Biochemical and molecular characterization of the polyhydroxybutyrate depolymerase of Comamonas acidovorans YM1609, isolated from freshwater

Comamonas acidovorans YM1609 secreted a polyhydroxybutyrate (PHB) depolymerase into the culture supernatant when it was cultivated on poly(3-hydroxybutyrate) [P(3HB)] or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] as the sole carbon source. The PHB depolymerase was purified from culture supernatant of C. acidovorans by two chromatographic methods, and its molecular mass was determined as 45,000 Da by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was stable at temperatures below 37 degrees C and at pH values of 6 to 10, and its activity was inhibited by diisopropyl fluorophosphonate. The liquid chromatography analysis of water-soluble products revealed that the primary product of enzymatic hydrolysis of P(3HB) was a dimer of 3-hydroxybutyric acid. Kinetics of enzymatic hydrolysis of P(3HB) film were studied. In addition, a gene encoding the PHB depolymerase was cloned from the C. acidovorans genomic library. The nucleotide sequence of this gene was found to encode a protein of 494 amino acids (M(r), 51,018 Da). Furthermore, by analysis of the N-terminal amino acid sequence of the purified enzyme, the molecular mass of the mature enzyme was calculated to be 48,628 Da. Analysis of the deduced amino acid sequence suggested a domain structure of the protein containing a catalytic domain, fibronectin type III module as linker, and a putative substrate-binding domain. Electron microscopic visualization of the mixture of P(3HB) single crystals and a fusion protein of putative substrate-binding domain with glutathione S-transferase demonstrated that the fusion protein adsorbed strongly and homogeneously to the surfaces of P(3HB) single crystals.

[1]  C. J. Lusty,et al.  Decomposition of Poly-β-Hydroxybutyrate by Pseudomonads , 1965 .

[2]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[3]  M. M. Bradford A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[4]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[5]  H. Birnboim,et al.  A RAPID ALKALINE EXTRACTION PROCEDURE FOR SCREENING RECOMBINANT DNA , 1979 .

[6]  H. Birnboim,et al.  A rapid alkaline extraction procedure for screening recombinant plasmid DNA. , 1979, Nucleic acids research.

[7]  R. Marchessault,et al.  Solid state properties of poly-β-hydroxybutyrate and of its oligomers , 1981 .

[8]  T. Saito,et al.  An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. , 1982, European journal of biochemistry.

[9]  P A Holmes,et al.  Applications of PHB - a microbially produced biodegradable thermoplastic , 1985 .

[10]  C. Yanisch-Perron,et al.  Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. , 1985, Gene.

[11]  T. Saito,et al.  Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei. , 1985, Biochimica et biophysica acta.

[12]  J. Vieira,et al.  Production of single-stranded plasmid DNA. , 1987, Methods in enzymology.

[13]  T. Narikawa,et al.  Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity. , 1988, Biochimica et biophysica acta.

[14]  S. Nakanishi,et al.  Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis , 1989, Journal of bacteriology.

[15]  A. Anderson,et al.  Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. , 1990, Microbiological reviews.

[16]  S. Wong,et al.  Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: general characteristics and active site studies. , 1991, Archives of biochemistry and biophysics.

[17]  B. Henrissat,et al.  Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. , 1991, Microbiological reviews.

[18]  J. Mergaert,et al.  Biodegradation of polyhydroxyalkanoates. , 1992, FEMS microbiology reviews.

[19]  C. K. Hansen,et al.  Fibronectin type III‐like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates , 1992, FEBS letters.

[20]  Y. Doi,et al.  Enzymatic degradation of poly(hydroxyalkanoates) by Pseudomonas pickettii. , 1993, International journal of biological macromolecules.

[21]  J. Mergaert,et al.  Microbial Degradation of Poly(3-Hydroxybutyrate) and Poly(3-Hydroxybutyrate-Co-3-Hydroxyvalerate) in Soils , 1994, Applied and environmental microbiology.

