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Applied and Environmental Microbiology, January 2003, p. 227-232, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.227-232.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

Tomás Bolumar, Yolanda Sanz, M.-Concepción Aristoy, and Fidel Toldrá*

Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Científicas, 46100 Burjassot (Valencia), Spain

Received 1 July 2002/ Accepted 21 September 2002

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg2+, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The Km for proline-7-amido-4-methylcoumarin was 40 µM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

* Corresponding author. Mailing address: Instituto de Agroquímica y Tecnología de Alimentos (C.S.I.C.), Apt. 73, 46100 Burjassot (Valencia), Spain. Phone: 34 96 3900022. Fax: 34 96 3636301. E-mail: ftoldra{at}iata.csic.es.

Applied and Environmental Microbiology, January 2003, p. 227-232, Vol. 69, No. 1
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.1.227-232.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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