Abstract: A hydroxyquinol 1,2-dioxygenase gene(pnpC) insertional inactivated mutant(DLL-ΔpnpC1) and a pnpC knock-out mutant(DLL-ΔpnpC) were constructed by homologous recombination from Pseudomonas putida DLL-E4.Insertional inactivated mutant DLL-ΔpnpC1 lost the p-nitrophenol(PNP) and hydroquinone(HQ) degradation ability.However,pnpC knock-out mutant DLL-ΔpnpC recovered the ability of utilizing PNP and HQ at a lower degradation rate compared with DLL-E4.Crude cell extracts of DLL-E4 and DLL-ΔpnpC induced by PNP and catechol were determined with catechol as substrate.The differences between crude enzyme′s activity of DLL-E4 and that of DLL-ΔpnpC on catechol in ammonium sulfate precipitation were compared.The results indicate that another dioxygenase was expressed in DLL-ΔpnpC which replaced pnpC with the same function in the catabolism of PNP.