title: Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285.
abstract: Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZRpiT1) consists of three functional domains to effectively degrade solid PHB materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate. We performed the directed evolution of PhaZRpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved p-nitrophenyl butyrate (pNPC4) activity. Mutated PhaZRpiT1 genes generated by error-prone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface. Some cells displaying the mutant enzymes showed a two- to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wild-type enzyme. These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five- to eightfold higher pNPC4 hydrolysis activity than the wild type. To further evaluate the mutation effects, unfused and undisplayed enzymes were prepared and applied to the hydrolysis of p-nitrophenyl esters having different chain lengths (pNPCn; n = 2-6) and PHB degradation. One specific second-generation mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis, although its PHB degradation efficiency at 1 μg/mL of enzyme concentration was approximately 3.5-fold lower than that of the wild type. Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved second-generation mutants, indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residues.
authors.affiliation: Bioengineering Laboratory, RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
authors.forename: Liu-Tzea
authors.initials: LT
authors.lastname: Tan
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Hiraishi
authors.forename: Kumar
authors.initials: K
authors.lastname: Sudesh
authors.forename: Mizuo
authors.initials: M
authors.lastname: Maeda
mesh_terms: D001216:Asparagine / Q000235:genetics* / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D061505:Cell Surface Display Techniques / Q000379:methods; D019020:Directed Molecular Evolution / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods; D049920:Ralstonia pickettii / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D011994:Recombinant Proteins; D001216:Asparagine; D002265:Carboxylic Ester Hydrolases; C022417:poly-beta-hydroxybutyrate depolymerase