Characterization of an extracellular polyhydroxyalkanoate depolymerase from Streptomyces sp. SFB5A.
A poly(-3-hydroxybutyrate) (PHB) degrading bacterium, Streptomyces sp. SFB5A, was isolated from hardwood mulch. It synthesized an extracellular poly(3-hydroxyalkanoate) (PHA) depolymerase during growth on PHB, poly(-3- hydroxyvalerate) (PHV), PHB-PHV copolymer (PHBV), or 3-hydroxybutyrate. The purified enzyme had a subunit relative molecular weight of 47,000 and a broad pH optimum of 7.0 to 8.5; was stimulated by Ca+2 and Mg+2; and was inhibited by ethylenediaminetetraacetic acid, dithiothreitol, and non-ionic detergents. The enzyme degraded PHB, PHV, and PHBV to a mixture of monomers, dimers, and trimers, with monomers predominating. The level of trimers peaked and then decreased over time, suggesting that they were produced early and were subsequently degraded to monomers and dimers. The enzyme did not degrade poly(3-hydroxyoctanoate), indicating that it was a short-chain-length PHA depolymerase. The PHA depolymerase gene was cloned and sequenced. The deduced amino acid sequence included three features typical of extracellular PHA depolymerases: a catalytic domain type 1, a fibronectin type III linker domain, and a substrate-binding domain. A mechanism accounting for the transient production of PHA trimers was proposed, based on modeling of the enzyme’s tertiary structure and amino acid sequence homology to related PHA depolymerases.