Advertisement

Journal of Industrial Microbiology

, Volume 5, Issue 4, pp 229–237 | Cite as

Mutation and screening to increase chymosin yield in a genetically-engineered strain ofAspergillus awamori

  • Michael Lamsa
  • Peggy Bloebaum
Original Papers

Summary

Through the course of five rounds of mutagenesis of a genetically-engineered strain ofAspergillus awamori, the yield of a heterologous protein (the acid protease, calf chymosin) increased four-fold. This was accomplished through the use of an agar plate screen incorporating the colony restrictor 2,6-dichloro-4-nitroaniline (dichloran) and the acid protease inhibitor diazoacetyl-norleucine methyl ester (DAN) to reduce high background concentrations of the native acid protease. A miniaturized liquid culture growth method using 24-well culture plates was an intermediate screen between agar plate and shake flask cultures. Analysis of broth samples for active calf chymosin was accomplished with a highly specific, 96-well microtiter plate turbidimetric assay.

Key words

Chymosin Acid protease Diazoacetyl-norleucine methyl ester Microculture Aspergillus awamori Heterologous protein 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Ernstrom, C.A. 1974. Milk clotting enzymes and cheese chemistry. In: Fundamental of Dairy Chemistry (Webb, B.H., A.H. Johnson, J.A. Alford, eds.), pp. 662–771 The AVI Publishing Company, Inc., Westport, CT.Google Scholar
  2. 2.
    Kobayashi, H., I. Kusakabe, S. Yokoyama and K. Murakami. 1985. Substrate specificity of the milk-clotting enzyme fromIrpex lacteus on kappa and beta-casein. Agric. Biol. Chem. 49: 1621–1631.Google Scholar
  3. 3.
    Kobayashi, H., I. Kusakabe and K. Murakami. 1985. Milkclotting enzyme fromIrpex Lacteus as a calf rennet substitute for cheddar cheese manufacture. Agric. Biol. Chem. 49: 1605–1609.Google Scholar
  4. 4.
    Zayas, J.F. 1987. Properties and quality characteristics of renning extracted by ultrasound. Biotechnol. Bioeng. 29: 969–975.Google Scholar
  5. 5.
    Cullen, D., G.L. Gray, L.J. Wilson, K.J. Hayenga M.H. Lamsa, M.W. Rey, S. Norton and R.M. Berka. 1987. Controlled expression and secretion of bovine chymosin inAspergillus nidulans. Bio/tech. 5: 369–376.Google Scholar
  6. 6.
    Rothe, G.A.L., N.H. Axelsen, P. Johnk and B. Foltmann. 1976. Immunochemical, chromatographic, and milk-clotting, activity measurements for quantification of milk-clotting enzymes in bovine rennets. J. Dairy Res. 43: 85–95.PubMedGoogle Scholar
  7. 7.
    Koburger, J.A., F.C. Chang and C.I. Wei. 1985. Evaluation of dichloran-rose bengal agar for enumeration of fungi in foods. J. Food Protection 48: 562–563.Google Scholar
  8. 8.
    Rajagopalan, T.G., W.H. Stein and S. Moore. 1966. The inactivation of pepsin by diazoacetyl-norleucine methyl ester. J. Biol. Chem. 241: 4295–4297.PubMedGoogle Scholar
  9. 9.
    Tang, J. 1971. Specific and irreversible inactivation of pepsin by substrate-like epoxides. J. Biol. Chem. 246: 4510–4517.PubMedGoogle Scholar
  10. 10.
    Carlton, B.C., B.J. Brown. 1981. Gene mutation. In: Manual of Methods for General Bacteriology. (Gerhardt, P., ed.), pp. 222–232, American Society for Microbiology, Washington, DC.Google Scholar
  11. 11.
    Berka, R.M., M. Ward, L.J. Wilson, K.J. Hayenga, K.K. Fong, L.C. Carlomagno and S.A. Thompson. 1989. Molecular characterization and deletion of the aspergillopepsin A gene fromAspergillus awamori. Gene. (Submitted for publication).Google Scholar
  12. 12.
    Esser, K. and F. Meinhardt. 1986. Genetics of strain improvement in filamentous fungi with respect to biotechnology. In: Overproduction of Microbial Metabolites (Vanek, Z. and Z. Hostalek, eds.), pp. 215–230, Butterworths Inc., Washington, DC.Google Scholar
  13. 13.
    Rowlands, R.T. 1984. Industrial strain improvement: mutagenesis and random screening procedures. Enzyme Microb. Technol. 6: 3–10.Google Scholar
  14. 14.
    Nolan, R.D. 1986. Partly automated systems in strain improvement and secondary metabolite detection. In: Overproduction of Microbial Metabolites (Vanek, Z. and Z. Hostalek, eds.), pp. 215–230. Butterworths Inc., Washington, D.C.Google Scholar
  15. 15.
    Stieglitz, B. and C.P. DeFelice. 1986. Mechanized systems for media dispensing, inoculation and replication of microorganisms. Biotechnol. Bioeng. 28: 1310–1317.Google Scholar
  16. 16.
    Ward, M. 1989. Chymosin production inAspergillus In: Molecular Industrial Mycology: Systems and Applications. (Leong, S.A. and Berka, R.M., eds.), Marcel Dekker, New York. (In press).Google Scholar
  17. 17.
    Hayenga, K.J., Crabb, D., Carlomango, L., Arnold, R., Heinsohn, H. and Lawlis, B. 1988. Protein chemistry and recovery of calf chymosin fromAspergillus nidulans andAspergillis awamori. The 18th Linderstrom-Lang Conference: Aspartic Proteinases: Biochemical, physiological and clinical aspects of pepsin, chymosin, renin and related proteinases. Elsinore, Denmark (abstract).Google Scholar

Copyright information

© Society for Industrial Microbiology 1990

Authors and Affiliations

  • Michael Lamsa
    • 1
  • Peggy Bloebaum
    • 1
  1. 1.Genencor, Inc.South San FranciscoUSA

Personalised recommendations