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

Area of Science:

• Biotechnology
• Enzyme Engineering
• Protein Engineering

Background:

• Poly[(R)-3-hydroxybutyrate] (PHB) is a biodegradable polymer requiring efficient depolymerases for degradation.
• Ralstonia pickettii T1 (PhaZRpiT1) possesses a PHB depolymerase with a catalytic domain responsible for ester bond cleavage.
• Improving the activity and specificity of PHB depolymerases is crucial for biotechnological applications.

Purpose of the Study:

• To enhance the catalytic activity of PhaZRpiT1 through directed evolution.
• To develop efficient screening methods for improved PHB depolymerase mutants.
• To investigate the impact of specific mutations on enzyme kinetics and PHB degradation.

Main Methods:

• Directed evolution targeting the catalytic domain of PhaZRpiT1.
• Cell surface display of mutant enzymes on Escherichia coli.
• Error-prone PCR and staggered extension process (StEP) for generating mutant libraries.
• Assays for p-nitrophenyl butyrate (pNPC4) hydrolysis and PHB degradation.

Main Results:

• Mutant PhaZRpiT1 enzymes displayed a two- to eightfold increase in pNPC4 hydrolysis activity.
• Second-generation mutants exhibited up to a tenfold increase in pNPC3 hydrolysis rate.
• One mutant showed a 3.5-fold decrease in PHB degradation efficiency at a specific enzyme concentration.
• N285D or N285Y mutations were identified in improved mutants, suggesting a role in substrate recognition.

Conclusions:

• Directed evolution combined with cell surface display is effective for improving PhaZRpiT1 activity.
• Specific mutations, particularly at Asn285, significantly influence enzyme kinetics and substrate specificity.
• Further optimization is needed to balance enhanced esterase activity with efficient PHB polymer degradation.