Phospholipase D (PLD) is a major virulence determinant of Corynebacterium pseudotuberculosis. Various studies have focused on the expression of recombinant PLD (rPLD) enzyme in different Escherichia coli strains, but generally a high yield of insoluble protein was reported. The aims of this study were to express soluble rPLD by different methods in E. coli. The rPLD and natural PLD (dPLD) enzymes were concentrated using an ultramembrane cassette system after the efficiencies of these concentrated enzymes were compared. The rPLD enzyme was expressed in One Shot®BL21(DE3) E. coli when induced by IPTG in TY medium. Soluble dPLD and rPLD enzyme hemolytic activities were determined using the reverse CAMP test. The nucleotide sequence of the rPLD gene was 99.7% similar to the PLD gene of C. pseudotuberculosis in the NCBI GenBank Database; these differences in nucleotides resulted in a difference in two amino acids. The rPLD protein concentration and the titer of hemolytic activity were 23.1 mg/mL and 1/256, respectively. Similarities in the enzyme characteristics were detected between rPLD and dPLD enzymes. These findings indicate that a protocol would be useful for the enhanced production of soluble rPLD in E. coli and a membrane cassette system for concentration the recombinant protein.