[22]  D. Jendrossek,et al.  Cloning and characterization of the poly(hydroxyalkanoic acid)-depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product. , 1993, European journal of biochemistry.

[23]  J. Mergaert,et al.  Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in soils , 1993, Applied and environmental microbiology.

[24]  D. Jendrossek,et al.  Degradation of poly(3-hydroxybutyrate), PHB, by bacteria and purification of a novel PHB depolymerase fromComamonas sp. , 1993 .

[25]  Y. Doi,et al.  Kinetics and mechanism of heterogeneous hydrolysis of poly[(R)-3-hydroxybutyrate] film by PHA depolymerases. , 1993, International journal of biological macromolecules.

[26]  Y. Doi,et al.  Enzymatic degradation of poly(hydroxyalkanoates) by a marine bacterium , 1993 .

[27]  Y. Doi Biodegradable Plastics and Polymers , 1994 .

[28]  Y. Doi,et al.  ENZYMATIC DEGRADATION OF POLY(R)-3-HYDROXYBUTYRATE BY COMAMONAS TESTOSTERONI ATSU OF SOIL BACTERIUM , 1994 .

[29]  H. Yukawa,et al.  Cloning and Nucleotide Sequencing of The Poly(3-hydroxybutyrate) Depolymerase Gene From Pseudomonas pickettii , 1994 .

[30]  H. Abe,et al.  Physical Properties and Enzymic Degradability of Poly(3-hydroxybutyrate) Stereoisomers with Different Stereoregularities , 1994 .

[31]  Y. Doi,et al.  Efficient hydrolysis of polyhydroxyalkanoates by Pseudomonas stutzeri YM1414 isolated from lake water , 1994 .

[32]  Bernhard Schmidt,et al.  Pseudomonas lemoignei has five poly(hydroxyalkanoic acid) (PHA) depolymerase genes: A comparative study of bacterial and eukaryotic PHA depolymerases , 1994 .

[33]  H. Yanase,et al.  Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122 , 1995, Applied and environmental microbiology.

[34]  Y. Inoue,et al.  Kinetics of surface hydrolysis of poly[(R)-3-hydroxybutyrate] film by PHB depolymerase from Alcaligenes faecalis T1 , 1995 .

[35]  D. Jendrossek,et al.  Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system , 1995, Journal of bacteriology.

[36]  H. Schrempf,et al.  Binding and substrate specificities of a Streptomyces olivaceoviridis chitinase in comparison with its proteolytically processed form. , 1995, European journal of biochemistry.

[37]  D. Jendrossek,et al.  Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp. and of its structural gene. , 1995, Canadian journal of microbiology.

[38]  D. Jendrossek,et al.  Enzymatic Degradation of Bacterial Poly(3-hydroxybutyrate) by a Depolymerase from Pseudomonas lemoignei , 1996 .

[39]  D. Jendrossek,et al.  Taxonomic identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene. , 1996, FEMS microbiology letters.

[40]  D. Jendrossek,et al.  Biodegradation of polyhydroxyalkanoic acids , 1996, Applied Microbiology and Biotechnology.

[41]  D. Jendrossek,et al.  Poly(3-hydroxybutyrate) depolymerases bind to their substrate by a C-terminal located substrate binding site. , 1996, FEMS microbiology letters.

[42]  D. Jendrossek,et al.  Splintering of Poly(3-hydroxybutyrate) Single Crystals by PHB-Depolymerase A from Pseudomonas lemoignei , 1996 .

[43]  T. Iwata,et al.  Cloning of the gene for poly(3-hydroxybutyric acid) depolymerase of Comamonas testosteroni and functional analysis of its substrate-binding domain. , 1997, FEMS microbiology letters.

[44]  Y. Doi,et al.  Enzymatic degradation of poly[(R)-3-hydroxybutyrate] : secretion and properties of PHB depolymerase from Pseudomonas stutzeri , 1997 .