Effects of mutation at position 285 of Ralstonia pickettii T1 poly[(R)-3-hydroxybutyrate] depolymerase on its activities.

Asn at position 285 (N285) in the catalytic domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 most likely participates in the cleavage of ester bonds as revealed by our previous evolutionary engineering study using the error-prone polymerase chain reaction (PCR) method. To exhaustively examine the effects of mutations at that position, we conducted site-directed saturation mutagenesis at that position and the resultant mutant enzymes (N285X) were evaluated in p-nitrophenyl ester (pNPCn) hydrolysis and PHB degradation. Kinetic studies demonstrated that the PHB-degrading activities of N285X were reciprocally related to their pNPCn-hydrolyzing activities, with the exception of N285A and N285G, and that His residue could functionally substitute for Asn285 on PHB degradation.

Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285.

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.

Display of functionally active PHB depolymerase on Escherichia coli cell surface.

The display of PHB depolymerase (PhaZ(RpiT1) ) from R. pickettii T1 on the surface of E. coli JM109 cells is realized using OprI of P. aeruginosa as the anchoring motif. The fusion protein is stably expressed and its surface localization is verified by immunofluorescence microscopy. The displayed PhaZ(RpiT1) retains its cleaving ability for soluble substrates as well as its ability to adsorb to the PHB surface, and also remains catalycically active in the degradation of insoluble polyester materials, in spite of the possible suppression of the enzyme movement on the polymer surface. The results demonstrate that PhaZ(RpiT1) -displaying E. coli shows potential for use as a whole-cell biocatalyst for the production of (R)-3-hydroxybutyrate monomers from insoluble PHB materials.