Identification and characterization of a novel intracellular poly(3-hydroxybutyrate) depolymerase from Bacillus megaterium.
A gene that codes for a novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase, designated PhaZ1, has been identified in the genome of Bacillus megaterium. A native PHB (nPHB) granule-binding assay showed that purified soluble PhaZ1 had strong affinity for nPHB granules. Turbidimetric analyses revealed that PhaZ1 could rapidly degrade nPHB granules in vitro without the need for protease pretreatment of the granules to remove surface proteins. Notably, almost all the final hydrolytic products produced from the in vitro degradation of nPHB granules by PhaZ1 were 3-hydroxybutyric acid (3HB) monomers. Unexpectedly, PhaZ1 could also hydrolyze denatured semicrystalline PHB, with the generation of 3HB monomers. The disruption of the phaZ1 gene significantly affected intracellular PHB mobilization during the PHB-degrading stage in B. megaterium, as demonstrated by transmission electron microscopy and the measurement of the PHB content. These results indicate that PhaZ1 is functional in intracellular PHB mobilization in vivo. Some of these features, which are in striking contrast with those of other known nPHB granule-degrading PhaZs, may provide an advantage for B. megaterium PhaZ1 in fermentative production of the biotechnologically valuable chiral compound (R)-3HB.
Chen
Hui-Ju
HJ
Institute of Biochemistry and Molecular Biology, School of Life Science, National Yang-Ming University, Taipei 112, Taiwan, ROC.
Pan
Shih-Chuan
SC
Shaw
Gwo-Chyuan
GC
D000595:Amino Acid Sequence; D001410:Bacillus megaterium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D000094122:Polyhydroxybutyrates
D001426:Bacterial Proteins; D002265:Carboxylic Ester Hydrolases; D004269:DNA, Bacterial; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C022417:poly-beta-hydroxybutyrate depolymerase