Isolation and characterization of an extracellular thermoalkanophilic P(3HB-co-3HV) depolymerase from Streptomyces sp. IN1

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• 作者：Adrian Douglas Allen^a ; W.A. Anderson^a ; F. Ayorinde^b ; B.E. Eribo^a ; ^b ; ^{beribo@howard.edu"" rel=""nofollow}
• 关键词：Biodegradation ; P(3HB-co-3HV) depolymerase ; Streptomyces sp. ; Esterase
• 刊名：International Biodeterioration and Biodegradation
• 出版年：2011
• 出版时间：September 2011
• 年：2011
• 卷：65
• 期：6
• 页码：777-785
• 全文大小：951 K

文摘

Here, we report on the biodegradation of the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by a novel thermoalkanophilic extracellular esterase from the soil isolate Streptomyces sp. IN1. Preliminary screening and isolation of the bacterium was done using polyhydroxyalkanoate latex medium (PHALM). The isolate was cultured with P(3HB-co-3HV) as the only carbon source and by-products of degradation were derivatized with [N,O-bis(trimethylsilyl)trifluroacetamide] (BSTFA). These products were identified by gas chromatography/mass spectrometry (GC–MS) as silylated hydroxybutyric acid (3HB) and hydroxyvaleric acid, suggesting extracellular depolymerase activity by the isolate. The depolymerase was isolated by (NH₄)₂SO₄ fractionation, dialyzed and purified using fast protein liquid chromatography (FPLC), and confirmed using P(3HB-co-3HV) as a sole source of carbon. The molecular mass of the FPLC purified enzyme occurred between 45 and 66 kDa (SDS–PAGE), but was confirmed by matrix assisted laser desorption ionization–time of flight mass spectrometry (MALDI–TOF MS) to be 62 kDa. Enzyme activity was significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), dithiothreitol (DTT), and Tween 80, but induced by azide (N³⁻). Sensitivity to PMSF, DTT, and Tween 80 suggests the involvement of serine as an active site amino acid with disulphide bonds contributing to the catalytic activity, as well as the presence of hydrophobic regions in the enzyme. Non-inhibition of activity by azide indicates that metal ions may not be required as cofactors for activity. This observation was further corroborated by the decrease in enzyme activity in the presence of metal ions such as Ca²⁺, Mg²⁺, Na⁺, and K⁺. The kinetic parameters, V_max and K_m, in the presence of p-nitrophenylbutyrate as substrate, were determined to be 5.06 × 10⁻¹ μmol min⁻¹ and 6.73 × 10⁻¹ mM, respectively.