Thermotolerant poly(3-hydroxybutyrate)-degrading bacteria from hot compost and characterization of the PHB depolymerase of Schlegelella sp. KB1a.
Eighteen gram-negative thermotolerant poly(3-hydroxybutyrate) (PHB)-degrading bacterial isolates ( T(max) approximately 60 degrees C) were obtained from compost. Isolates produced clearing zones on opaque PHB agar, indicating the presence of extracellular PHB depolymerases. Comparison of physiological characteristics and determination of 16S rRNA gene sequences of four selected isolates revealed a close relatedness of three isolates (SA8, SA1, and KA1) to each other and to Schlegelella thermodepolymerans and Caenibacterium thermophilum. The fourth strain, isolate KB1a, showed reduced similarities to the above-mentioned isolates and species and might represent a new species of Schlegelella. Evidence is provided that S. thermodepolymerans and C. thermophilum are only one species. The PHB depolymerase gene, phaZ, of isolate KB1a was cloned and functionally expressed in Escherichia coli. Purified PHB depolymerase was most active around pH 10 and 76 degrees C. The DNA-deduced amino acid sequence of the mature protein (49.4 kDa) shared significant homologies to other extracellular PHB depolymerases with a domain substructure: catalytic domain type 2-linker domain fibronectin type 3-substrate-binding domain type 1. A catalytic triad consisting of S(20), D(104), and H(138) and a pentapeptide sequence (GLS(20)AG) characteristic for PHB depolymerases (PHB depolymerase box, GLSXG) and for other serine hydrolases (lipase box, GXSXG) were identified.
Romen
Fabian
F
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550 Stuttgart, Germany.
Reinhardt
Simone
S
Jendrossek
Dieter
D
D020816:Amino Acid Motifs; D020562:Betaproteobacteria / Q000145:classification* / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D003001:Cloning, Molecular; D042621:Comamonadaceae / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D020459:Genes, rRNA; D006863:Hydrogen-Ion Concentration; D006885:Hydroxybutyrates / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D011091:Polyesters / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012987:Soil; D012988:Soil Microbiology; D013696:Temperature; D000094122:Polyhydroxybutyrates
D002265:Carboxylic Ester Hydrolases; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D006885:Hydroxybutyrates; D011091:Polyesters; D012336:RNA, Ribosomal, 16S; D011994:Recombinant Proteins; D012987:Soil; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C022417:poly-beta-hydroxybutyrate depolymerase