Surface Modification of Cellulose Acetate with Cutinase and ...

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Tailoring Cutinase Activity Towards Polyethylene Terephthalate and Polyamide 6,6 Fibers surface of polyamide and polyester fibers. Concentrations of PET hydrolysis products were calculated after 24 h of PET fabric incubation with the enzyme, in the linear area of substrate conversion. Again L182A was the most active enzyme. The relative ratios of activities were similar for PET fibers and p-NPB, except for L189A and N84A. Cutinase is also able to biodegrade polyamide 6,6 substrates, but the designed mutations failed to give a clear increase of activity (Table III). Just 19% of increase was found for L182A. These results tend not to be in agreement with the modeling studies of the free energy of stabilization of TI for all mutant enzymes for polyamide 6,6 (Table II). Given that PET and PA 6,6 fibers are mostly hydrophobic, the adsorption properties of native and mutant unbound enzymes were modeled considering an analysis based on the total enzyme hydrophobic surface. Modified enzymes tested have an equal or lower percentage of hydrophobic surface in comparison with the native, as it was expected (Table IV), given that large hydrophobic residues were changed by smaller ones (with the exception of N84, which is polar). Our studies predict that L182A, N84A and L81A do not significantly affect the adsorption by the hydrophobic fibers, but V184A and L189A show a decrease in hydrophobic area, suggesting that fiber adsorption is reduced in this order. The modeling studies predictions are not in total agreement with the experimental results. N84A, L81A, V184A and L189A displayed different behavior of protein adsorption levels for both fibers when compared with the modeling studies. On the other hand, the L182A seems to maintain the same adsorption properties as the native enzyme for PET fibers and a slight decrease in the case of PA 6,6. Given that the biotransformation of a fiber is a heterogeneous reaction, a pre-adsorption of the enzyme on the solid substrate is assumed before the catalysis can proceed. By looking at adsorption data it is possible to verify that cutinase adsorption levels are higher for PET than for PA 6,6 (Table III). For the same concentrations of native cutinase, PET fibers seems to be fully covered with enzyme (± 95%) while this level was not reached for PA 6,6 fibers (± 30%). Despite the stabilization of TI for several mutant cutinases, other adsorption and substrate recognition issues seem to play a major role on the enzymatic hydrolysis of solids substrates by cutinase. According to these results, L182A was considered to be the most promising enzyme for future studies. 75
Subchapter 2.2 Table IV. Hydrophobic surface percentage (hydrophobic surface/total surface) with SE of native and genetically modified enzymes (Eisenhaber et al., 1995). 76 Enzyme Hydrophobic surface Standard (hydrophobic/total) (%) error (SE) Native 40.710 0.096 L81A 40.780 0.192 N84A 40.650 0.243 L182A 40.610 0.147 V184A 40.560 0.154 L189A 40.410 0.185 We further measured the activity of the native and L182A cutinases towards aliphatic polyesters, which resemble the original cutin substrate. Apparently, the native form seems to have a decrease of activity and protein adsorption levels as the hydrophobicity of the substrate increases (Table V). The opposite seems to happen with L182A, which presents a lower hydrophobic area (Table IV). However, the space created by the substitution of leucine by alanine close to the active site, appears to be enough to better “accommodate” hydrophobic aliphatic substrates. These results seem to indicate that cutinase is designed to recognize aliphatic chains, being one of the reasons why this enzyme shows activity towards the aliphatic structure of polyamide 6,6. In summary, we have obtained cutinases with enhanced activity towards polyester fibers, namely L81A and L182A. The increase in activity of these mutations can be explained by a higher stabilization of TI and a better accommodation of the substrate, as has been shown by our modeling studies. Furthermore, the L182A mutation does not affect the adsorption levels. Regarding polyamide treatment, our findings suggest that these fibers can be more efficiently modified when L182A cutinase is used. Being cutinase an esterase, it seems unlikely that it will biodegrade polyamide substrates. However, our findings suggest that the similarity of polyamide structure with cutin and the diversified substrate recognition of cutinase, might explain the ability of this enzyme to modify the surface of these fibers, showing however a slow enzymatic kinetics (Silva and Cavaco-Paulo, 2004).
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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 48 and 49: 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 and 96: Subchapter 2.2 3. Results and discu
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Page 107 and 108: Subchapter 2.3 Abstract Two polyami
Page 109 and 110: Subchapter 2.3 aliphatic polyester.
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Page 113 and 114: Subchapter 2.3 2.7. Enzymatic hydro
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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 125 and 126: Subchapter 2.3 Table II. Time of wa
Page 127 and 128: Subchapter 2.3 104 Abs 60 50 40 30
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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
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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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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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