1887

Microbiology Society logo

Volume 139, Issue 4

Purification and characterization of an alkaline isoamylase from an alkalophilic strain of Free

Abstract

Alkaline isoamylase (glycogen 6-glucanohydrolase, EC 3.2.1.68) activity was detected in the culture medium of an alkalophilic strain of sp., designated KSM-3309, which was isolated from a soil sample. This novel enzyme was purified to homogeneity from the culture filtrate by precipitation with ammonium sulphate, chromatography on DEAE-cellulose and DEAE-Bio-Gel A, and gel filtration on Sephacryl S-200. The purified enzyme had a pH optimum of approximately 9·0, and displayed maximum catalytic activity at 55 °C. The enzyme had a molecular mass of 65 kDa, as determined by both SDS-polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200. The isoelectric point was 4·2. This enzyme cleaved the branching points of both amylopectin and glycogen, and incubation of the enzyme with these glucans caused large increases in coloration of the iodine reagent. Amylose, pullulan and maltose were practically insensitive to the enzyme. The enzyme activity was inhibited by Hg ions and by -bromosuccinimide, but the thiol inhibitors iodoacetate, 4-chloromercuribenzoate and -ethylmaleimide had either no effect or a slightly inhibitory effect. -Cyclodextrin, an inhibitor of pullulanase, was not inhibitory.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology 1993
Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-139-4-781
1993-04-01
2024-05-03
Loading full text...

Full text loading...

/deliver/fulltext/micro/139/4/mic-139-4-781.html?itemId=/content/journal/micro/10.1099/00221287-139-4-781&mimeType=html&fmt=ahah

