Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Oct;61(10):3580–3585. doi: 10.1128/aem.61.10.3580-3585.1995

Purification and characterization of an extracellular pectate lyase from an Amycolata sp.

F Brühlmann 1
PMCID: PMC167654  PMID: 7486993

Abstract

The extracellular pectate lyase (EC 4.2.2.2) of a nonsporulating Amycolata sp. was purified to homogeneity by anion- and cation-exchange chromatographies followed by hydrophobic interaction chromatography. The enzyme cleaved polygalacturonate but not highly esterified pectin in a random endolytic transeliminative mechanism that led to the formation of a wide range of 4,5-unsaturated oligogalacturonates. As shown by high-performance anion-exchange chromatography and pulsed amperometric detection, these unsaturated oligogalacturonates were further depolymerized by the enzyme to the unsaturated dimer and trimer as final products. The pectate lyase had a molecular weight of 31,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a molecular mass of 30,000 Da determined by matrix-assisted laser desorption ionization mass spectrometry. The isoelectric point of the protein was 10. Maximum activity occurred at pH 10.25. Calcium was essential for activity, and EDTA inactivated the enzyme under standard assay conditions. Interestingly, EDTA did not inhibit the ability of the enzyme to cleave the native pectin (protopectin) of ramie (Boehmeria nivea) fibers. The Km value with sodium polygalacturonate as the substrate was 0.019 g liter-1. The purified enzyme lost its activity after a 1-h incubation at 50 degrees C but was stabilized by calcium or polygalacturonate. The N-terminal sequence showed high similarity within a stretch of 13 amino acids to the N-terminal sequences of pectate lyases PLa and PLe from Erwinia chrysanthemi. The Amycolata sp. did not produce additional isozymes of pectate lyase but produced further activities of pectinesterase, xylanase, and carboxymethyl cellulase when grown in a medium with decorticated bast fibers from ramie as the sole carbon source.

Full Text

The Full Text of this article is available as a PDF (276.5 KB).

Selected References

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

  1. ALBERSHEIM P., KILLIAS U. Studies relating to the purification and properties of pectin transeliminase. Arch Biochem Biophys. 1962 Apr;97:107–115. doi: 10.1016/0003-9861(62)90050-4. [DOI] [PubMed] [Google Scholar]
  2. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Brühlmann F., Kim K. S., Zimmerman W., Fiechter A. Pectinolytic enzymes from actinomycetes for the degumming of ramie bast fibers. Appl Environ Microbiol. 1994 Jun;60(6):2107–2112. doi: 10.1128/aem.60.6.2107-2112.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Keen N. T., Tamaki S. Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli. J Bacteriol. 1986 Nov;168(2):595–606. doi: 10.1128/jb.168.2.595-606.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kobayashi Y., Komae K., Tanabe H., Matsuo R. Approach to maceration mechanism in enzymatic pulping of bast fibers by alkalophilic pectinolytic enzymes produced by Erwinia species. Biotechnol Adv. 1988;6(1):29–37. doi: 10.1016/0734-9750(88)90572-1. [DOI] [PubMed] [Google Scholar]
  6. Lietzke S. E., Yoder M. D., Keen N. T., Jurnak F. The Three-Dimensional Structure of Pectate Lyase E, a Plant Virulence Factor from Erwinia chrysanthemi. Plant Physiol. 1994 Nov;106(3):849–862. doi: 10.1104/pp.106.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Nielsen B. L., Brown L. R. The basis for colored silver-protein complex formation in stained polyacrylamide gels. Anal Biochem. 1984 Sep;141(2):311–315. doi: 10.1016/0003-2697(84)90047-2. [DOI] [PubMed] [Google Scholar]
  8. Preston J. F., 3rd, Rice J. D., Ingram L. O., Keen N. T. Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16. J Bacteriol. 1992 Mar;174(6):2039–2042. doi: 10.1128/jb.174.6.2039-2042.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Reverchon S., Huang Y., Bourson C., Robert-Baudouy J. Nucleotide sequences of the Erwinia chrysanthemi ogl and pelE genes negatively regulated by the kdgR gene product. Gene. 1989 Dec 21;85(1):125–134. doi: 10.1016/0378-1119(89)90472-1. [DOI] [PubMed] [Google Scholar]
  10. Ried J. L., Collmer A. Activity stain for rapid characterization of pectic enzymes in isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gels. Appl Environ Microbiol. 1985 Sep;50(3):615–622. doi: 10.1128/aem.50.3.615-622.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Sakamoto T., Hours R. A., Sakai T. Purification, characterization, and production of two pectic transeliminases with protopectinase activity from Bacillus subtilis. Biosci Biotechnol Biochem. 1994 Feb;58(2):353–358. doi: 10.1271/bbb.58.353. [DOI] [PubMed] [Google Scholar]
  12. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  13. Stutzenberger F. J. Inducible thermoalkalophilic polygalacturonate lyase from Thermomonospora fusca. J Bacteriol. 1987 Jun;169(6):2774–2780. doi: 10.1128/jb.169.6.2774-2780.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Tamaki S. J., Gold S., Robeson M., Manulis S., Keen N. T. Structure and organization of the pel genes from Erwinia chrysanthemi EC16. J Bacteriol. 1988 Aug;170(8):3468–3478. doi: 10.1128/jb.170.8.3468-3478.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Taniguchi Y., Ono A., Sawatani M., Nanba M., Kohno K., Usui M., Kurimoto M., Matuhasi T. Cry j I, a major allergen of Japanese cedar pollen, has pectate lyase enzyme activity. Allergy. 1995 Jan;50(1):90–93. doi: 10.1111/j.1398-9995.1995.tb02489.x. [DOI] [PubMed] [Google Scholar]
  16. Yoder M. D., Keen N. T., Jurnak F. New domain motif: the structure of pectate lyase C, a secreted plant virulence factor. Science. 1993 Jun 4;260(5113):1503–1507. doi: 10.1126/science.8502994. [DOI] [PubMed] [Google Scholar]
  17. Zacharius R. M., Zell T. E., Morrison J. H., Woodlock J. J. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem. 1969 Jul;30(1):148–152. doi: 10.1016/0003-2697(69)90383-2. [DOI] [PubMed] [Google Scholar]

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

RESOURCES