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A recombinant polyester-degrading hydrolase from Thermobifida sp. BCC23166 targeting on aliphatic-aromatic copolyester (rTfH) was produced in Streptomyces rimosus R7. rTfH was expressed by induction with thiostrepton as a C-terminal His(6) fusion from the native gene sequence under the control of tipA promoter and purified from the culture supernatant to high homogeneity by a single step affinity purification on Ni-Sepharose matrix. The enzyme worked optimally at 50-55 degrees C and showed esterase activity on C3-C16 p-nitrophenyl alkanoates with a specific activity of 76.5 U/mg on p-nitrophenyl palmitate. Study of rTfH catalysis on surface degradation of polyester films using surface plasmon resonance analysis revealed that the degradation rates were in the order of poly-epsilon-caprolactone > Ecoflex > polyhydroxybutyrate. Efficient hydrolysis of Ecoflex by rTfH was observed in mild alkaline conditions, with the highest activity at pH 8.0 and ionic strength at 250 mM sodium chloride, with the maximal specific activity of 0.79 mg(-1)min(-1)mg(-1) protein. Under the optimal conditions, rTfH showed a remarkable 110-time higher specific activity on Ecoflex in comparison to a lipase from Thermomyces lanuginosus, while less difference in degradation efficiency of the two enzymes was observed on the aliphatic polyesters, suggesting greater specificities of rTfH to the aliphatic-aromatic copolyester. This study demonstrated the use of streptomycetes as an alternative expression system for production of the multi-polyester-degrading enzyme of actinomycete origin and provided insights on its catalytic properties on surface degradation contributing to further biotechnological application of this enzyme.

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A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His(6)-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline-serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V(max) and K(m) of 3.63 +/- 0.16 micromol min(-1) mg(-1) and 0.79 +/- 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50-55 degrees C and pH 7-8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm(-2) h(-1) for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.