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An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis

T Tanio, T Fukui, Y Shirakura

    European Journal of Biochemistry
    |May 1, 1982
    PubMed
    Summary

    Alcaligenes faecalis T1 excretes an extracellular poly(3-hydroxybutyrate) depolymerase. This enzyme breaks down poly(3-hydroxybutyrate) from the hydroxyl terminus, releasing dimer units.

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    Area of Science:

    • Microbiology
    • Biochemistry
    • Polymer Science

    Background:

    • Poly(3-hydroxybutyrate) (PHB) is a biodegradable polymer produced by various bacteria.
    • Understanding the enzymatic degradation of PHB is crucial for its potential applications and environmental fate.
    • Extracellular depolymerases play a key role in the microbial breakdown of PHB.

    Purpose of the Study:

    • To isolate and characterize an extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.
    • To investigate the enzyme's substrate specificity and mode of action.
    • To determine kinetic parameters for the enzymatic hydrolysis of PHB and its derivatives.

    Main Methods:

    • Isolation and purification of the extracellular enzyme from Alcaligenes faecalis T1 culture medium.

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  • Determination of enzyme molecular weight using Sephadex G-100 filtration and SDS-PAGE.
  • Enzyme activity assays at optimal pH (7.5) using purified PHB and oligomeric esters.
  • Analysis of hydrolytic products to elucidate the depolymerization mechanism.

    • Main Results:

    • An extracellular poly(3-hydroxybutyrate) depolymerase was successfully purified from Alcaligenes faecalis T1.
    • The enzyme exhibited a molecular weight of approximately 48,000-50,000 Da and had a pH optimum of 7.5.
    • The enzyme efficiently depolymerized purified PHB but did not degrade native PHB granules.
    • Kinetic analysis revealed specific substrate affinities and a mode of hydrolysis from the hydroxyl terminus, releasing dimer units.

    Conclusions:

    • Alcaligenes faecalis T1 produces a specific extracellular poly(3-hydroxybutyrate) depolymerase.
    • The enzyme's ability to degrade purified PHB suggests its potential role in PHB turnover.
    • The enzyme's specificity towards purified PHB and its defined hydrolytic mechanism provide insights into microbial PHB degradation pathways.