Surface Modification of Cellulose Acetate with Cutinase and ...

Subchapter 3.3
in the cellular lysate (Figure 1B). Preparations of purified chimeric enzyme were used
for wool treatment experiments.
Figure 1. SDS-PAGE of A) soluble fraction from two different clones of E. coli
BL21(DE3) transformed with pET25:prosubtilisin-VPAVG220 and B) purified
subtilisin-VPAVG220 polymer stained with copper chloride. The solid arrows indicate
the position of recombinant protein.
3.2. Effect of subtilisin size
210
200.0
200.0
116,0
116.0
97.4
97.4
66.2 66.2
45.0
31.0
22.5
3.2.1. On diffusion trought the fibre
Native subtilisins have a molecular mass of approximately 30 kDa, which is the major
drawback on the application of subtilisins for wool treatment. Due to their relatively
small size the enzymes can diffuse into the fibre cortex causing the degradation of the
internal parts of wool structure. We postulate that an increase of more than four-fold on
subtilisin E molecular weight would prevent diffusion on enzyme into wool. To follow
the diffusion of the enzymes into yarns, they were fluorescently labelled with a
fluorescent dye, FITC. After the covalent coupling of enzymes to FITC, wool yarns
were incubated in these solutions for 24 h. After enzymatic treatment, the yarns were
entrapped in a non-fluorescent resin and sliced in thin layers with a microtome.
Fluorescence microscopy images show that native subtilisin penetrates completely
45.0
31.0
22.5
A B