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Production of Active Bacillus licheniformis Alpha-Amylase in Tobacco and its Application in Starch Liquefaction

Abstract

As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis α-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the re-combinant α-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with α-amylase from Bacillus licheniformis.

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References

  1. Hiatt, A., Cafferkey, R. and Bowdish, K. 1989. Production of antibodies in transgenic plants. Nature 342: 76–78.

    Article  CAS  PubMed  Google Scholar 

  2. Düring, K., Hippe, S., Kreuzaler, F. and Schell, J. 1990. Synthesis and self-assembly of a functional monoclonal antibody in transgenic Nicotiana tabacum . Plant Mol. Biol. 15: 281–293.

    Article  PubMed  Google Scholar 

  3. Vandekerckhove, J., VanDamme, J., Van Lijsebettens, M., Botterman, J., De Block, M., Vandewiele, M., De Clercq, A., Leemans, J., Van Montagu, M. and Krebbers, E. 1989. Enkephalins produced in transfenic plants using modified 2S seed storage proteins. Bio/Technology 7: 929–932.

    CAS  Google Scholar 

  4. Krebbers, E. and VandeKerckhove, J. 1990. Production of peptides in plant seeds. TIBTECH 8: 1–3.

    Article  CAS  Google Scholar 

  5. DeZoeten, G.A., Penswick, J.R., Horisberger, M.A., Ahl, P., Schultze, M. and Hohn, T. 1989. The expression, localization and effect of a human interferon in plants. Virology 172: 213–222.

    Article  CAS  Google Scholar 

  6. Sijmons, P.C., Dekker, B.M.M., Schrammeijer, B., Verwoerd, T.C., VandenElzen, P.J.M and Hoekema, A. 1990. Production of correctly processed human serum albumin in transgenic plants. Bio/Technology 8: 217–221.

    CAS  Google Scholar 

  7. Schwardt, E. 1990. Production and use of enzymes degrading starch and some other polysaccharkles. Food Biotechnol. 4: 337–351.

    Article  CAS  Google Scholar 

  8. Whitaker, J.R. 1990. New and future uses of enzymes in food processing. Food Biotechnol 4: 669–697.

    Article  CAS  Google Scholar 

  9. Peppler, H.J. and Reed, G. 1987. Enzymes in food and feed processing, p. 547–603. In: Biotechnology, Vol. 7A. Kennedy, J.F. (Ed.). VGH, Weinheim, F.R.G.

    Google Scholar 

  10. Kennedy, J.F., Cabalda, V.M. and White, C.A. 1988. Enzymic starch utilization and genetic engineering. TIBTECH 6: 184–189.

    Article  CAS  Google Scholar 

  11. Yuuki, T., Nomura, T., Tezuka, H., Tsuboi, A., Yamagata, H., Tsukagoshi, N. and Udaka, S. 1985. Complete nucleotide sequence of a gene coding for heat- and pH-stable α-amylase of Bacillus licheniformis: Comparison of the amino acid sequences of three bacterial liquefying α-amylases deduced from the cDNA sequences. J. Biochem. 98: 1147–1156.

    Article  CAS  PubMed  Google Scholar 

  12. Guilley, H., Dudley, R.K., Jonard, G., Balasz, E. and Richards, K.E. 1982. Transcription of Cauliflower Mosaic Virus DNA: detection of promoter sequences and characterization of transcripts. Cell 30: 763–773.

    Article  CAS  PubMed  Google Scholar 

  13. Brederode, F.T., Koper-Zwarthoff, E.C. and Bol, J.F. 1980. Complete nucleotide sequence of alfalfa mosaic virus RNA4. Nucl. Acids Res. 8: 2213–2223.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Cornelissen, B.J.C., Hooft van Huijsduijnen, R.A.M. and Bol, J.F. 1986. A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin. Nature 321: 531–532.

    Article  CAS  PubMed  Google Scholar 

  15. Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. 1983. A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium lumefaciens Ti-plasmid. Nature 303: 179–180.

    Article  CAS  Google Scholar 

  16. Reilly, P.J. 1985. Enzymic degradation of starch, p. 101–142. In: Starch Conversion Technology. Van Beynum, G.M.A. and Roels, J.A. (Eds.). Marcel Dekker Inc., NY.

    Google Scholar 

  17. Kovaleva, I.E., Novikova, L.A. and Luzikov, V.N. 1989. Synthesis and secretion of bacterial α-amylase by the yeast Saccharvmyices cerevisiae . FEES Lett. 251: 183–186.

    Article  CAS  Google Scholar 

  18. Van Ee, J.H. 1987. Regulatory region cloning and analysis plasmid for Bacillus. E. P. 0224294.

  19. Marsh, J.L., Erfle, M. and Wykes, E.J. 1984. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32: 481–485.

    Article  CAS  PubMed  Google Scholar 

  20. Horsch, R.B., Fry, J.E., Hoffmann, N.L., Eichholtz, D., Rogers, S.G. and Fraley, R.T. 1985. A simple and general method for transferring genes into plants. Science 227: 1229–1231.

    Article  CAS  Google Scholar 

  21. Ditta, G., Stanfield, S., Corbiu, D. and Helinski, D. 1980. Broad host range DNA cloning system for gram-negative bacteria: construction of a genebank of Rhizobium meliloti . Proc. Natl. Acad. Sci. USA 77: 7347–7351.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. 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.

    Article  CAS  PubMed  Google Scholar 

  23. Saito, N. 1973. A thermophilic extracellular α-amylase from Bacillus licheniformis Arch. Biochem. Biophys. 155: 290–298.

    Article  CAS  PubMed  Google Scholar 

  24. Lacks, S.A. and Springhorn, S.S. 1980. Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J. Biol. Chem. 255: 7467–7473.

    CAS  PubMed  Google Scholar 

  25. Kocken, C. 1989. Recombinant fusion proteins of herpes simplex virus type 1 glycoprotein D. Thesis R.U. Groningen.

    Google Scholar 

  26. Hendriks, T., Van den Berg, B.M. and Schram, A.W. 1985. Cellular location of peroxidase isozymes in leaf tissue of Petunia and their affinity for Concavalin A-Sepharosc. Planta 164: 89–95.

    Article  CAS  PubMed  Google Scholar 

  27. Edge, A.S.B., Faltynek, C.R., Hof, L., Reichert, L.F. Jr. and Weber, P. 1981. Deglycosylation of glycoproteins by trifluoromethanesulfonic acid. Anal. Biochem. 118: 131–137.

    Article  CAS  PubMed  Google Scholar 

  28. Caspar, T., Huber, S.C. and Somerville, C. 1986. Alterations in growth, photosynthesis, and respiration in a starch mutant of Arabidopsis thaliana (L.) Heynh deficient in chloroplast phosphoglucomutase activity. Plant Physiol. 79: 11–17.

    Article  Google Scholar 

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Pen, J., Molendijk, L., Quax, W. et al. Production of Active Bacillus licheniformis Alpha-Amylase in Tobacco and its Application in Starch Liquefaction. Nat Biotechnol 10, 292–296 (1992). https://doi.org/10.1038/nbt0392-292

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