Surface Modification of Cellulose Acetate with Cutinase and ...
Surface Modification of Cellulose Acetate with Cutinase and ...
Surface Modification of Cellulose Acetate with Cutinase and ...
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Enzymatic Hydolysis <strong>of</strong> Wool <strong>with</strong> a Genetically Modified Subtilisin E<br />
The plasmid construct was verified by restriction <strong>with</strong> LguI <strong>and</strong> by DNA sequencing,<br />
following the method <strong>of</strong> Sanger (Sanger et al., 1977), using an ABI PRISM 310 Genetic<br />
Analyzer. DNA cloning <strong>and</strong> manipulation were performed according to the st<strong>and</strong>ard<br />
protocols (Sambroock et al., 1998). The recombinant plasmids were then transformed<br />
into the expression strain Escherichia coli BL21(DE3) (Novagen).<br />
2.4. Protein expression <strong>and</strong> purification<br />
Expression host strain BL21(DE3) transformed <strong>with</strong> pET25:proSubtilisin-VPAVG220<br />
was used for protein expression. Bacterial cultures were grown at 30 ºC in Novagen<br />
Auto-induction medium, containing 100 μg/mL ampicilin. After fermentation time,<br />
(usually overnight) the cells were harvested by centrifugation, washed <strong>with</strong> phosphate<br />
buffered saline solution (10 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, 3 mM KCl,<br />
pH 7.4) <strong>and</strong> lysed by ultrasonic disruption (Ultrasonic Processor GEX 400).<br />
Supernantant was reserved for protein purification <strong>and</strong> the pellet, insoluble debris, was<br />
ressuspended in phosphate buffered saline solution <strong>and</strong> reserved for analysis by sodium<br />
dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE.<br />
Recombinant protein was purified using an IMAC system <strong>with</strong> a HisPrep FF 16/10<br />
column (GE Healthcare) already prepacked <strong>with</strong> pre-charged Ni Sepharose 6 Fast Flow.<br />
Column equilibration was performed <strong>with</strong> 10 mM imidazole, 0.5 M NaCl, 20 mM<br />
phosphate buffer pH 7.6. Samples were applied onto the column at a flow rate <strong>of</strong> 2<br />
ml.min -1 . Elution was performed <strong>with</strong> a buffer containing 80 mM imidazole, 0.5 M<br />
NaCl <strong>and</strong> 20 mM phosphate buffer, pH 7.6.<br />
Proteins were detected by SDS-PAGE using a Tris-SDS-glycine buffer system<br />
(Laemmli, 1970). SDS PAGE gel images were acquired <strong>with</strong> Molecular Imager<br />
ChemiDoc XRS system <strong>and</strong> Quantity One s<strong>of</strong>tware from Biorad. Protein detection was<br />
done by Coomassie Brilliant Blue R250.<br />
2.5. Enzyme assay <strong>and</strong> protein concentration<br />
The activity <strong>of</strong> commercial Esperase <strong>and</strong> chimeric subtilisin was measured, according to<br />
Silva et al., (2004) using casein as substrate. One unit <strong>of</strong> activity is defined as the<br />
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