Anjulal_2026_Enzyme.Microb.Technol_193_110767

The ability of Nocardiopsis dassonvillei NCIM 5124 to synthesize polyhydroxybutyrate depolymerase (PHBD) was recently reported. In this investigation, in vitro codon optimized gene synthesis, overexpression, and biochemical characterization of this enzyme along with molecular docking studies are presented. The sequence of the PHBD was inserted in pET-28a(+) along with the PelB_Signal and His(6) tag to generate the recombinant vector pET-Nd-pelB_PHBD. The transformed Escherichia coli BL21(DE3) could produce active PHBD. This enzyme was purified using Ni-NTA affinity chromatography, producing a product with a molecular weight of roughly 50 kDa. The optimum temperature and pH of the recombinant enzyme were 35 degreesC and 8.0, respectively. Triton X100 and Tween 20 inhibited the enzyme activity by 90 %, indicating the role of hydrophobic residues in the active site of the enzyme, as also noted during docking studies. On the basis of the Michaelis-Menten equation, apparent K(m) and V(max) of recombinant PHBD were found to be 1.782 mg/mL and 4.79 U/mL/min, respectively. Molecular docking studies indicated that the hydrophobic amino acids Cys 39, Ala 40, Cys 77, Phe 158, Met 161, Val 201, Ala 272, and Tyr 273 present in the catalytic site were providing the necessary hydrophobic environment for binding of the ligand. The purified enzyme could also degrade films of PHB and poly (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate (PHBVH). As far as we are aware, this is the first report on the overexpression of PHBD from Nocardiopsis sp.