Surface Modification of Cellulose Acetate with Cutinase and ...

Subchapter 3.2
activity is defined to be the amount of enzyme required to produce an absorbance
change of 1.0 in a 1 cm cuvette, under the conditions of the assay.
3. Results
3.1. Expression of recombinant proteins using pET25b (+) expression system
All the constructions reported in this work were originally cloned by PCR from the
vector pET11a:pro-subtilisin (Hu et al., 1994). The final constructions that are
described in table 2 were transformed into the appropriate strains. Depending on the
vector used, the expression of recombinant proteins was induced with IPTG or with
arabinose, as well as, by varying the cell cultures temperature incubation, as described
in Material and Methods.
E. coli BL21(DE3) cells transformed with pET25b (+) constructions (table II), grown at
37 ºC, were able to express prosubtilisinE, pro2subtilisinE and pro4subtilisinE in the
presence of 1 mM IPTG at 30 ºC at a high level (figure 2). The molecular mass of
prosubtilisinE, estimated as 45.0 kDa, is not in agreement with the expected molecular
weight for mature subtilisinE, (30 kDa) (Ikemura et al., 1987). The chimeric proteins
pro2subtilisinE and pro4subtilisinE showed a molecular mass near 67 and 125 kDa,
respectively, probably due to the unprocessed pelB-prosubtilisinE, pelB-pro2subtilisinE
and pelB-pro4subtilisinE. The theoretical molecular mass of the processed proteins
according to Expasy is near 58 and 116 kDa. pelB leader signal peptide directs the
recombinant proteins to the periplasmic space, where they were mostly expected to be
found. However, recombinant proteins were only found in the insoluble fractions
(pellets) (figure 2). The same results were obtained for expression strains
BL21(DE3)pLysS and Tuner using the same fermentation conditions (data not shown).
In order to increase the solubility of the recombinant proteins, all bacterial strains were
grown under different temperatures (30 and 25 ºC) and induction phase was performed
with lower concentrations of IPTG (0.5 and 0.3 mM) at lower incubation temperatures
(18 and 4 ºC). It was assumed that combining decreasing of temperature with lower
concentrations of inducer would prevent overloading the E. coli periplasmic transport
system and recombinant enzymes would be able to fold properly. However, none active
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