Recombinant Expression and Characterization of an Arginine Deiminase from Pseudomonas sp. LJY.

Arginine deiminase (ADI) is an Arg-degrading enzyme of the guanidino-group-modifying enzymes (GME) superfamily, which can hydrolyze L-Arg to L-Cit and ammonia through guanidine deamination. It is one of the key enzymes of the ADI pathway in microorganisms. In this study, ADI coding gene arcA of Pseudomonas sp. LJY isolated from the intestine of swamp eel was cloned. Subsequently, the prokaryotic expression vector pET28a-arcA was constructed and transformed into Escherichia coli BL21 (DE3). The recombinant protein ADI (rPS-ADI) was induced by IPTG and purified by Ni-NTA His·Bind Resin affinity column. The rPS-ADI was detected by SDS-PAGE and Western blot. The activity of rPS-ADI was examined by TLC assay, and the enzymatic properties of rPS-ADI were characterized. The arcA gene of Pseudomonas sp. LJY had 1257 bp and coded 417 amino acids. Multiple sequence alignments showed that Pseudomonas sp. LJY ADI (PS-ADI) has high similarity with other Pseudomonas bacteria, and its amino acid sequence contains a highly conserved catalytic triplet Cys406-His268-Glu224. TLC assay showed that rPS-ADI catalyzed L-Arg to generate L-Cit. The optimal reaction temperature and pH of rPS-ADI are 45°C and 2.0, respectively. Moreover, Al3+, Cd2+, Zn2+ and EDTA can promote the activity of rPS-ADI. The KM and Vmax of rPS-ADI for L-Arg were 22.42 mM and 68.49 μmol/min/mg, respectively. This study provides promising foundation to further understanding the amino acid metabolism and ADI function of Pseudomonas sp. LJY. [ABSTRACT FROM AUTHOR]