An Extracellular Poly(3‐Hydroxybutyrate) Depolymerase from Alcaligenes faecalis

Abstract

A strain of Alcaligenes faecalis T I , which was isolated from activated sludge, excreted an extracellular poly(3-hydroxybutyrate) depolymerase as it grew in a medium containing poly(3-hydroxybutyrate) as the sole carbon source. The molecular weight of the enzyme, purified from the culture medium to electrophoretic homogeneity, was 48 000 as determined by Sephadex G-I00 filtration, and 50000 by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The pH optimum for the enzyme reaction was 7.5. The purified enzyme depolymerized poly(3-hydroxybutyrate) purified from Zoogloea ramigeva 1-1 6-M, but did not attack the bacterial native poly(3-hydroxybutyrate)-containing granules. K, values were 13.3 pg/ml (= 0.78 pM, based on an estimated average molecular weight of 17000) for poly(3-hydroxybutyrate) and 5.4mM for the trimeric ester of I I ( --)-3-hydroxybutyric acid. Analysis of hydrolytic products of poly(3-hydroxybutyrate), several oligomeric esters of I )( --)-3-hydroxybutyric acid, and the methyl ester of the trimeric ester indicated that the enzyme hydrolyzed these substrates from the free hydroxyl terminus, releasing 1>(-)-3-hydroxybutyrate dimer units one at a time.Poly(3-hydroxybutyrate) is a unique intracellular reserve of organic carbon and/or chemical energy found in a wide variety of microorganisms [I, 21. In contrast to the fairly extensive studies on the biosynthesis of poly(3-hydroxybutyrate) [3-91, relatively little is known about the mechanism of intracellular degradation of poly(3-hydroxybutyrate) owing to its complexity, e.g. the dependence of the depolymerase activity on the structural integrity of poly(3-hydroxybutyrate)-containing granules and on the presence of a thermostable activator and a protein inhibitor [3][4][5] 101. On the other hand, some bacteria secrete extracellular poly(3-hydroxybutyrate) depolymerase which degrades purified poly(3-hydroxybutyrate) [I 1 -141 devoid of a granular membrane component [15].Recently we isolated a poly(3-hydroxybutyrate)-depolymerizing bacterium, Alcaligenes faecalis T which grew on a poly(3-hydroxybutyrate)-supplemented medium. It seems important to investigate the mechanisms of enzymatic depolymerization of the extremely hydrophobic poly(3-hydroxybutyrate) molecule. In this communication we describe the purification and some of the properties of poly(3-hydroxybutyrate) depolymerase from A . faecalis T,. EXPERIMENTAL PROCEDURES Poly(3-hydroxybutyrate)-Degrading BacteriumActivated sludge, obtained from the Toba sewagetreatment plant (Kyoto, Japan), was incubated in a mediumAbh~eviutions. Dimer, n(-)-3-[i~(-)-.l-hydroxybutyryloxy]butyric acid; trimer, trimeric ester of 1>(-)-3-hydroxybutyric acid; tetramer, tetrameric ester of 11(-)-3-hydrowybutyric acid; pentamer, pentameric ester of n(-)-3-hydroxybutyric acid; TEAE-cellulose, triethylaminoethyl cellulose.Enzymes.

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“…On the other hand, the PHB depolymerase activity found in the halophilic of N. aegyptia sp. nov., was about twice that obtained from a strain of Alcaligenes faecalis (Tanio et al, 1982).…”

Bioremediation - A Progressive Approach toward Reducing Plastic Wastes

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Exploring biodegradable polymer production from marine microbes

“…The rapid hydrolyzation of polymers is done with the help of extracellular depolymerases of bacteria; in aerobic condition the polymers are converted into water and carbon dioxide whereas in anaerobic condition they are converted into methane that can be utilized as a carbon source for the growth [3]. The polymer degrading microorganisms secrete the extracellular enzyme and convert the PHAs into molecular building blocks; a wide variety of extracellular depolymerase enzymes were identified from variety of microbes and the mechanism is well understood [208][209][210][211][212].…”