Enzyme and Microbial Technology 38 (2006) 893–898 Evaluation of the presence of aspartic proteases from Centaurea calcitrapa during seed germination Sofia Matos Salvador, Carlos Novo, Ana Domingos ∗ UTPAM, Departamento de Biotecnologia, Instituto Nacional de Engenharia Tecnologia e Inova¸ c˜ ao, Estrada do Pa¸ co do Lumiar, 22, 1649-038 Lisboa, Portugal Received 2 February 2005; received in revised form 14 June 2005; accepted 21 June 2005 Abstract Aspartic proteinases are present in a variety of organisms including plants. Common features of aspartic proteases include an active site cleft that contains two catalytic aspartic residues, acid pH optima for enzymatic activity, inhibition by pepstatin A. Plant aspartic proteinases occur in seeds and may be involved in the processing of storage proteins. Many of them have been purified and characterized. The presence of aspartic proteases in seeds of Centaurea calcitrapa during germination was investigated by measuring the activity on enzyme extracts. The aspartic proteases are present mainly in the beginning of seed germination suggesting that they could initiate the degradation of protein reserves in germinating seeds. These proteases were purified by salt precipitation followed by anion-exchange chromatography. Purified aspartic proteases have an optimal pH between 3.5 and 4.5, using FTC-hemoglobin as substrate and an optimal temperature at 52 ◦ C. The ability of seed extracts for milk clotting was tested and the clotting time that was achieved is in the same range found for flower extracts appropriated for special cheeses in which weak clotting agents are required. © 2006 Elsevier Inc. All rights reserved. Keywords: Centaurea calcitrapa; Aspartic proteases; Seed germination; Milk clotting 1. Introduction Proteinases play an important role in biotechnology since proteolysis modifies the chemical, physical, biological and immunological properties of proteins. Some plant proteinases are used in the food industry, in manufacturing cheeses and drinks, meat tenderizing, cookie baking and the production of protein hydrolysates [1]. Almost all enzymes employed commercially in milk coagu- lation are aspartic proteinases (APs; EC 3.4.23); they are most active at acidic pH, are specifically inhibited by pepstatin A and contain two aspartic residues indispensable for catalytic activity [2]. APs are widely distributed in a variety of organisms such as viruses, some bacteria, yeast, fungi, plants and ani- mals [2,3]. According to the MEROPS database (http://www. merops.ac.uk), created by Rawlings and Barrett [4], APs are grouped into 14 different families, based on their amino acid ∗ Corresponding author. Tel.: +351 210924712; fax: +351 217163636. E-mail address: ana.domingos@ineti.pt (A. Domingos). sequence homology, which in turn are assembled into 6 differ- ent clans based on their evolutionary relationship and tertiary structure. Plant APs have been distributed among families A1, A3, A11 and A12 of clan AA, and family A22 of clan AD. The majority of plant APs belongs to the A1 family, together with pepsin-like enzymes from many different origins. The three-dimensional structure of two plant aspartic pro- teinases has been determined, sharing significant structural similarity with other known structures of mammalian aspartic proteinases [5,6]. With few exceptions, the majority of plant aspartic proteinases identified so far are synthesized with a prepro-domain and subsequently converted to mature enzymes. A characteristic feature of the majority of plant aspartic pro- teinase precursors is the presence of an extra protein domain of about 100 amino acids known as the plant-specific insert [7]. Plant APs have been detected and purified from many dif- ferent plant species. However, their biological functions are not as well assigned or characterized as those of their mammalian, microbial or viral counterparts that were shown to perform many different and diverse functions. For the great majority of plant APs, no definitive role has been defined and the biological func- tions are still hypothetical. In general, plant APs have been 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2005.06.025