Enzyme and Microbial Technology 31 (2002) 242–249 Growth and proteolytic activity of hairy roots from Centaurea calcitrapa: effect of nitrogen and sucrose Pedro M.L. Lourenço, Susana de Castro, Tiago M. Martins, Alda Clemente, Ana Domingos ∗ Departamento de Biotecnologia, Instituto Nacional de Engenharia e Tecnologia Industrial, Est. do Paço do Lumiar, 1649 038 Lisboa, Portugal Received 13 July 2001; received in revised form 7 March 2002; accepted 8 March 2002 Abstract Centaurea calcitrapa hairy root cultures were established by infection with Agrobacterium rhizogenes strain LBA 9402. The liquid medium hairy root cultures exhibited a biomass doubling time of approximately 1.5 days in the 20 days exponential growth phase. The effect of the initial sucrose and nitrogen concentrations in biomass and proteinase production of the liquid medium cultures was studied. The highest values for both proteolytic activity and fresh weight were attained between 30 and 50 g/l of sucrose. A low ammonium:nitrate ratio favoured the development of hairy roots and proteolytic activity. The best results for both parameters in terms of nitrogen nutrition were obtained with nitrate (24.7 mM) as the only nitrogen source. The maximum proteolytic activity was found to be at pH 4.0, within the pH range for aspartic proteinases (APs), and the inhibition studies showed that only pepstatin A, specific for that class of enzymes, revealed a significant inhibitory effect. The C. calcitrapa aspartic proteinase (cenprosin) gene was detected in hairy roots using specific polymerase chain reaction (PCR) primers. The specific proteolytic activity present in hairy roots seems to be lower than the reported for flowers but similar to the existent in the untransformed roots and cell suspension cultures. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Centaurea calcitrapa; Hairy roots; Proteolytic activity 1. Introduction Proteolytic enzymes are by far the most important group of enzymes produced commercially and are used in many areas of application, such as detergent, brewing, meat, pho- tographic, leather, and dairy industries [1]. Aspartic proteinases (APs) (EC 3.4.23) constitute one of the four superfamilies of proteolytic enzymes showing acidic pH optima for enzymatic activity, inhibition by pepstatin A, are widely distributed in a variety of organisms such as viruses, some bacteria, yeast, fungi, plants, and animals [2]. The high price of AP from animal origin, normally used in dairy industries, and ethical considerations associated with their use, have enhanced research toward alternative milk co- agulants. In Iberian Peninsula, aqueous crude extracts from the flowers of the thistles Cynara scolymus, C. humilis and mostly C. cardunculus, have been traditionally used for the manufacture of raw ovine and/or caprine milk cheese, a tra- ditional and highly regarded dairy product [3–5]. ∗ Corresponding author. Tel.: +351-21-716-5141; fax: +351-21-716-3636. E-mail address: ana.domingos@mail.ineti.pt (A. Domingos). Centaurea calcitrapa, a plant widely distributed in Por- tugal, accumulates APs in all parts of the plant, particularly in flowers [6,7], also showing milk clotting activity [8]. A comparison between the degradation patterns of milk caseinates using purified C. calcitrapa AP (cenprosin) and the patterns obtained using commercial rennet consisting of chymosin and pepsin was previously reported [9]. The pro- teolytic activity of cenprosin is higher than the activity of commercial rennet and exhibits higher specificity towards ovine and caprine caseinates, suggesting that it can be used as an alternative rennet to produce high quality cheeses from caprine and/or ovine milk [9]. The major limitations for the widespread use of APs from C. calcitrapa on cheese making are the low yield of the final product, the heterogeneity in proteinase profile of the flowers and the fact that flowering is seasonal. An al- ternative for intensive production of these proteinases could be the use of in vitro plant cell/tissue culture for biomass production. The presence of plant proteinases in cell suspension cultures has been reported in Sylibum marianum [10], C. cardunculus [11,12], Onopordum turcicum [13] and C. calcitrapa [14,15], however, the low proteinase yields 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0141-0229(02)00117-5