Abstract
Active bacterial xanthine oxidase (XOD) has not been functionally expressed in Escherichia coli, limiting protein engineering of this industrial molybdoflavoprotein. Here, the first production of active XOD from Cellulosimicrobium sp. TH20 in E. coli BW25113 is reported. The key factor was identified as the plasmid pBADp15, combining a low-copy P15A origin with the tightly regulated araBAD promoter, with which an enzymatic activity of 0.2 U/mL was obtained-a 6.7-fold improvement over the high-copy vector pBAD18. Activity was completely abolished upon deletion of xodC from the expression cassette, consistent with its established role as an essential maturation chaperone. The purified enzyme exhibited a specific activity of 8.0 U/mg, a Km of 92 μM for xanthine, and optimal activity at pH 7.0 and 50 °C. It is demonstrated that active bacterial XOD can be produced in E. coli when plasmid copy number and promoter stringency are properly balanced, providing a platform for future protein engineering.