Purification and Characterization of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Depolymerase from Thermophilic Actinomadura sp. TF1

Abstract

Actinomadura sp. strain TF1, a thermophilic actinomycete bacterium that produces an enzyme capable of degrading the bioplastic copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), was cultured in basal medium under optimized conditions at pH 8, 45 °C, and 0.4% (w/v) PHBV substrate concentration, as previously studied, to achieve the highest production of PHBV depolymerase. The enzyme was purified through 80% (w/v) ammonium sulphate precipitation, dialysis, and Diethylaminoethyl cellulose (DEAE) column chromatography, resulting in a specific activity of 463.7 U/mg protein, 49.9% recovery, and a 2.8-fold purification. The molecular weight of the purified PHBV depolymerase was determined to be 35 kDa by SDS-PAGE. The enzyme exhibited optimal activity at 45 °C and demonstrated thermal stability within the range of 35-45 °C. The optimal pH for activity was 8, with stability observed between pH 8 and 9. PHBV depolymerase displayed strong activity against substrates such as PHBV, polyhydroxybutyrate (PHB), and polybutylene succinate (PBS), with the highest degradation activity observed for PHBV. Additionally, the enzyme hydrolyzed p-nitrophenol esters, confirming its classification as an esterase. The activity of PHBV depolymerase was enhanced by the presence of Ca²⁺, Na⁺, and K⁺ ions but was inhibited by Fe²⁺, Mg²⁺, Mn²⁺, Zn²⁺, Cu²⁺, and Hg²⁺ ions. Dithiothreitol (DTT) exhibited a significant inhibitory effect. In conclusion, this study is the first to report the purification and characterization of PHBV depolymerase from Actinomadura sp. strain TF1.