Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422


The cheese industry is seeking novel sources of enzymes for cheese production. Microbial rennets have several advantages over animal rennets. (1) They are easy to generate and purify and do not rely on the availability of animal material. (2) The production of microbial clotting enzymes may be improved by biotechnological techniques. In this work, the biochemical characterization of a novel milk-clotting extracellular enzyme from Myxococcus xanthus strain 422 and a preliminary evaluation of its cheese-producing ability are reported. Strain 422 was selected from four M. xanthus strains as the best producer of extracellular milk-clotting activity, based on both its enzyme yield and specific milk-clotting activity, which also afforded lower titration values than enzymes from the three other M. xanthus strains. The active milk-clotting enzyme from M. xanthus strain 422 is a true milk-clotting enzyme with a molecular mass of 40 kDa and a pI of 5.0. Highest milk-clotting activity was at pH 6 and 37 °C. The enzyme was completely inactivated by heating for 12 min at 65 °C. The crude enzyme preparation was resolved by anion-exchange chromatography into two active fractions that were tested in cheese production assays of compositional (dry matter, fat content, fat content/dry-matter ratio, and moisture-non-fat content) and physicochemical properties (firmness, tensile strength, pH and Aw) of the milk curds obtained. Purified protein fraction II exhibited a significantly higher milk-clotting ability than either protein fraction I or a total protein extract, underlining the potential usefulness of M. xanthus strain 422 as a source of rennet for cheese production.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2a, b
Fig. 3
Fig. 4a, b


  1. 1.

    Carias J-R, Raingeaud J, Mazaud C, Vachon G, Lucas N, Cenatiempo Y, Julien R (1990) A chymosin-like extracellular acidic endoprotease from Myxococcus xanthus DK101. A potential tool for protein engineering. FEBS Lett 262:97─100

  2. 2.

    Coletta PL, Miller PGG (1986) The extracellular proteases of Myxococcus xanthus. FEMS Microbiol Lett 37:203─207

  3. 3.

    Dworkin M (1996) Recent advantages in the social and developmental biology of the myxobacteria. Microbiol Rev 60:70─102

  4. 4.

    Fontes M, Kaiser D (1999) Myxococcus cells respond to elastic forces in their substrate. Proc Natl Acad Sci USA 96:8052─8057

  5. 5.

    Guespin-Michel JF, Letouvet-Pawlak B, Petit F (1993) Protein secretion in Myxobacteria. In: Myxobacteria II. American Society for Microbiology, Washington DC, pp 235─255

  6. 6.

    Guinee TP, Auty MAE, Fenelon MA (2000) The effect of fat content on the rheology, microstructure and heat-induced functional characteristics of cheddar cheese. Int Dairy J 10: 277─288

  7. 7.

    Kunitz M (1947) Crystalline soybean trypsin inhibitor. J Gen Physiol 30:291–310

  8. 8.

    Lowry OH, Rosenbrough NJ, Fare AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:263─275

  9. 9.

    Lucas N, Mazaud-Aujard C, Bremaud L, Cenatiempo Y, Julien R (1994) Protein purification, gene cloning and sequencing of an acidic endoprotease from Myxococcus xanthus DK101. Eur J Biochem 222:247─254

  10. 10.

    Madsen JS, Ardo YH (2001) Exploratory study of proteolysis, rheology and sensory properties of Danbo cheese with different fat contents. Int Dairy J 11:423─431

  11. 11.

    Mala Rao B, Aparna Tanksale M, Mohini Ghatge S, Vasanti Deshpande V (1998) Molecular and biotechnological aspects of microbial proteases. Microbiol Mol Biol Rev 62:597─635

  12. 12.

    North MJ (1982) Comparative biochemistry of proteinases of eukaryotic microorganisms. Microbiol Rev 46:308─340

  13. 13.

    Petit F, Guespin-Michel JF (1992) Production of an extracellular milk-clotting activity during development in Myxococcus xanthus. J Bacteriol 174:5136─5140

  14. 14.

    Plamann L, Kuspa A, Kaiser D (1992) Proteins that rescue A-signal-defective mutants of Myxococcus xanthus. J Bacteriol 174:3311─3318

  15. 15.

    Poza M, de Miguel T, Sieiro C, Villa TG (2001) Characterization of a broad pH range protease of Candida caseinolytica. J Appl Microbiol 91:916─921

  16. 16.

    Villa TG, Notario V, Villanueva JR (1975) β-glucanases of the yeast Pichia polymorpha. Arch Microbiol 104:201─206

  17. 17.

    Walsh MK, Li X (2000) Thermal stability of acid proteinases. J Dairy Res 67:637─640

  18. 18.

    Wendin K (2001) Sensory dynamics in emulsion products differing in fat content. SIK Report 679, viii

  19. 19.

    Whitaker JR (1963) Determination of molecular weights of proteins by gel filtration on Sephadex. Anal Chem 35:1950─1956

Download references


The authors express their gratitude to the Spanish Ministry of Science and Technology for a FEDER grant (no. PIFD97–0046). They also extend their appreciation to the Fundación Ramón Areces for their financial contribution to the pilot-plant fermentation unit.

Author information

Correspondence to T. G. Villa.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Poza, M., Sieiro, C., Carreira, L. et al. Production and characterization of the milk-clotting protease of Myxococcus xanthus strain 422. J IND MICROBIOL BIOTECHNOL 30, 691–698 (2003). https://doi.org/10.1007/s10295-003-0100-y

Download citation


  • Myxococcus xanthus
  • Milk-clotting proteases
  • Microbial coagulants
  • Industrial fermentations
  • Cheese making
  • Milk curds