Study on expression system of catechol 2,3-dioxygenase gene in e.coli

A 924bp fragment encoding catechol 2,3-dioxygenase gene was got from the Pseudomonas sp. S-47 DNA by polymerase chain reaction (PCR) method. The PCR product was digested with BamHI and Sad, and inserted into the pBluescript SK vector. The sequencing result indicated that the nucleotide sequence of the xylE gene was the same as that reported by Kim in 2000. The gene was inserted into a vector pG251 with gentamicin resistance gene promoter and tlt2 terminator, and into vectors pYF395 and pYF5254 respectively with ompA and EAP signal sequences, and Ipp and Ga~r promoters, and so the constitutive expression vector pG251 and the secretion vectors pYFXl and pYFX2 were constructed. The determination of enzyme activity showed that pG251 was 665 mu/mL, pYFXl 1815 mu/mL and pYFX2 1015mu/mL. The SDS-PAGE revealed that the expression product of xylE gene was about 35 kD protein.