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 3.3. Expression of recombinant proteins using pET11b expression system Since B. subtilis subtilsinE was previously expressed in E. coli pET11 system (Hu et al., 1994), the genes coding for the native enzyme, as well as, the three chimeric enzymes, pro2subtilisinE, pro4subtilisinE and prosubtilisinE-SPDnd were cloned into this vector, using E. coli BL21(DE3) as host strain. The transformants carrying pET11 vectors were able to produce all the constructs at a level of almost 80 % of total cellular proteins in the presence of 1 mM IPTG (Figure 7). After cell disruption, by ultra-sonic treatment, the products isolated in the pellets, were collected by low-speed centrifugation, indicating that the proteins aggregated to form inclusion bodies. Purification and refolding of native and chimeric enzymes were carried out with the isolated inclusion bodies as described under Materials and Methods. All samples were assayed for azocasein activity. Except for native subtilisinE, (3.2 U), no activity was detected for chimeric enzymes pro2subtilisinE, pro4subtilisinE and prosubtilisinE-SPDnd (data not shown). Mature subtilisinE and pro2subtilisinE purified and renatured from inclusion bodies were used for circular dicroism analysis. Compared to active mature subtilisinE, chimeric pro2subtilisinE presented only 30% of secondary structure (data not shown) which suggests that in vitro renaturation of chimeric enzymes do not result in the correct folding necessary for enzymatic activity. 3.4. Prosequence mediated folding E. coli LMG194 cells carrying the construction pBAD-prosequence, grown at 30 ºC and induced at 18 ºC, were able to express the prosequence at high level in the soluble, as well as, in the insoluble fractions (Figure 8). Soluble fractions were purified by IMAC using a nickel column, and used for refolding procedures. Prosequence-mediated folding was performed by addition of prosequence to recombinant proteins purified from inclusion bodies in a 1:1 molar ratio and incubation at 4 ºC for 12 h. All the experiments were repeated three times. In previous work developed by Shinde and its collaborators (1993) the refolding of Gdn-HCldenatured subtilisinE was achieved by incubation with prosequence in the same 193
Subchapter 3.2 conditions described above. In all the assays performed refolding of prosubtilisinE was attained, since activity was recovered, but refolding of chimeric pro2subtilisinE, pro4subtilisinE and prosubtilisinE-SPDnd enzymes was not achieved (data not shown). Figure 7. SDS-PAGE of insoluble fractions from E coli BL21(DE3) cell cultures grown at 37 ºC, induced with IPTG 1 mM at 37 ºC. Lane 1: pET11-prosubtilisinE, lane 2: pET11-pro2subtilisinE and lane 3: pET11-pro4subtilisinE. MW: SDS-PAGE Standard, Broad Range (Bio-Rad). The solid arrowheads indicate the position of recombinant proteins. 194 116.5 66.2 45.0 MW 1 2 3 MW 1 2 Figure 8. SDS-PAGE of proteins from E. coli LMG194 transformed with pBAD C*prosequence. Lane 1: soluble fraction and lane 2: insoluble fraction. MW: SDS-PAGE Standard, Broad Range (Bio-Rad). The solid arrowhead indicates the position of recombinant prosequence.
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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 166 and 167: Surface Modification of Cellulose A
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Page 226 and 227: Enzymatic Hydolysis of Wool with a
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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