Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis.
Purification of poly(3-hydroxybutyrate) depolymerase (EC 3.1.1.75) from Paucimonas lemoignei is complicated because the bacterium produces several isoenzymes which are difficult to separate from each other. The phaZ5 gene of P. lemoignei encoding extracellular poly(3-hydroxybutyrate) depolymerase A was functionally expressed from the constitutive P43 promoter of pWB980 in a multiple protease-negative mutant of Bacillus subtilis (strain WB800) and secreted to the culture medium. The depolymerase (apparent M(r), 42 kDa; 1.9 mg purified protein per liter culture) was purified from cell-free culture fluid to homogenity by applying only one chromatography step in comparison to at least two necessary steps if poly(3-hydroxybutyrate) depolymerases are purified from P. lemoignei. The recombinant depolymerase lacked any carbohydrate content in contrast to the glycosylated depolymerase of the wild-type. Glycosylation was not essential for activity but enhanced the thermal stability of the enzyme at high temperature. Overexpression of poly(3-hydroxybutyrate) depolymerase in B. subtilis is more efficient than in Escherichia coli.
Braaz
Reinhard
R
Institut für Mikrobiologie, Universität Stuttgart, Allmandring 31, 70550, Germany.
Wong
Sui-Lam
SL
Jendrossek
Dieter
D
D001412:Bacillus subtilis / Q000235:genetics*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics*; D006885:Hydroxybutyrates / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism; D000094122:Polyhydroxybutyrates
D002265:Carboxylic Ester Hydrolases; D004274:DNA, Recombinant; D006885:Hydroxybutyrates; D011091:Polyesters; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C077226:poly(3-hydroxyalkanoic acid) depolymerase