Surface Modification of Cellulose Acetate with Cutinase and ...

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