Hydrophobilization of esterase by genetic combination with polyproline or polytyrosine at the carboxyl terminal.
Polyproline (Poly-Pro) that is an amphiphilic polypeptide, or polytyrosine (Poly-Tyr) that is a hydrophobic polypeptide, were connected to the carboxy terminus of Pseudomonas sp. esterase by the recombinant DNA technique. The hydrophobicity of the esterase was enhanced by the introduction of Poly-Pro or Poly-Tyr, and also by increasing chain length of the polypeptides. Poly-Tyr increased the hydrophobicity of esterase more than Poly-Pro. Poly-Tyr induced significant conformational change of fusion esterase, but Poly-Pro did not. Consequently, the introduction of Poly-Tyr led to loss of activity of the fusion enzyme to a negligible level. On the other hand, the Poly-Pro-fusion-esterase retained enzymatic activity and the hydrolytic activity (kcat/Km) of the fusion esterase carrying 40 proline residues (esterase-Pro40) relative to that of the wild-type esterase with the substrates p-nitrophenyl-propionate, -pentanoate, and -hexanoate was 1.76, 1.95, and 4.7, respectively. The results could be explained in terms of easier access of long-chain carboxylate to the fusion esterase compared to the wild-type esterase in aqueous solution.
Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan.
O H
OH
Kwon
Y
Y
Ito
M
M
Ueda
A
A
Tanaka
Y
Y
Imanishi
D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics*; D011549:Pseudomonas / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D010455:Peptides; D011993:Recombinant Fusion Proteins; C011083:polyproline; C017663:polytyrosine; D002265:Carboxylic Ester Hydrolases