References

  1. Akai H., Yokobayashi K., Misaki A., Harada Y. 1971; Complete hydrolysis of branching linkages in glycogen by pseudomonas isoamylase: distribution of linear chains. Biochimica et Biophysica Acta 237:422–429
     [Google Scholar]
  2. Amemura A., Chakraborty R., Fujita M., Noumi T., Futai M. 1988; Cloning and nucleotide sequence of the isomylase gene from Pseudomonas amyloderamosa SB-15. Journal of Biological Chemistry 263:9271–9275
     [Google Scholar]
  3. Ara K., Igarashi K., Saeki K., Kawai S., Ito S. 1992; Purification and some properties of an alkaline pullulanase from alkalophilic Bacillus sp. KSM-1876. Bioscience, Biotechnology, and Biochemistry 56:62–65
     [Google Scholar]
  4. Bender H., Wallenfels K. 1961; Untersuchungen an Pullulan. II. Spezifischer Abbau durch ein bakterielles Enzym. Biochemische Zeitschrift 334:79–95
     [Google Scholar]
  5. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
     [Google Scholar]
  6. Claus D., Berkeley R. C. W. 1986; Genus Bacillus. In Bergey’s Manual of Systematic Bacteriology 2 pp. 1105–1139 Edited by Sneath P. H. A., Mair N. S., Sharpe M. E., Holt J. G. Baltimore: Williams & Wilkins;
     [Google Scholar]
  7. Eriksson K.-E., Pettersson G. 1968; Studies on cellulolytic enzymes. V. Some structural properties of the cellulase from Penicillium notatum. Archives of Biochemistry and Biophysics 124:160–166
     [Google Scholar]
  8. Gordon R. E., Haynes W. C., Pang C. H. 1973; The Genus Bacillus. Washington, DC: US Department of Agriculture;
     [Google Scholar]
  9. Gunja Z. H., Manners D. J., Maung K. 1961; Studies on carbohydrate-metabolizing enzymes. VII. Yeast isoamylase. Biochemical Journal 81:392–398
     [Google Scholar]
  10. Gunja-Smith Z., Marshall J. J., Mercier C., Smith E. E., Whelan W. J. 1970a; A glycogen-debranching enzyme from Cytophage. FEBS Letters 12:96–100
     [Google Scholar]
  11. Gunja-Smith Z., Marshall J. J., Mercier C., Smith E. E., Whelan W. J. 1970b; A reversion of the Mayer-Bernfeld model of glycogen and amylopectin. FEBS Letters 12:101–104
     [Google Scholar]
  12. Harada T., Yokobayashi K., Misaki A. 1968; Formation of isoamylase by Pseudomonas. Applied Microbiology 16:1439–1444
     [Google Scholar]
  13. Harada T., Misaki A., Akai K., Yokobayashi K., Sugimoto K. 1972; Characterization of Pseudomonas isoamylase by its actions on amylopectin and glycogen: comparison with Aerobacter pullulanase. Biochimica et Biophysica Acta 268:497–505
     [Google Scholar]
  14. Hizukuri S., Kaneno T., Takeda I. 1983; Measurement of the chain length of amylopectin and its relevance to the origin of crystalline polymorphism of starch granules. Biochimica et Biophysica Acta 760:188–191
     [Google Scholar]
  15. Igarashi K., Ara K., Saeki K., Ozaki K., Kawai S., Ito S. 1992; Nucleotide sequence of the gene that encodes a neopullu-lanase from an alkalophilic Bacillus. Bioscience, Biotechnology, and Biochemistry 56:514–516
     [Google Scholar]
  16. Ishizuka Y., Taniguchi H., Maruyama Y., Nakamura M. 1983; Debranching enzymes of potato tubers (Solanum tuberosum L.). I. Purification and some properties of potato isoamylase. Agricultural and Biological Chemistry 47:771–779
     [Google Scholar]
  17. Jeanningros R., Creuzet N., Frixon C., Cattaneo J. 1975; A debranching enzyme in Escherichia coli. Biochemical Society Transactions 3:336–337
     [Google Scholar]
  18. Kitagawa H., Amemura A., Harada T. 1975; Studies on the inhibition and molecular properties of crystalline Pseuodomonas isoamylase. Agricultural and Biological Chemistry 39:989–994
     [Google Scholar]
  19. Marshall J. J. 1973; Inhibition of pullulanase by Schardinger dextrins. FEBS Letters 37:269–273
     [Google Scholar]
  20. Maruo B., Kobayashi T. 1951; Enzymic scission of the branch links in amylopectin. Nature, London 167:606–607
     [Google Scholar]
  21. Miller G. L., Blum R., Glennon W. E., Burton A. L. 1960; Measurement of carboxymethylcellulase activity. Analytical Biochemistry 2:127–132
     [Google Scholar]
  22. Nakamura N., Sashihara N., Nagayama H., Horikoshi K. 1987; Production of a thermostable pullulanase by Thermus sp. Journal of the Japanese Society of Starch Science 34:38–44
     [Google Scholar]
  23. Norman B. E. 1982; A novel debranching enzyme for application in the glucose syrup industry. Starch/Stärke 34:340–346
     [Google Scholar]
  24. Oakley B. R., Kirsch D. R., Morris N. R. 1980; A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry 105:361–363
     [Google Scholar]
  25. Okoshi H., Ozaki K., Shikata S., Oshino K., Kawai S., Ito S. 1990; Purification and characterization of multiple caroxy-methylcellulases from Bacillus sp. KSM-522. Agricultural and Biological Chemistry 54:83–89
     [Google Scholar]
  26. Sato H. H., Park Y. K. 1980; Purification and characterization of extracellular isoamylase from Flavobacterium sp. Starch/Stärke 32:132–136
     [Google Scholar]
  27. Spencer-Martins I. 1982; Extracellular isoamylase produced by the yeast Lipomyces kononenkoae. Applied and Environmental Microbiology 44:1253–1257
     [Google Scholar]
  28. Stominska L., Maczynski M. 1985; Studies on the application of pullulanase in starch saccharification process. Starch/Stärke 37:386–390
     [Google Scholar]
  29. Tognoni A., Carrera P., Galli G., Lucchese G., Camerini B., Grandi G. 1989; Cloning and nucleotide sequence of the isoamylase gene from a strain of Pseudomonas sp. Journal of General Microbiology 135:37–45
     [Google Scholar]
  30. Trevelyan W. E., Procter D. P., Harrison J. S. 1950; Detection of sugars on paper chromatograms. Nature, London 166:444–445
     [Google Scholar]
  31. Urlaub H., Wober G. 1975; Identification of isoamylase, a glycogen-debranching enzyme, from Bacillus amyloliquefaciens. FEBS Letters 57:1–4
     [Google Scholar]
  32. Walker G. J. 1968; Metabolism of the reserve polysaccharide of Streptococcus mitis. Biochemical Journal 108:33–40
     [Google Scholar]
  33. Wrigley C. W. 1971; Gel electrofocusing. Methods in Enzymology 22:559–564
     [Google Scholar]
  34. Yokobayashi K., Misaki A., Harada T. 1970; Purification and properties of pseudomonas isoamylase. Biochimica et Biophysica Acta 212:458–469
     [Google Scholar]
  35. Yoshimatsu T., Ozaki K., Shikata S., Ohta Y., Koike K., Kawai S., Ito S. 1990; Purification and characterization of alkaline endo-14-yff-glucanases from alkalophilic Bacillus sp. KSM-635. Journal of General Microbiology 136:1973–1979
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-139-4-781
Loading
Purification and characterization of an alkaline isoamylase from an alkalophilic strain of Bacillus
Microbiology 139, 781 (1993); https://doi.org/10.1099/00221287-139-4-781
/content/journal/micro/10.1099/00221287-139-4-781
/content/journal/micro/10.1099/00221287-139-4-781
Loading

Data & Media loading...

Most cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error