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. 1997 May;179(10):3324–3330. doi: 10.1128/jb.179.10.3324-3330.1997

Cloning and characterization of the gene for amylosucrase from Neisseria polysaccharea: production of a linear alpha-1,4-glucan.

V Büttcher 1, T Welsh 1, L Willmitzer 1, J Kossmann 1
PMCID: PMC179114  PMID: 9150231

Abstract

The gene for the amylosucrase from Neisseria polysaccharea (ATCC 43768) was cloned by use of a functional expression system in Escherichia coli XL1-Blue. The deduced amino acid sequence of the protein has homology to the sequences of the alpha-amylase class of enzymes, with the highest similarities being found to the sequences of the trehalose synthase from Pimelobacter sp. strain R48 (17) and amylomaltase from Thermotoga maritima (11). However, the regions of highest homology within the alpha-amylase class of enzymes, which are essential for the catalytic activity, are only scarcely found in the sequence of amylosucrase. By using the enzyme isolated from culture supernatants of transformed E. coli cells, it is possible to synthesize linear alpha-1,4-glucans from sucrose, indicating that the enzyme is not capable of producing alpha-1,6-glycosidic linkages on its own.

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Selected References

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  1. Adhya S., Schwartz M. Phosphoglucomutase mutants of Escherichia coli K-12. J Bacteriol. 1971 Nov;108(2):621–626. doi: 10.1128/jb.108.2.621-626.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Büttcher V., Rühlmann A., Cramer F. Improved single-stranded DNA producing expression vectors for protein manipulation in Escherichia coli. Nucleic Acids Res. 1990 Feb 25;18(4):1075–1075. doi: 10.1093/nar/18.4.1075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  5. Hockney R. C. Recent developments in heterologous protein production in Escherichia coli. Trends Biotechnol. 1994 Nov;12(11):456–463. doi: 10.1016/0167-7799(94)90021-3. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Liebl W., Feil R., Gabelsberger J., Kellermann J., Schleifer K. H. Purification and characterization of a novel thermostable 4-alpha-glucanotransferase of Thermotoga maritima cloned in Escherichia coli. Eur J Biochem. 1992 Jul 1;207(1):81–88. doi: 10.1111/j.1432-1033.1992.tb17023.x. [DOI] [PubMed] [Google Scholar]
  8. MacKenzie C. R., Johnson K. G., McDonald I. J. Glycogen synthesis by amylosucrase from Neisseria perflava. Can J Microbiol. 1977 Sep;23(9):1303–1307. doi: 10.1139/m77-196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. MacKenzie C. R., McDonald I. J., Johnson K. G. Glycogen metabolism in the genus Neisseria: synthesis from sucrose by amylosucrase. Can J Microbiol. 1978 Apr;24(4):357–362. doi: 10.1139/m78-060. [DOI] [PubMed] [Google Scholar]
  10. MacKenzie C. R., Perry M. B., McDonald I. J., Johnson K. G. Structure of the D-glucans produced by Neisseria perflava. Can J Microbiol. 1978 Nov;24(11):1419–1422. doi: 10.1139/m78-227. [DOI] [PubMed] [Google Scholar]
  11. Meyer T. S., Lamberts B. L. Use of coomassie brilliant blue R250 for the electrophoresis of microgram quantities of parotid saliva proteins on acrylamide-gel strips. Biochim Biophys Acta. 1965 Aug 24;107(1):144–145. doi: 10.1016/0304-4165(65)90403-4. [DOI] [PubMed] [Google Scholar]
  12. Nishimoto T., Nakano M., Nakada T., Chaen H., Fukuda S., Sugimoto T., Kurimoto M., Tsujisaka Y. Purification and properties of a novel enzyme, trehalose synthase, from Pimelobacter sp. R48. Biosci Biotechnol Biochem. 1996 Apr;60(4):640–644. doi: 10.1271/bbb.60.640. [DOI] [PubMed] [Google Scholar]
  13. Okada G., Hehre E. J. New studies on amylosucrase, a bacterial alpha-D-glucosylase that directly converts sucrose to a glycogen-like alpha-glucan. J Biol Chem. 1974 Jan 10;249(1):126–135. [PubMed] [Google Scholar]
  14. Preiss J., Romeo T. Molecular biology and regulatory aspects of glycogen biosynthesis in bacteria. Prog Nucleic Acid Res Mol Biol. 1994;47:299–329. doi: 10.1016/s0079-6603(08)60255-x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Schatz P. J., Beckwith J. Genetic analysis of protein export in Escherichia coli. Annu Rev Genet. 1990;24:215–248. doi: 10.1146/annurev.ge.24.120190.001243. [DOI] [PubMed] [Google Scholar]
  17. Svensson B. Protein engineering in the alpha-amylase family: catalytic mechanism, substrate specificity, and stability. Plant Mol Biol. 1994 May;25(2):141–157. doi: 10.1007/BF00023233. [DOI] [PubMed] [Google Scholar]
  18. von Heijne G., Abrahmsén L. Species-specific variation in signal peptide design. Implications for protein secretion in foreign hosts. FEBS Lett. 1989 Feb 27;244(2):439–446. doi: 10.1016/0014-5793(89)80579-4. [DOI] [PubMed] [Google Scholar]

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