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Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei.
Biochimica Et Biophysica Acta
|January 21, 1985
Summary
Pseudomonas lemoignei secretes four extracellular poly(3-hydroxybutyrate) depolymerase isozymes. These enzymes efficiently degrade poly(3-hydroxybutyrate) and its oligomers, with specific cleavage patterns.
Area of Science:
- Microbiology
- Biochemistry
- Enzymology
Background:
- Poly(3-hydroxybutyrate) is a biodegradable polymer with potential industrial applications.
- Microbial enzymes capable of degrading poly(3-hydroxybutyrate) are of significant interest for bioremediation and recycling.
- Pseudomonas lemoignei is known to produce extracellular enzymes involved in polymer degradation.
Purpose of the Study:
- To purify and characterize the extracellular poly(3-hydroxybutyrate) depolymerase from Pseudomonas lemoignei.
- To investigate the enzymatic properties and substrate specificities of the purified isozymes.
- To elucidate the hydrolytic mechanism of the depolymerase isozymes.
Main Methods:
- Purification of extracellular poly(3-hydroxybutyrate) depolymerase using CM-Sepharose CL-6B chromatography.
- Separation of the enzyme into four isozymes (A1, A2, B1, B2).
- Determination of molecular weights by SDS-PAGE and isoelectric points by isoelectric focusing.
- Enzyme activity assays using poly(3-hydroxybutyrate) and oligomeric esters of D-(-)-3-hydroxybutyrate.
Main Results:
- Four distinct isozymes (A1, A2, B1, B2) of poly(3-hydroxybutyrate) depolymerase were isolated.
- Isozymes A1/A2 had a molecular weight of 54,000 Da, while B1/B2 had a molecular weight of 58,000 Da.
- Isoelectric points ranged from pH 9.7 to 10.6, indicating alkaline properties.
- All isozymes hydrolyzed poly(3-hydroxybutyrate) and oligomers but not dimers.
- Analysis revealed specific cleavage sites on oligomeric substrates, dependent on chain length.
Conclusions:
- Pseudomonas lemoignei possesses multiple extracellular poly(3-hydroxybutyrate) depolymerase isozymes with distinct biochemical properties.
- These isozymes exhibit specific hydrolytic mechanisms on poly(3-hydroxybutyrate) and its oligomers.
- The findings contribute to understanding microbial degradation of polyhydroxyalkanoates and their potential biotechnological applications.