Monday, May 4, 2009
5-102
Production by solid-state fermentation and structural modeling of a lipase from Aspergillus parasiticus
Laila C. Cortás, Valéria F. Soares, Bruno C.P. Santos, Melissa L. E. Gutarra, Rodrigo V. Almeida, and Denise M.G. Freire. Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
The lipase from the filamentous fungi Aspergillus parasiticus was produced by solid-state fermentation and demonstrated high esterification activity, making it a potentially feasible catalyst for biodiesel and biolubricant production.
The present investigation reports on the production, characterization and lipase modeling of an Aspergillus parasiticus strain. Initially, kinetic experiments were carried out using babassu cake as the basal solid medium, moistened with water or sugar cane molasses supplement. The highest activity was reached in the medium without supplement after 72h of fermentation at 30°C. The influence of the inoculum concentration, temperature and moisture on enzyme production was evaluated employing statistical experimental design, and an empirical model was adjusted to the experimental data. The last two variables were found to be the most significant in the process. It was shown that higher lipase activity could be achieved at lower temperatures and moisture levels. Maximum lipase activity was obtained at 28°C and 65% moisture (w/v). The substrate specificity of the crude lipase was determined using several p-nitrophenyls as substrates. Higher Vmax and lower Km were obtained with p-nitrophenyl butyrate, indicating that this enzyme presents a high affinity to short-chain esters.
In order to better characterize the specificity of this enzyme, a structural model was constructed using Thermomyces lanuginosus lipase as template. Analyzing the model, we identified the catalytic triad: Ser145, His260 and Asp198. We also had seen that model was considered satisfactory with 97.2% of the residue in regions allowed in the Rammachandran’s plot.
The present investigation reports on the production, characterization and lipase modeling of an Aspergillus parasiticus strain. Initially, kinetic experiments were carried out using babassu cake as the basal solid medium, moistened with water or sugar cane molasses supplement. The highest activity was reached in the medium without supplement after 72h of fermentation at 30°C. The influence of the inoculum concentration, temperature and moisture on enzyme production was evaluated employing statistical experimental design, and an empirical model was adjusted to the experimental data. The last two variables were found to be the most significant in the process. It was shown that higher lipase activity could be achieved at lower temperatures and moisture levels. Maximum lipase activity was obtained at 28°C and 65% moisture (w/v). The substrate specificity of the crude lipase was determined using several p-nitrophenyls as substrates. Higher Vmax and lower Km were obtained with p-nitrophenyl butyrate, indicating that this enzyme presents a high affinity to short-chain esters.
In order to better characterize the specificity of this enzyme, a structural model was constructed using Thermomyces lanuginosus lipase as template. Analyzing the model, we identified the catalytic triad: Ser145, His260 and Asp198. We also had seen that model was considered satisfactory with 97.2% of the residue in regions allowed in the Rammachandran’s plot.