TY - JOUR
T1 - Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119
AU - Thumarat, Uschara
AU - Nakamura, Ryota
AU - Kawabata, Takeshi
AU - Suzuki, Hideyuki
AU - Kawai, Fusako
N1 - Funding Information:
Acknowledgements The study was supported by funds from the Institute for Fermentation, Osaka (Japan) to F. K. We appreciate Prof. Y. Yamamoto, Institute for Plant Resources, Okayama University, for her kind help in atomic absorption spectrometry measurements. We are grateful to Dr. T. Nakajima-Kambe, University of Tsukuba, for his kind supply of PBSA (Bionolle™EM-301). This work is linked with the Asia Core Program supported by JSPS (Japan) and NRCT (Thailand).
PY - 2012/7
Y1 - 2012/7
N2 - Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β- sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca 2+ and was stable up to 60 °C with 150 mM Ca 2+. Another identical gene was located in tandem in the upstream of est119.
AB - Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β- sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca 2+ and was stable up to 60 °C with 150 mM Ca 2+. Another identical gene was located in tandem in the upstream of est119.
KW - Ca-dependent cutinase
KW - Cutinase-type polyesterase
KW - Polyester degradation
KW - Thermobifida alba AHK119
KW - Thermostable polyesterase
UR - http://www.scopus.com/inward/record.url?scp=84864717982&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84864717982&partnerID=8YFLogxK
U2 - 10.1007/s00253-011-3781-6
DO - 10.1007/s00253-011-3781-6
M3 - Article
C2 - 22183084
AN - SCOPUS:84864717982
SN - 0175-7598
VL - 95
SP - 419
EP - 430
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
IS - 2
ER -