PRODUCTION AND ACTIVITY ANALYSIS OF THE Trichoderma longibrachiatum PROTEASES
ROSSI, B.C.1; RODRIGUES, A.1; BROCHETTO-BRAGA, M.R.1;TAUK-TORNIZZIELLO, S.2; CARMONA, E.C.3; DA SILVA, G.P.1
1 Departmento de Biologia - IB - Unesp -RC.
2 Centro de Estudos Ambientais - CEA – IB - Unesp -RC. 3 Departmento de Bioquímica e Microbiologia - IB - UNESP - RC.
Trichoderma is a natural fungus of the soil where, due to its well-known ability to produce a great number of metabolic, it plays an important role for the nutrients recycling. This way, and also for its pathogenic action, the genera Trichoderma exhibits high industrial potential, being used for production of several antibiotics and enzymes such as, the proteases. In this work, we established the best conditions to the fungus cultivation (nitrogen sources) and to the enzymatic assays (substrate, temperature and pH) for both, the protease production and determination of the acid, neutral and alkaline protease activities in Trichoderma longibrachiatum. The fungus was grown, with agitation, in Vogel media at pH 6,5 supplemented with casein, gelatin or albumin during 24, 48 and 72 hours. The proteolytic activity was determined in aliquots of the filtrates, which were incubated in McIlvaine buffer at pHs 4,5, 7,0 and 9,5 for 1 hour at 40ºC or 50ºC in presence of the substrates casein, albumin or gelatin. The reactions were interrupted with trichloroacetic acid, centrifuged and, the free amino acids determined in the supernatants. One unit of activity was defined as the amount of the enzyme to liberate 1 mg of free amino acids per mg protein per hour. From all the nitrogen sources tested, casein showed not to be a good inducer. Whereas the gelatin was the best inducer to the acid, neutral, and alkaline proteases, which exhibited the highest activity levels in the time cultivation filtrates of the 48, 24 and 48 hours respectively using gelatin as substrate, being the reactions incubated at 50, 40 and 40 ºC, respectively. Although the albumin has also induced the production of acid and neutral proteases with peaks of activity in 48 hour of time cultivation, the observed levels were much smaller than those obtained with gelatin. In all the tested conditions, the gelatin was shown to be the best inducer to the proteases production and the best substrate to the reactions. The results obtained here confirm this fungus potentiality in biotechnological purposes.
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