Surface Modification of Cellulose Acetate with Cutinase and ...

Tailoring Cutinase Activity Towards Polyethylene Terephthalate and Polyamide 6,6 Fibers
Table I. Primers used for site-directed mutagenesis of cutinase gene from Fusarium
solani pisi. The codons corresponding to the specific mutations introduced are indicated
in bold. The overlapping regions (22 bp) of primers forward (F) and reverse (R) are
underlined.
Mutation Primer (5´→ 3´) Bp GC %
L81A F1 CTCTCCCTCGCGGAACCTCTAGCGCCGCAATCAGGGAGA 39 64.1
R1 CTAGAGGTTCCGCGAGGGAGAGCATTGTCTCCGGCAGTGGCTCGGTAGGCAC 52 63.5
N84A F2 GCGGAACCTCTAGCGCCGCAATCAGGGAGATGCTCGGTC 39 64.1
R2 ATTGCGGCGCTAGAGGTTCCGCGAGGGAGAGCGGCGTCTCCAAGAGTGGCTC 52 65.4
L182A F3 CACCTCACTTGGCTTATGGTCCTGATGCTCGTGGCCCTG 39 59.0
R3 GGACCATAAGCCAAGTGAGGTGCAGCAACGATGGCGCTACCAGTACAAACGA 52 53.8
V184A F4 ACTTGGCTTATGGTCCTGATGCTCGTGGCCCTGCCCCTG 39 61.5
R4 GCATCAGGACCATAAGCCAAGTGAGGTGCAGCGGCGATCAAGCTACCAGTAC 52 55.8
L189A F5 CTGATGCTCGTGGCCCTGCCCCTGAGTTCCTCATCGAGA 39 61.5
R5 GGGGCAGGGCCACGAGCATCAGGACCATAAGCGGCGTGAGGTGCAGCAACG 51 66.7
The pET25b (+) carrying native and genetically modified cutinases were first
established in E. coli strain XL1 Blue, according to the SEM method (Inoue et al.,
1990), and the presence of each specific mutation was confirmed by sequencing. DNA
cloning and manipulation were performed according to the standard protocols
(Sambroock et al., 1989). T7 expression host strain BL21(DE3) was used for protein
expression.
Strains were grown at 37 ºC in Luria-Broth medium, supplemented with 50 μg/ml
ampicillin until an absorbance A600 nm of 0.6 was reached. Cells were then induced by
adding isopropyl-1-thio-β-galactopyranoside (IPTG) (final concentration 1 mM),
followed by 16 h at 18 ºC. The cells were harvested by centrifugation (5000 rpm for 10
min) and washed twice with phosphate buffered saline solution (10 mM Na2HPO4,
2 mM KH2PO4, 137 mM NaCl, 3 mM KCl, pH 7.4), supplemented with a mixture of
protease inhibitors. Ultrasonic treatment of bacterial cells was performed at 20 KHz
with a 13-mm probe in an Ultrasonic Processor GEX 400. Four 2-min pulses with 2 min
in ice between each pulse were performed. The lysate was centrifuged for 30 min at
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