Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Dec;63(12):4844–4852. doi: 10.1128/aem.63.12.4844-4852.1997

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

K Kasuya 1, Y Inoue 1, T Tanaka 1, T Akehata 1, T Iwata 1, T Fukui 1, Y Doi 1
PMCID: PMC168810  PMID: 9406404

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (400.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson A. J., Dawes E. A. Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiol Rev. 1990 Dec;54(4):450–472. doi: 10.1128/mr.54.4.450-472.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Behrends A., Klingbeil B., Jendrossek D. Poly(3-hydroxybutyrate) depolymerases bind to their substrate by a C-terminal located substrate binding site. FEMS Microbiol Lett. 1996 Oct 1;143(2-3):191–194. doi: 10.1111/j.1574-6968.1996.tb08479.x. [DOI] [PubMed] [Google Scholar]
  3. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blaak H., Schrempf H. Binding and substrate specificities of a Streptomyces olivaceoviridis chitinase in comparison with its proteolytically processed form. Eur J Biochem. 1995 Apr 1;229(1):132–139. doi: 10.1111/j.1432-1033.1995.tb20447.x. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Brucato C. L., Wong S. S. Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: general characteristics and active site studies. Arch Biochem Biophys. 1991 Nov 1;290(2):497–502. doi: 10.1016/0003-9861(91)90572-z. [DOI] [PubMed] [Google Scholar]
  7. CHOWDHURY A. A. POLY-BETA-HYDROXYBUTTERSAEURE ABBAUENDE BAKTERIEN UND EXOENZYM. Arch Mikrobiol. 1963 Dec 10;47:167–200. [PubMed] [Google Scholar]
  8. Delafield F. P., Doudoroff M., Palleroni N. J., Lusty C. J., Contopoulos R. Decomposition of poly-beta-hydroxybutyrate by pseudomonads. J Bacteriol. 1965 Nov;90(5):1455–1466. doi: 10.1128/jb.90.5.1455-1466.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fukui T., Narikawa T., Miwa K., Shirakura Y., Saito T., Tomita K. Effect of limited tryptic modification of a bacterial poly(3-hydroxybutyrate) depolymerase on its catalytic activity. Biochim Biophys Acta. 1988 Jan 29;952(2):164–171. doi: 10.1016/0167-4838(88)90112-4. [DOI] [PubMed] [Google Scholar]
  10. Gilkes N. R., Henrissat B., Kilburn D. G., Miller R. C., Jr, Warren R. A. Domains in microbial beta-1, 4-glycanases: sequence conservation, function, and enzyme families. Microbiol Rev. 1991 Jun;55(2):303–315. doi: 10.1128/mr.55.2.303-315.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hansen C. K. Fibronectin type III-like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates. FEBS Lett. 1992 Jun 29;305(2):91–96. doi: 10.1016/0014-5793(92)80871-d. [DOI] [PubMed] [Google Scholar]
  12. Jendrossek D., Backhaus M., Andermann M. Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase of Comamonas sp. and of its structural gene. Can J Microbiol. 1995;41 (Suppl 1):160–169. doi: 10.1139/m95-183. [DOI] [PubMed] [Google Scholar]
  13. Jendrossek D., Frisse A., Behrends A., Andermann M., Kratzin H. D., Stanislawski T., Schlegel H. G. Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system. J Bacteriol. 1995 Feb;177(3):596–607. doi: 10.1128/jb.177.3.596-607.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jendrossek D., Müller B., Schlegel H. G. Cloning and characterization of the poly(hydroxyalkanoic acid)-depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product. Eur J Biochem. 1993 Dec 1;218(2):701–710. doi: 10.1111/j.1432-1033.1993.tb18424.x. [DOI] [PubMed] [Google Scholar]
  15. Jendrossek D., Schirmer A., Schlegel H. G. Biodegradation of polyhydroxyalkanoic acids. Appl Microbiol Biotechnol. 1996 Dec;46(5-6):451–463. doi: 10.1007/s002530050844. [DOI] [PubMed] [Google Scholar]
  16. Kita K., Ishimaru K., Teraoka M., Yanase H., Kato N. Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122. Appl Environ Microbiol. 1995 May;61(5):1727–1730. doi: 10.1128/aem.61.5.1727-1730.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klingbeil B., Kroppenstedt R. M., Jendrossek D. Taxonomic identification of Streptomyces exfoliatus K10 and characterization of its poly(3-hydroxybutyrate) depolymerase gene. FEMS Microbiol Lett. 1996 Sep 1;142(2-3):215–221. doi: 10.1111/j.1574-6968.1996.tb08433.x. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Lusty C. J., Doudoroff M. Poly-beta-hydroxybutyrate depolymerases of Pseudomonas lemoignei. Proc Natl Acad Sci U S A. 1966 Sep;56(3):960–965. doi: 10.1073/pnas.56.3.960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mergaert J., Anderson C., Wouters A., Swings J., Kersters K. Biodegradation of polyhydroxyalkanoates. FEMS Microbiol Rev. 1992 Dec;9(2-4):317–321. doi: 10.1111/j.1574-6968.1992.tb05853.x. [DOI] [PubMed] [Google Scholar]
  21. Mergaert J., Webb A., Anderson C., Wouters A., Swings J. Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in soils. Appl Environ Microbiol. 1993 Oct;59(10):3233–3238. doi: 10.1128/aem.59.10.3233-3238.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mukai K., Yamada K., Doi Y. Kinetics and mechanism of heterogeneous hydrolysis of poly[(R)-3-hydroxybutyrate] film by PHA depolymerases. Int J Biol Macromol. 1993 Dec;15(6):361–366. doi: 10.1016/0141-8130(93)90054-p. [DOI] [PubMed] [Google Scholar]
  23. Nakayama K., Saito T., Fukui T., Shirakura Y., Tomita K. Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei. Biochim Biophys Acta. 1985 Jan 21;827(1):63–72. doi: 10.1016/0167-4838(85)90101-3. [DOI] [PubMed] [Google Scholar]
  24. Saito T., Suzuki K., Yamamoto J., Fukui T., Miwa K., Tomita K., Nakanishi S., Odani S., Suzuki J., Ishikawa K. Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. J Bacteriol. 1989 Jan;171(1):184–189. doi: 10.1128/jb.171.1.184-189.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shinomiya M., Iwata T., Kasuya K., Doi Y. Cloning of the gene for poly(3-hydroxybutyric acid) depolymerase of Comamonas testosteroni and functional analysis of its substrate-binding domain. FEMS Microbiol Lett. 1997 Sep 1;154(1):89–94. doi: 10.1111/j.1574-6968.1997.tb12628.x. [DOI] [PubMed] [Google Scholar]
  27. Tanio T., Fukui T., Shirakura Y., Saito T., Tomita K., Kaiho T., Masamune S. An extracellular poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis. Eur J Biochem. 1982 May;124(1):71–77. doi: 10.1111/j.1432-1033.1982.tb05907.x. [DOI] [PubMed] [Google Scholar]
  28. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  29. Yamada K., Mukai K., Doi Y. Enzymatic degradation of poly(hydroxyalkanoates) by Pseudomonas pickettii. Int J Biol Macromol. 1993 Aug;15(4):215–220. doi: 10.1016/0141-8130(93)90040-s. [DOI] [PubMed] [Google Scholar]
  30. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES