Characterization of an extracellular poly(3-hydroxy-5-phenylvalerate) depolymerase from Xanthomonas sp. JS02

Area of Science:

• Microbiology
• Biochemistry
• Polymer Science

Background:

• Polyhydroxyalkanoates (PHAs) are biodegradable polymers with diverse applications.
• Medium-chain-length PHAs (PHA(MCL)) like poly(3-hydroxy-5-phenylvalerate) (PHPV) require specific enzymes for degradation.
• Isolation of microorganisms capable of PHA(MCL) degradation is crucial for bioremediation and recycling.

Purpose of the Study:

• To isolate and identify bacteria capable of degrading PHPV.
• To purify and characterize the extracellular enzyme responsible for PHPV hydrolysis.
• To determine the enzymatic properties and substrate specificity of the purified depolymerase.

Main Methods:

• Isolation of bacterium JS02 from wastewater-treatment sludge.
• Purification of extracellular PHPV depolymerase using a series of chromatographic techniques (Sephadex G-75, QAE-Sephadex A-50, hydroxyapatite).
• Enzyme characterization including molecular mass estimation, pH and temperature optima, and substrate specificity assays using PHPV and p-nitrophenyl (PNP)-esters.

Main Results:

• Bacterium JS02, identified as a Xanthomonas species, was isolated and shown to degrade PHPV.
• An extracellular PHPV depolymerase with an estimated molecular mass of 41.7 kDa was purified.
• The purified enzyme hydrolyzed PHPV and PNP-esters of fatty acids, with optimal activity at pH 8.0-9.0 and 60°C.
• The enzyme exhibited specificity for PNP-hexanoate (Km = 10.9 µM) and PNP-octanoate (Km = 0.88 µM), but did not degrade short-chain-length PHAs.

Conclusions:

• Xanthomonas sp. JS02 produces an extracellular depolymerase capable of degrading aromatic medium-chain-length polyhydroxyalkanoates.
• The purified PHPV depolymerase is a distinct enzyme with specific activity and characterized kinetic properties.
• This enzyme has potential applications in the biodegradation and processing of PHA(MCL) polymers.