Molecular analysis of a NCIMB 7423 gene encoding 4-α-glucanotransferase and characterization of the recombinant enzyme produced in Free

Abstract

An clone was detected in a NCIMB 7423 plasmid library capable of degrading soluble amylose. Deletion subcloning of its recombinant plasmid indicated that the gene(s) responsible for amylose degradation was localized on a 1·8 kb fragment. This region was sequenced in its entirety and shown to encompass a large ORF capable of encoding a protein with a calculated molecular mass of 57184 Da. Although the deduced amino acid sequence showed only weak similarity with known amylases, significant sequence identity was apparent with the 4-α-glucanotransferase enzymes of Streptococcus pneumoniae (46.9%), potato (42.9%) and (16.2%). The clostridial gene (designated ) was followed by a second ORF which, through its homology to the equivalent enzymes of and , was deduced to encode maltodextrin phosphorylase (MalP). The translation stop codon of overlapped the translation start codon of the putative gene, suggesting that the two genes may be both transcriptionally and translationally coupled. 4-α-Glucanotransferase catalyses a disproportionation reaction in which single or multiple glucose units from oligosaccharides are transferred to the 4-hydroxyl group of acceptor sugars. Characterization of the recombinant enzyme demonstrated that glucose, maltose and maltotriose could act as acceptor, whereas of the three only maltotriose could act as donor. The enzyme therefore shares properties with the MalQ protein, but differs significantly from the glucanotransferase of , which is unable to use maltotriose as donor or glucose as acceptor. Physiologically, the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase provides with a mechanism of utilizing amylose/maltodextrins with little drain on cellular ATP reserves.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-143-10-3287
1997-10-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/143/10/mic-143-10-3287.html?itemId=/content/journal/micro/10.1099/00221287-143-10-3287&mimeType=html&fmt=ahah

References

  1. Barker H.A., Smyth R.D., Wilson R.M., Weissbach H. 1959; The purification and properties of β-methylaspartase. J Biol Cbem 234:320–328
    [Google Scholar]
  2. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
    [Google Scholar]
  3. Chambers S.P., Prior S.E., Barstow D.A., Minton N.P. 1988; The pmtlmc-cloning vectors. I. improved polylinker regions to facilitate the use of sonicated dna for nucleotide sequencing. Gene 68:139–149
    [Google Scholar]
  4. Cummins C.S., Johnson J.L. 1973; Taxonomy of the clostridia: Wall composition and. J Gen Microbiol 67:33–46
    [Google Scholar]
  5. Hermann M., Fayolle F., Marchal R., Podvin L., Sebald M., Vandecasteele J.P. 1985; Isolation and characterisation of butanol resistant mutants of clostridium cetobutylicum . Appl Environ Microbiol 50:1238–1243
    [Google Scholar]
  6. Holmes D.S., Quigley M. 1981; A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197
    [Google Scholar]
  7. Lacks S.A., Dunn J.J., Greenberg B. 1982; Identification of base mismatches recognised by the heteroduplex-dna-repair system of streptococcus pneumoniae . Cell 31:327–336
    [Google Scholar]
  8. Liebl W., Feil R., Gabelsberger J., Kellermann J., Schleifer K.-H. 1992; Purification and characterisation of a novel thermostable 4-α-glucanotransferase of thermotoga maritima cloned in escherichia coli . Eur J Biochem 207:81–88
    [Google Scholar]
  9. Lin Y.L., Blaschek H.P. 1983; Butanol production by a butanol-tolerant strain of clostridium acetobutylicum in extruded corn broth. Appl Environ Microbiol 45:966–973
    [Google Scholar]
  10. Marmur J. 1961; A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218
    [Google Scholar]
  11. Minton N.P., Clarke D.J. 1989 Biotechnology Handbook vol. 3 Clostridia New York: Plenum;
    [Google Scholar]
  12. Minton N.P., Atkinson T., Sherwood R.F. 1983; Molecular cloning of the pseudomonas carboxypeptidase g2 gene and its expression in escherichia coli and pseudomonas putida . J Bacteriol 156:1222–1227
    [Google Scholar]
  13. Minton N.P., Bullman H.M.S., Atkinson T., Scawen M.D., Gilbert H.J. 1986; Nucleotide sequence of the erwinia chrysanthemi 1066 l-asparaginase gene. Gene 46:25–35
    [Google Scholar]
  14. Minton N.P., Brehm J.K., Swinfield T.J., Whelan S.M., Rodger M.L., Bodsworth N., Oultram J.D. 1993; Clostridial cloning vectors. In The Clostridia and Biotechnology pp. 119–150 Edited by Woods D. T. New York: Butterworths;
    [Google Scholar]
  15. Palmer T.N., Ryman B.E., Whelan W.J. 1976; The action pattern of amylomaltase from Escherichia coli . Eur J Biochem 69:105–115
    [Google Scholar]
  16. Papoutsakis E.T., Bennett G.N. 1993; Cloning, structure, and expression of acid and solvent pathway genes of clostridium acetobutylicum . The Clostridia and Biotechnology157–199 Edited by Woods D. T. New York: Butterworths;
    [Google Scholar]
  17. Pugsley A.P., Dubreuil C. 1988; Molecular characterisation of malq, the structural gene for the escherichia coli enzyme amylomaltase. Mol Microbiol 2:473–479
    [Google Scholar]
  18. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual Cold Spring Harbor,NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  19. Sanger F., Coulson A.R., Barrell B.G., Smith A.J.H., Roe B.A. 1980; Cloning in single-stranded bacteriophage as an aid to rapid dna sequencing. J Mol Biol 143:161–178
    [Google Scholar]
  20. Schwartz M. 1987; The maltose regulon. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology pp. 1482–1502 Edited by Neidhardt F. C., Ingraham J. L., Brooks Low K., Magasanik B., Schaechter M., Umbarger H. E. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  21. Tabor S., Richardson C.C. 1987; Dna sequence analysis with modified t7 dna polymerase. Proc Natl Acad Sci USA 84:4767–4771
    [Google Scholar]
  22. Takaha T., Yanase M., Okada S., Smith S.M. 1993; Disproportionating enzyme of potato (4-a-glucanotransferase, ec 2.4.1.25). Eur J Biochem 268:1391–1396
    [Google Scholar]
  23. Whelan S.M., Elmore M.J., Bodsworth N.J., Brehm J.K., Atkinson T., Minton N.P. 1992; Molecular cloning of the clostridium botulinum structural gene encoding the type b neurotoxin and determination of its entire nucleotide sequence. Appl Environ Microbiol 58:2345–2354
    [Google Scholar]
  24. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved m13 phage cloning vectors and host strains: Nucleotide sequences of m13mpl8 and puc19 vectors. Gene 33:103–119
    [Google Scholar]
  25. Young M., Minton N.P., Staudenbauer W.L. 1989; Recent advances in clostridial genetics. FEMS Microbiol Rev 63:1–25
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-143-10-3287
Loading
/content/journal/micro/10.1099/00221287-143-10-3287
Loading

Data & Media loading...

Most cited Most Cited RSS feed