PROSPECTION OF CELLULOLYTIC ENZYMES IN TWO ARGENTINIAN NATIVE TERMITE SPECIES

BEN GUERRERO, E; ARNEODO J; GIOFFRE A; CAMPOS E; ETCHEVERRY C; GODOY C; SALUM T; TALIA P

Rosario

Congreso; IX Congreso Argentino de Microbiología General; 2013

Sociedad Argentina de Microbiología General- SAMIGE

The interest in lignocellulosic ethanol has been increasing over the last years due mainly to the currentcrisis of global warming and the high cost of petroleum. In this sense, the availability of sustainable and environmentally clean biofuels is crucial. Many insects, such as termites, can use lignocellulosic substrates as their main food source and are highly efficient at degrading cellulose to glucose, with the help of their intestinal microbiota. The aim of this study was to detect and to evaluate the cellulolytic activity in two South American termite species with different nutritional habits: Nasutitermes aquilinus (wood) and Cortaritermes fulviceps (soil). Specimens (workers) were field-collected in Corrientes province (Argentina), and stored at -20°C until processing. Insects were surface sterilized in 70% ethanol and dissected under binocular microscope. The guts were extracted and grounded in bidistilled water or 5 mM Tris-HCl pH 7.6. These homogenates, containing insect endosymbiotic bacteria, were grown on minimal medium (MM) with different cellulosic substrates: carboxymethyl cellulose (CMC), Avicel, filter paper, sugarcane bagasse and Napier grass (Pennisetum purpureum), as the only carbon source. Bacterial growth was observed in every condition assayed, suggesting the presence of microorganisms expressing the enzymes needed to breakdown cellulosic substrates. So far, CMCase activity was measured quantitatively in the culture supernatants of both termites grown on CMC, using the dinitrosalicylic acid (DNS) method. Enzymatic activity was 0.060 ± 0.003 and 0.009 ± 0.0014 IU/ml for N. aquilinus and C. fulviceps respectively. In addition, the CMCase activity was assessed in the culture supernatant of sugarcane bagasse (0.076 ± 0.019 and 0.027 ± 0.005 IU/ml in N. aquilinus and C. fulviceps, respectively) and elephant grass (0.059 ± 0.009 IU/ml in N. aquilinus). Also, a modified substrate agar plate assay (Teather and Wood, 1982) was used to assess b-1,4 endoglucanase activity in gut extracts and culture supernatants. Cellulolytic activity was evidenced for the two species by a zone of clearance around the sample drop, indicating CMC degradation. N. aquilinus showed a hydrolysis diameter greater than C. fulviceps. To further elucidate cellulases in N. aquilinus and C. fulviceps, gut extract samples and culture supernatants were analyzed. The zymograms indicated the presence of at least one endoglucanase of 55 kDa in gut extract samples from the two termite species. In the culture supernatants (MM-CMC) from N. aquilinus, two major proteins (about 55-60 kDa) with cellulolytic activity were observed. In this study, we have identified and evaluated for the first time the cellulolytic activity of enzymes present in two native termites. Further research will focus on the characterization of these insect cellulolytic systems and its application on lignocellulosic biodegradation for biofuel production.