[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-012] Functional Expression and Biochemical Characterization of Oxalate Decarboxylase from Bacillus pumilus and Virgibacillus halophilus

○Jeong Ah Yu¹, In Hwan Jang¹, Jun Yeong Jung¹, Yu Jin Park¹, Su Been Im¹, Tae-Yoon Kim¹, Tae-Jip Kim¹ (1. Chungbuk National University, Division of Animal, Horticultural and Food Sciences (Korea))

Keywords:

Oxalate decarboxylases (ODCs),Bacillus pumilus,Virgibacillus halophilus

Oxalate decarboxylase (ODC; EC 4.1.1.2) is an enzyme that catalyzes the decarboxylation of oxalate into formate and CO₂ in the presence of manganese ions. This enzyme belongs to the cupin superfamily and is classified as a bicupin protein with two manganese-binding domains. A representative bacterial ODC, Bacillus subtilis ODC (BsODC), has been reported, but studies on ODC enzymes remain limited. In this study, three novel ODC genes were identified from bacterial genome databases, expressed in Escherichia coli, and their enzymatic properties were characterized. Using the amino acid sequence of BsODC (PDB ID: 1J58) for BLAST sequence similarity analysis, three candidate ODC genes were identified from Bacillus pumilus (BpODC, 385 amino acids), Cohnella mopanensis (CmODC, 397 amino acids), and Virgibacillus halophilus (VhODC, 386 amino acids). The amino acid sequences of these enzymes showed 60.0%, 59.7%, and 65.3% identity to BsODC, respectively. Structural predictions using the AlphaFold server indicated that an overall three-dimensional bicupin fold similar to BsODC was shared by all enzymes. Conserved amino acid residues at structurally equivalent positions suggested the presence of Mn²⁺ binding sites, indicating that these proteins likely function as ODC enzymes. To verify enzymatic function, each putative ODC gene was cloned into the pET-21a(+) vector with a C-terminal 6xHistidine tag, to generate pEBsODC, pEBpODC, pEVhODC, and pECmODC. The resulting recombinant plasmids were transformed into E. coli BL21(DE3) for inducible gene expression. While CmODC (45.0 kDa) was overexpressed in an insoluble form, BsODC (44.6 kDa), BpODC (43.5 kDa), and VhODC (44.0 kDa) were successfully overexpressed in soluble form. The activity of ODC was determined by measuring the amount of formate produced from oxalate. The generated formate was quantified by monitoring the increase in absorbance derived from the reduction of NAD⁺ to NADH by formate dehydrogenase. VhODC and BpODC exhibited optimal activity in the pH range of 3.0–4.0, which is lower than the optimal pH range of BsODC (4.0–5.0). Under their respective optimal pH conditions, specific activities were measured as follows: BsODC (1.25 U/mg), VhODC (0.73 U/mg), and BpODC (0.48 U/mg). This study expands the known diversity of ODC enzymes and provides biochemical data that may support various industrial applications, including enzyme engineering.