title: Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119.
abstract: 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.
authors.affiliation: Division of Applied Biology, Graduate School of Science and Technology, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto, Japan.
authors.forename: Uschara
authors.initials: U
authors.lastname: Thumarat
authors.forename: Ryota
authors.initials: R
authors.lastname: Nakamura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kawabata
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Suzuki
authors.forename: Fusako
authors.initials: F
authors.lastname: Kawai
mesh_terms: D000192:Actinomycetales / Q000201:enzymology*; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D020536:Enzyme Activators; D050505:Mutant Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D002118:Calcium