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 3.2. Expression of recombinant proteins using pBAD expression systems The E. coli pBAD expression system has been described to express B. subtilis subtilisinE (Sroga and Dordick, 2001; Sroga and Dordick, 2002), where the entire preprosubtilisinE gene was used and full functional enzyme was efficiently targeted to the periplasmic space. The pBAD plasmid has a geneIII signal sequence that can be used for periplasmic expression of recombinant enzymes. SDM by recombinant PCR was performed into pBAD C vector in order to allow in frame integration of inserts. The native and chimeric genes were cloned into pBAD C* expression system and E. coli strains TOP10 and LMG194 were used. TOP 10 E. coli did not express any of recombinant enzymes (data not shown). LMG194 cells, carrying pBAD C* constructions, produced the recombinant proteins in the insoluble fraction in all the conditions tested. Figure 5 shows representative results of these assays. Unlike Sroga and its collaborators (2002) we were not able to produce active and soluble subtilsin E using pBAD expression system, since no azocasein activity could be detected in soluble fractions, derived both from cytoplasm or periplasmic space. As previously observed for pET25b (+) pelB leader sequence, also pBAD C geneIII leader sequence did not revealed to be a suitable signal peptide to export these recombinant proteins to the periplasmic space. It was assumed that these facts might be explained by the absence of native B. subtilis presequence. Following this hypothesis, in all the pBAD C* constructions, the original B. subtilis prepeptide was introduced in frame upstream the pBAD geneIII signal sequence. Cells LMG194 harboring the pBAD-preprosubtilisinE, pBAD-pre-pro2subtilisinE, pBAD-pre-pro4subtilisinE and pBAD-preprosubtilisinE-SPDnd, were grown and induced as previously described. The results demonstrated that no production of active and soluble recombinant protein using pBAD/gIII expression system was achieved, even in the presence of the original leader sequence of B. subtilis (figure 6). 191
Subchapter 3.2 Figure 5. SDS-PAGE of proteins from E. coli LMG194 cells grown at 37 ºC, induced with 0.2% arabinose at 18 ºC. Lanes 1 to 5: soluble fractions of negative control pBAD, pBAD-prosubtilisinE, pBAD-pro2subtilisinE, pBAD-pro4subtilisinE and pBAD- prosubtilisinE-SPDnd respectively and lanes 6 to 10: insoluble fractions of negative control pBAD, pBAD-prosubtilisinE, pBAD-pro2subtilisinE, pBAD-pro4subtilisinE and pBAD-prosubtilisinE-SPDnd. MW: SDS-PAGE Standard, Broad Range (Bio-Rad). The solid arrowheads indicate the position of recombinant proteins. 192 MW 1 2 3 4 5 6 7 8 9 10 MW MW 1 2 3 4 5 6 Figure 6. SDS-PAGE of proteins from E coli LMG194 cells grown at 30 ºC, induced 0.2% arabinose at 18 ºC. Lanes 1 to 3: soluble fractions of pBAD-pre-prosubtilisinE, pBAD-pre-pro2subtilisinE and pBAD-pre-pro4subtilisinE respectively and lanes 4 to 6: insoluble fractions of pBAD-pre-prosubtilisinE, pBAD-pre-pro2subtilisinE and pBADpre-pro4subtilisinE. MW: SDS-PAGE Standard, Broad Range (Bio-Rad). The solid arrowheads indicate the position of recombinant proteins.
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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 2.3 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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Page 164 and 165: Surface Modification of Cellulose A
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Page 228 and 229: 1. Introduction Enzymatic Hydolysis
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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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