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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<strong>Surface</strong> <strong>Modification</strong> <strong>of</strong> <strong>Cellulose</strong> <strong>Acetate</strong> <strong>with</strong> <strong>Cutinase</strong> <strong>and</strong> <strong>Cutinase</strong> Fused to Carbohydrate-binding Modules<br />
2.10. Wide Angle X-ray Scattering<br />
The X-ray diffraction patterns were obtained for the CDA <strong>and</strong> CTA fabric samples<br />
treated during 24 hours <strong>with</strong> cutinase <strong>and</strong> respective controls. The X-ray diffraction<br />
experiments were undertaken in a Philips PW1710 apparatus, using Cu Kα radiation<br />
<strong>and</strong> operating at a 40 KV voltage <strong>and</strong> 30 mA current. The patterns were continuously<br />
recorded in the diffraction angular range 2θ from 4º to 40º, <strong>with</strong> a step size <strong>of</strong> 0.02º at<br />
0.6ºmin -1 . The non linear fitting <strong>of</strong> the diffraction patterns was performed using the<br />
Pseudo-Voigt peak function from OriginPro 7.5 (Origin Lab Corporation, USA)<br />
considering the cellulose acetate structural polymorphism II (Cerqueira et al., 2006).<br />
The crystallinity index was determined according to the equation (1)<br />
I<br />
A<br />
= c<br />
(1)<br />
( Ac<br />
+ a)<br />
C A<br />
Ac is the total area <strong>of</strong> the crystalline peaks <strong>and</strong> Aa is the total area <strong>of</strong> the amorphous<br />
peaks. The peaks that were considered crystalline were at the diffraction angles 2º 11º<br />
<strong>and</strong> 17º, for CDA, <strong>and</strong> 8º, 10º, 13º, 17º, 21º <strong>and</strong> 23º, for CTA (Chen et al., 2002;<br />
Hindeleh <strong>and</strong> Johnson, 1972).<br />
2.11. Cloning <strong>and</strong> expression <strong>of</strong> cutinase fused to carbohydrate-binding modules<br />
2.11.1. Bacteria, plasmids <strong>and</strong> genes<br />
The bacterial hosts used for cloning <strong>and</strong> expression <strong>of</strong> cutinase fusion genes were the<br />
Escherichia coli strain XL1-Blue <strong>and</strong> strain BL21 (DE3), respectively. The plasmid<br />
pGEM ® -T Easy (Promega Corporation, Madison, USA) was used to clone <strong>and</strong> sequence<br />
the PCR products. The plasmid pCWT (pET25b(+) carrying native cutinase gene from<br />
F. solani pisi, (Araújo et al., 2007) was used to insert the genes coding for the CBMs at<br />
the 3’ end <strong>of</strong> the cutinase gene <strong>and</strong> to express the fusion proteins.<br />
The DNA coding the wild type linker <strong>and</strong> wild type CBM <strong>of</strong> T. reesei CBH I,<br />
wtCBMCBHI, was synthesized <strong>and</strong> purchased from Epoch Biolabs (Missouri City, USA),<br />
as well as, the DNA fragment coding for a smaller linker <strong>and</strong> the wild type CBM,<br />
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