Surface Modification of Cellulose Acetate with Cutinase and ...

Strategies Towards the Functionalization of Bacillus subtilis Subtilisin E for Wool Finishing Applications
LMG194 were grown in RM medium. In both cases ampicillin was used at the
concentration of 100 μg/μL. Cells were induced according to conditions described in
table IV.
Table III. Primers used to generate and amplify B. subtilis presequence. XhoI
restriction site is underlined.
Gene Primer (5´→ 3´) bp GC
%
Presequence
Fpres CTC GAG TGA GAA GCA AAA AAT TGT GGA TCA GCT TGT
TGT TTG CGT TAA CGT TAA TCT TTA CGA
63 38.1
Rpres CTC GAG CCT GCG CAG ACA TGT TGC TGA ACG CCA TCG
TAA AAG TTA ACG TTA AGC CAA ACA ACA
63 47.6
Famp CCC TCG AGT GAG AAG CAA AA 20 50
Ramp CCC TCG AGC CTG CGC AGA CA 20 70
Table IV. Fermentations conditions performed for E. coli pET25b (+) and pBAD
vectors.
E. coli strain/ Vector
Temperature
of growth (ºC)
[Inducer]
Temperature of
induction (ºC) /
time
BL21(DE3)/ pET25b (+) 37; 30; 25 IPTG 1.0; 0.5; 0.3 (mM) 30;18; 4 h
BL21(DE3)pLysS/ pET 25b (+) 30; 25 IPTG 1.0; 0.5; 0.3 (mM) 18 h
Tuner/ pET25b (+) 30; 25 IPTG 1.0; 0.5; 0.3 (mM) 18 h
TOP10 and LMG194/ pBAD C* 37; 30 Arabinose 0.2; 0.1(%) 18 h / ON and 3 h
2.6. Cell fractionation
Overnight cell cultures from all the strains transformed with pET25 and pBAD C*
constructions were harvested by centrifugation (5000 rpm for 15 minutes) and
resuspended in Osmotic Solution I (OS I: 20 mM Tris-HCl pH 8.0, 2.5 mM EDTA, 2
mM CaCl2, sucrose 20%, w/v) to an OD600 of 5.00. Cells resuspended in OS I were
incubated on ice for 10 min and centrifuged at 4 ºC. Supernatants were decanted and the
cell pellets resuspended in the same volume of Osmotic Solution II (OS II: 20 mM Tris-
HCl pH 8.0, 2.5 mM EDTA, 2 mM CaCl2). The suspension was incubated for 20 min
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