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 Figure 1. Strategy used for the construction of chimeric gene pro2subtilisinE in pGEM T-easy. PCR was carried out with pET11a:prosubtilisin (Hu et al., 1994) containing the native prosubtilisinE as template. PCR products were subcloned into pGEM T-easy. Construction pGEM-subtilisinE was digested with BamHI and BglII to recover the BamHI-subtilisinE-BglII fragment, which was then ligated to the BglII linearized pGEM-prosubtilisinE. BamHI and BglII recognize different restriction sites generating compatible sticky-ends. The original Bacillus subtilis presequence, described by Ikemura and collaborators (1987) was synthesized in vitro using self-annealing oligonucleotides Fpres and Rpres, overlapping in 33 bp (table III). The full DNA sequence was obtained in a PCR of 20 s at 94 ºC and 20 s at 72 ºC, for Accuzyme (Bioline, Germany) extension, for 30 cycles. Primers Famp and Ramp (flanked with XhoI restriction site) were further used to amplify the presequence (table III). The PCR product was digested with XhoI, purified from a 2% (w/v) agarose gel electrophoresis and cloned into the XhoI digested and dephosphorilated pBAD C* constructions, resulting in the final expression vectors pBAD-pre-prosubtilisinE, pBAD-pre-pro2subtilisinE, pBAD-pre-pro4subtilisinE and pBAD-pre-prosubtilsinE-SPDnd (table 2). Table I. Primers used to amplify the genes prosequence, subtilisinE and prosubtilisinE Gene Primer (5´→ 3´) bp GC % Prosequence ProF CGC GGA TCC CAT GGC CGG AAA AAG CAG TAC AG 32 59.4 ProR GGA AGA TCT CCA TAT TCA TGT GCA ATA TGA T 31 35.5 SubtilisinE SubF CGC GGA TCC CAT GGC GCA AAG CTT TCC TTA TG 32 56.3 SubR GGA AGA TCT CCT TGT GCA GCT GCT TGT ACG TTG 33 51.5 proSubtilisinE ProF CGC GGA TCC CAT GGC CGG AAA AAG CAG TAC AG 32 59.4 SubR GGA AGA TCT CCT TGT GCA GCT GCT TGT ACG TTG 33 51.5 183
Subchapter 3.2 Table II. Heterologous protein expression systems used: E. coli strains and recombinant vectors. 184 E. coli strain/ vector Constructs BL21(DE3)/ pET25b (+) Tuner/ pET25b (+) BL21(DE3)pLysS/ pET25b (+) BL21(DE3)/ pET11b TOP10/ pBAD C* LMG194/ pBAD C* 2.5. Induction conditions for protein expression prosubtilisinE pro2subtilisinE pro4subtilisinE prosubtilisinE pro2subtilisinE pro4subtilisinE prosubtilisinE pro2subtilisinE pro4subtilisinE prosubtilisinE pro2subtilisinE pro4subtilisinE prosubtilisinE-SPDnd prosubtilisinE pro2subtilisinE pro4subtilisinE prosubtilisinE-SPDnd Prosequence prosubtilisinE pro2subtilisinE pro4subtilisinE prosubtilisinE-SPDnd pre-prosubtilisinE pre-pro2subtilisinE pre-pro4subtilisinE pre-prosubtilisinE-SPDnd Expression host strains BL21(DE3), BL21(DE3)pLysS and Tuner, transformed with pET25 constructions, were used for protein expression. Cells were grown in Luria- Broth, (LB), medium containing 100 μg/μL ampicillin, and induced according to the conditions described in table IV. Cells of the strain BL21(DE3) containing pET11 constructions were grown in LB medium/100 μg/μl ampicillin, at 37 ºC and induced with isopropyl β-D-1thiogalactopyranoside (IPTG) 1 mM at 18 ºC. E. coli strains TOP10 carrying the pBAD C* constructions were grown in Complete Minimal (CM) medium supplemented with 20 amino acids (40 μg/mL) and vitamin B1 (5 mg/L). Glycerol was used at a concentration of 0.20% (w/v). Cells of the strain
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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 (%)
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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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Surface Modification of Cellulose A
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Page 182 and 183: Chapter 3 Enzymatic Modification of
Page 184 and 185: SubChapter 3.1 Introduction
Page 186 and 187: 1. Properties of Wool Enzymatic Mod
Page 188 and 189: Enzymatic Modification of Wool Surf
Page 190 and 191: 3.3. Bleaching Enzymatic Modificati
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Page 198 and 199: Strategies Towards the Functionaliz
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Page 220 and 221: References Strategies Towards the F
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
Page 248 and 249: Appendix
Page 250 and 251: SOB TE buffer Autoclave Tryptone 2%
Page 252: Dissolve the nucleic acids in 30 μ
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