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Applied and Environmental Microbiology, May 2004, p. 2588-2595, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2588-2595.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cloning of the Authentic Bovine Gene Encoding Pepsinogen A and Its Expression in Microbial Cells

Rosario Muñoz,1 José L. García,2 Alfonso V. Carrascosa,1 and Ramon Gonzalez1*

Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC),1 Department of Molecular Microbiology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain2

Received 22 October 2003/ Accepted 15 January 2004

Bovine pepsin is the second major proteolytic activity of rennet obtained from young calves and is the main protease when it is extracted from adult animals, and it is well recognized that the proteolytic specificity of this enzyme improves the sensory properties of cheese during maturation. Pepsin is synthesized as an inactive precursor, pepsinogen, which is autocatalytically activated at the pH of calf abomasum. A cDNA coding for bovine pepsin was assembled by fusing the cDNA fragments from two different bovine expressed sequence tag libraries to synthetic DNA sequences based on the previously described N-terminal sequence of pepsinogen. The sequence of this cDNA clearly differs from the previously described partial bovine pepsinogen sequences, which actually are rabbit pepsinogen sequences. By cloning this cDNA in different vectors we produced functional bovine pepsinogen in Escherichia coli and Saccharomyces cerevisiae. The recombinant pepsinogen is activated by low pH, and the resulting mature pepsin has milk-clotting activity. Moreover, the mature enzyme generates digestion profiles with {alpha}-, ß-, or {kappa}-casein indistinguishable from those obtained with a natural pepsin preparation. The potential applications of this recombinant enzyme include cheese making and bioactive peptide production. One remarkable advantage of the recombinant enzyme for food applications is that there is no risk of transmission of bovine spongiform encephalopathy.

* Corresponding author. Mailing address: Department of Microbiology, Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain. Phone: 34-915622900, ext. 359. Fax: 34-915644853. E-mail: rgonzalez{at}ifi.csic.es.

Applied and Environmental Microbiology, May 2004, p. 2588-2595, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2588-2595.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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