Surface Modification of Cellulose Acetate with Cutinase and ...

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