Abstract
The cDNA encoding the precursor of theRhizomucor miehei triglyceride lipase was inserted in anAspergillus oryzae expression vector. In this vector the expression of the lipase cDNA is under control of theAspergillus oryzae α-amylase gene promoter and theAspergillus niger glucoamylase gene terminator. The recombinant plasmid was introduced intoAspergillus oryzae, and transformed colonies were selected and screened for lipase expression. Lipase-positive transformants were grown in a small fermentor, and recombinant triglyceride lipase was purified from the culture broth. The purified enzymatically active recombinant lipase (rRML) secreted fromA. oryzae was shown to have the same characteristics with respect to mobility on reducing SDS-gels and amino acid composition as the native enzyme. N-terminal amino acid sequencing indicated that approximately 70% of the secreted rRML had the same N-terminal sequence as the nativeRhizomucor miehei enzyme, whereas 30% of the secreted rRML was one amino acid residue shorter in the N-terminal. The recombinant lipase precursor, which has a 70 amino acid propeptide, is thus processed in and secreted fromAspergillus oryzae. We have hereby demonstrated the utility of this organism as a host for the production of recombinant triglyceride lipases.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
Abbreviations
- bp:
-
base pair
- kb:
-
kilobase
- SDS:
-
sodium dodecyl sulphate
- LU:
-
lipase units
- RML:
-
Rhizomucor miehei lipase
- rRML:
-
recombinant-RML synthesized inA. oryzae
References
Schipper, M.A.A. (1978)Studies in Mycology, No. 17, pp. 53–71, Institute of the Royal Netherlands Academy of Sciences and Letters, Amsterdam, The Netherlands.
Huge-Jensen, B., Galluzzo, D.R., and Jensen, R.G. (1987)Lipids 22, 559–565.
Boel, E., Huge-Jensen, B., Christensen, M., Thim, L., and Fill, N.P. (1988)Lipids 23, 701–706.
Christensen, T., Woeldike, H., Boel, E., Mortensen, S.B., Hjortshöj, K., Thim, L., and Hansen, M.T. (1988)BIO/TECHNOLOGY 6, 1419–1422.
Boel, E., Beck, A-M., Randrup, K., Dræger, B., Fiil, N.P., and Foltmann, B. (1986)Proteins 1, 363–369.
Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Vieira, H., and Messing, J. (1982)Gene 19, 259–268.
Boel, E., Hansen, M.T., Hjort, I., Höegh, I., and Fiil, N.P. (1984)Eur. Mol. Biol. Org. J. 3, 1581–1585.
Kelly, J.M., and Hynes, M. (1985)Eur. Mol. Biol. Org. J. 4, 475–479.
Thom, C., and Raper, K.B. (1945)A Manual of the Aspergilli, p. 39, Williams & Wilkins, Baltimore.
Thim, L., Hansen, M.T., and Soerensen, A.R. (1987)FEBS Lett. 212, 307–312.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951)J. Biol. Chem. 193, 265–275.
Pâquet, D., Lorient, D., Mejean, L., Alais, C. (1981)Neth. Milk Dairy J. 35, 358–360.
Antonian, E. (1988)Lipids 23, 1101–1106.
Author information
Authors and Affiliations
About this article
Cite this article
Huge-Jensen, B., Andreasen, F., Christensen, T. et al. Rhizomucor miehei triglyceride lipase is processed and secreted from transformedAspergillus oryzae . Lipids 24, 781–785 (1989). https://doi.org/10.1007/BF02544584
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02544584