Recombinant expression of BTA hydrolase in Streptomyces rimosus and catalytic analysis on polyesters by surface plasmon resonance
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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 His6 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°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-ε-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−1min−1mg−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.
KeywordsAliphatic-aromatic copolyester Ecoflex® Hydrolase Streptomyces rimosus Surface plasmon resonance Thermobifida sp.
This project was supported by the Thailand Research Fund (TRF). The authors would like to thank Dr. Phillip James Shaw for manuscript proofreading. N.S. was granted YSTP senior project studentship from NSTDA. T.W. was granted the Development and Promotion of Science and Technology Talents Project (DPST) Fund.
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