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Phthalate esters are known to be endocrine disrupting chemicals and are documented to pollute environments. Enzymatic degradation of PAEs is a potential bioremedial strategy to manage contamination. Thermostable bioremedial enzymes have advantages in enzyme manufacturing and storage. In this study, we identified, overexpressed, and characterised a moderately thermostable para-nitrobenzyl esterase from whole genome sequencing of a Bacillus velezensis NP05 from the Great Artesian Basin , capable of sequential 2-step hydrolysis of diisobutyl phthalate. The pnbA enzyme has a molecular weight of 55.14 kDa and pI of 5.31. It preferentially degrades para-nitrophenyl butanoate and has an optimal pH of 7-8. The pnbA esterase has an optimal temperature of 55 °C with a half-life of 4 h. Using HPLC we found that pnbA (0.122 U) can hydrolyse 0.83 mM of DIBP within 25 min. Lastly, pnbA is potentially a more economically viable candidate for enzymatic bioremediation of diisobutyl phthalate as a free enzyme., Competing Interests: None., (© 2024 The Authors. Published by Elsevier B.V.)