Assay of poly(3-hydroxybutyrate) depolymerase activity and product determination.

Area of Science:

• Biochemistry
• Polymer Science
• Analytical Chemistry

Background:

• Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer with significant industrial applications.
• Accurate determination of PHB depolymerase activity is crucial for understanding its degradation pathways.
• Existing methods for analyzing PHB hydrolysis products have limitations in sensitivity and scope.

Purpose of the Study:

• To develop and validate novel methods for quantifying PHB depolymerase activity.
• To establish techniques for the precise identification and quantification of PHB hydrolysis products.
• To apply these methods to study PHB hydrolysis by various depolymerases.

Main Methods:

• Online titration using a pH-stat apparatus to measure NaOH consumption for neutralizing 3-hydroxybutyric acid (3HB) and oligomers.
• Derivatization of 3HB oligomers into bromophenacyl derivatives followed by high-performance liquid chromatography (HPLC) separation.
• Application of developed methods to analyze hydrolysis of different PHB types by selected PHB depolymerases.

Main Results:

• The online titration method allows sensitive (1 nmol/min) and wide-range (6 orders of magnitude) determination of hydrolysis rates.
• Specific hydrolysis rates of native PHB granules from Ralstonia eutropha H16 were determined for the first time.
• The HPLC method enables separation and quantification of 3HB and its oligomers up to the octamer.

Conclusions:

• The developed methods provide accurate and versatile tools for studying PHB depolymerase activity and hydrolysis products.
• These advancements facilitate a deeper understanding of PHB biodegradation mechanisms.
• The methods are applicable to a broad range of polyhydroxyalkanoates and ester-containing molecules.