Monday, May 4, 2009
5-99
Optimization of lipase extraction conditions obtained by solid-state fermentation
Jaqueline N. Silva, Melissa L. E. Gutarra, and Denise M. G. Freire. Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
The aim of this work is to select recovery methods for lipase produced by Penicillium simplicissimum in solid-state fermentation (SSF), using babassu cake as the culture medium. The parameters for this enzyme recovery were studied using Plackett-Burman (PB) and Central Composite Rotatable (CCRD) experimental designs.
In our observations of lipase recovery kinetics, an increase in enzyme extraction was identified until 20 minutes, after which time there was no improvement in lipase recovery. The following extraction conditions were evaluated: sodium phosphate buffer (100mM, pH: 7.0) with or without NaCl (0.6%(w/v)), Tween 80 (0.1%(w/v)), Triton X-100 (0.5%(w/v)) and glycerol 20%(w/v). Extraction with Tween and NaCl were the best conditions, yielding lipase activity of 85.7U/g and 65.7U/g, respectively.
In the PB experimental design it was observed that pH and Tween had a positive impact on enzyme extraction, while temperature and buffer molarity had a negative effect. NaCl, stirring and volume presented no statistically significant effects, meaning that any value for these parameters could be used (in the studied range). A drop in the buffer molarity could harmfully reduce the buffer capacity, for which reason it was fixed at a minimum value. Later, a CCRD was employed, which yielded a 70% increase in lipase activity in the crude extract. Through the CCRD results analysis, a quadratic model was built, from which it was concluded that the maximum predicted lipase activity (160 U/g) was obtained at 25ºC, Tween 0.5%(w/v), pH 8.0 and extraction medium volume of 7mL per gram of fermented solids.
In our observations of lipase recovery kinetics, an increase in enzyme extraction was identified until 20 minutes, after which time there was no improvement in lipase recovery. The following extraction conditions were evaluated: sodium phosphate buffer (100mM, pH: 7.0) with or without NaCl (0.6%(w/v)), Tween 80 (0.1%(w/v)), Triton X-100 (0.5%(w/v)) and glycerol 20%(w/v). Extraction with Tween and NaCl were the best conditions, yielding lipase activity of 85.7U/g and 65.7U/g, respectively.
In the PB experimental design it was observed that pH and Tween had a positive impact on enzyme extraction, while temperature and buffer molarity had a negative effect. NaCl, stirring and volume presented no statistically significant effects, meaning that any value for these parameters could be used (in the studied range). A drop in the buffer molarity could harmfully reduce the buffer capacity, for which reason it was fixed at a minimum value. Later, a CCRD was employed, which yielded a 70% increase in lipase activity in the crude extract. Through the CCRD results analysis, a quadratic model was built, from which it was concluded that the maximum predicted lipase activity (160 U/g) was obtained at 25ºC, Tween 0.5%(w/v), pH 8.0 and extraction medium volume of 7mL per gram of fermented solids.