31.10.2012 Views

Surface Modification of Cellulose Acetate with Cutinase and ...

Surface Modification of Cellulose Acetate with Cutinase and ...

Surface Modification of Cellulose Acetate with Cutinase and ...

SHOW MORE
SHOW LESS

You also want an ePaper? Increase the reach of your titles

YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.

Enzymatic Hydolysis <strong>of</strong> Wool <strong>with</strong> a Genetically Modified Subtilisin E<br />

described above. Volumes <strong>of</strong> phosphate buffer solution (pH 7.6, 0.01 M) <strong>and</strong> protein<br />

stock solution were added to the sorbent so that every flask contained the same total<br />

volume (50 ml) <strong>and</strong> the same units <strong>of</strong> enzyme activity. Then, the flasks were closed <strong>and</strong><br />

rotated at 90 rpm for 24 h at 37 ºC in a shaking water bath. Several controls were run<br />

simultaneously: a control test <strong>with</strong> wool <strong>with</strong>out pretreatment <strong>and</strong> <strong>with</strong>out protein (C), a<br />

control test <strong>with</strong> surfactant washed wool (C, S) <strong>and</strong> <strong>with</strong>out protein <strong>and</strong> a control test<br />

<strong>with</strong> bleaching washed wool (C, S+B) <strong>and</strong> <strong>with</strong>out protein. After incubation, wool yarns<br />

were removed <strong>and</strong> washed for tensile strength, felting <strong>and</strong> pilling evaluation.<br />

2.8.1. Tensile strength<br />

Tensile strength resistance was determined by using a tensile tester machine,<br />

accordingly to ASTMD5035-90. The samples were conditioned before testing in a<br />

st<strong>and</strong>ard atmosphere.<br />

The tensile strength resistance values are given as the mean <strong>of</strong> an n≥10 replicates,<br />

together <strong>with</strong> the st<strong>and</strong>ard deviation (the coefficient <strong>of</strong> variation was bellow 10% for all<br />

cases).<br />

2.8.2. Felting <strong>and</strong> pilling<br />

Felting <strong>and</strong> pilling were visually evaluated after repeated washing (3 times) at 50 ºC, for<br />

60 min <strong>and</strong> 20 rpm.<br />

3. Results<br />

3.1. Expression <strong>and</strong> purification <strong>of</strong> recombinant protein<br />

E. coli BL21(DE3) transformants carrying pET25:proSubtilisin-GAG220 grown <strong>and</strong><br />

induced at 30 ºC were able to express the quimeric enzyme at high level in the soluble<br />

fraction (Figure 1A). The SDS-PAGE analysis <strong>of</strong> the cellular lysate revealed the<br />

presence <strong>of</strong> a soluble chimeric protein <strong>with</strong> a high molecular weight (> 116 kDa). This<br />

chimeric protein was efficiently purified from all other contaminating proteins present<br />

209

Hooray! Your file is uploaded and ready to be published.

Saved successfully!

Ooh no, something went wrong!