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|Title:||Recombinant expression of BTA hydrolase in Streptomyces rimosus and catalytic analysis on polyesters by surface plasmon resonance|
Thailand National Center for Genetic Engineering and Biotechnology
|Keywords:||Biochemistry, Genetics and Molecular Biology;Immunology and Microbiology|
|Citation:||Applied Microbiology and Biotechnology. Vol.86, No.6 (2010), 1775-1784|
|Abstract:||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 His6fusion 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-1protein. 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. © 2010 Springer-Verlag.|
|Appears in Collections:||Scopus 2006-2010|
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