Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis

Area of Science:

• Biotechnology
• Enzymology
• Microbial Genetics

Background:

• Poly(3-hydroxybutyrate) depolymerases are crucial for biodegrading polyhydroxyalkanoates.
• Purifying these enzymes from Paucimonas lemoignei is challenging due to multiple, difficult-to-separate isoenzymes.
• The phaZ5 gene encodes extracellular poly(3-hydroxybutyrate) depolymerase A.

Purpose of the Study:

• To achieve efficient functional expression and secretion of P. lemoignei poly(3-hydroxybutyrate) depolymerase A in a heterologous host.
• To develop a simplified purification strategy for the recombinant enzyme.
• To compare the properties of the recombinant, non-glycosylated enzyme with the wild-type enzyme.

Main Methods:

• Functional expression of the phaZ5 gene in a protease-negative Bacillus subtilis mutant (WB800) using the pWB980 vector.
• Purification of the secreted recombinant depolymerase from culture supernatant via a single chromatography step.
• Characterization of the recombinant enzyme's molecular weight, activity, and thermal stability.
• Comparison with the wild-type enzyme from P. lemoignei.

Main Results:

• The recombinant poly(3-hydroxybutyrate) depolymerase was successfully expressed and secreted in B. subtilis.
• Purification required only one chromatography step, yielding 1.9 mg/L of enzyme.
• The recombinant enzyme was non-glycosylated, unlike the wild-type enzyme.
• Glycosylation was not essential for enzymatic activity but contributed to thermal stability.

Conclusions:

• Bacillus subtilis is an efficient host for overexpressing poly(3-hydroxybutyrate) depolymerase, simplifying purification compared to P. lemoignei.
• Recombinant expression provides a more accessible source of the enzyme for research and potential applications.
• The study elucidates the role of glycosylation in enzyme stability, offering insights for enzyme engineering.