TABLE 2.
Site-directed mutagenesis of catB from P. putida PRS2000 and the respective changes on the DNA and protein levels
| Amino acid sub- stitution | Nucleotide substitution in catBa | Mutagenesis oligonucleotideb |
|---|---|---|
| I54V | ATC→GTC | 5′-CAGGCCACCGACGGTGGTGG-3′ |
| Y59W | TAT→TGG | 5′-CGTAGCCCCAGGCCAGGCCAC-3′ |
| A271S | GCC→TCC | 5′-CGATTTTCAGGGAAAAGATGC-3′ |
| K276N | AAG→AAC | 5′-GCCGCCGTTGTTGGCGATTTTC-3′ |
| F329I | TTC→ATC | 5′-CAGCGGGCCGATCAGCTCGG-3′ |
| L333V | CTG→GTG | 5′-CGGTCAGCACCAGCGGGCCG-3′ |
| I54V-Y59W | ATC→GTC and TAT→TGG | 5′-GCCCCAGGCCAGGCCACCGACG GTG-3′ |
a
Bases in boldface were exchanged.
b
Sequences of mutagenesis oligonucleotides were complementary, in an inverted orientation, to single-stranded templates. Characters in boldface indicate nucleotide changes compared to the wild-type sequence.