Surface Modification of Cellulose Acetate with Cutinase and ...

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Tailoring Cutinase Activity Towards Polyethylene Terephthalate and Polyamide 6,6 Fibers A B Figure 2. Cutinase surface rendering of a representative configuration of an equilibrated simulation: Native (A), and genetically modified cutinase L182A (B). L182A mutation is responsible for the highest increase of TI stabilization and enzymatic activity toward the 1,2-ethanodiol dibenzoate substrate. The 1,2-ethanodiol dibenzoate TI model is rendered in sticks. Table II. Stabilization free energy of the model substrate TI estimated for the genetically modified enzymes. Free energies are calculated relatively to the native enzyme. ∆∆G (kJ/mol) Mutation PA 6.6 PET L182A -3.80 -4.81 L189A -2.38 -2.83 V184A -2.44 -1.81 L81A -1.77 -2.35 N84A 15.95 14.64 73
Mutation Subchapter 2.2 Table III. Activity of cutinases towards PET and PA 6,6 and protein adsorption levels. Values are normalized to the native enzyme. 74 p- Nitrophenyl butyrate (p- NPB) CH3CH2CH2 O C O % (Umg -1 ) NO 2 Terephthalic acid formed after incubation with PET fibers (24 h)* O O C C O CH2CH2 O % (mM) PET PA 6,6 n Protein adsorption levels after incubation (24h) (%) Amines formed in solution with PA66 fibers (48 h)* O C (CH 2)4 O C N N % (mM) (Ch 2)6 H N n Protein adsorption levels after incubation Native 100 (210) 100 (0.024) 95 100 (0.094) 30 (± 1.5) L182A 465 (± 1.8) 528 95 119 (± 2.5) 25 (± 2) L189A 147 (±1.3) 78 45 94 (± 3) 37 (± 2) V184A 289 (± 2.2) 203 95 98 (± 3) 21 (± 2.5) L81A 125 (± 1.8) 399 75 98 (± 2.8) 43 (± 2) N84A 45 (± 2.6) 170 95 83 (± 3) 15 (± 3) *See references (O’Neill et al., 2004) (Silva et al., 2004) for experimental details. The activity of cutinases towards PET and PA 6,6 fibers was expressed as mmolar (mM) of soluble terephthalic acid and soluble amines, respectively, obtained after a certain period of time. Due to the fact that these substrates are solid, no proper Michaelis–Menten kinetic could be calculated. Since we wanted to compare performances of each mutant enzyme based on equal amounts of protein, we expressed those estimated activities in mM of hydrolysis products. The experimental hydrolytic activity on p-NPB was higher for all the mutant enzymes, when compared with the native cutinase, with the exception of N84A, which is explained by the modeling studies on basis of the favorable interaction of the aspargine with the oxianion hole (Table III). Modified cutinases L182A and V184A have shown a remarkable increase in activity on p-NPB. Hydrolytic activity of L182A form increased more than four-fold. This seems to be a promising mutation to modify the hydrophobic (48h) (%)
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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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Page 46 and 47: General Introduction: Application o
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Page 56 and 57: References General Introduction: Ap
Page 72: Chapter 2 Surface Modification of S
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Page 77 and 78: Subchapter 2.1 major material of co
Page 79 and 80: Subchapter 2.1 End uses include swe
Page 81 and 82: Subchapter 2.1 References Battistel
Page 87 and 88: Subchapter 2.2 Abstract Cutinase fr
Page 89 and 90: Subchapter 2.2 2. Materials and met
Page 91 and 92: Subchapter 2.2 Scheme 1. Thermodyna
Page 93 and 94: Subchapter 2.2 14000 rpm at 4 ºC.
Page 95: Subchapter 2.2 3. Results and discu
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Page 101 and 102: Subchapter 2.2 References Ansaldi M
Page 107 and 108: Subchapter 2.3 Abstract Two polyami
Page 109 and 110: Subchapter 2.3 aliphatic polyester.
Page 111 and 112: Subchapter 2.3 2.3. Determination o
Page 113 and 114: Subchapter 2.3 2.7. Enzymatic hydro
Page 115 and 116: Subchapter 2.3 The crystallinity va
Page 117 and 118: Subchapter 2.3 Table I. Protein ads
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Page 121 and 122: Subchapter 2.3 98 Protein in soluti
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Page 127 and 128: Subchapter 2.3 104 Abs 60 50 40 30
Page 129 and 130: Subchapter 2.3 Acknowledgments The
Page 131 and 132: Subchapter 2.3 Lau Y, Bruice C. 199
Page 135 and 136: Subchapter 2.4 Abstract The effect
Page 137 and 138: Subchapter 2.4 A balance between en
Page 139 and 140: Subchapter 2.4 flask with stirring
Page 141 and 142: Subchapter 2.4 118 TPA (mM) TPA (mM
Page 143 and 144: Subchapter 2.4 of native cutinase a
Page 145 and 146: Subchapter 2.4 122 K/S Increase (%)
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Subchapter 2.4 References Araújo R
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Subchapter 2.4 126
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Surface Modification of Cellulose A
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Chapter 3 Enzymatic Modification of
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SubChapter 3.1 Introduction
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1. Properties of Wool Enzymatic Mod
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Enzymatic Modification of Wool Surf
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3.3. Bleaching Enzymatic Modificati
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Subchapter 3.2 Strategies Towards t
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Strategies Towards the Functionaliz
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1. Introduction Strategies Towards
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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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