RESUMO
Approximately 1â¯million tons of agave plants are processed annually by the Mexican tequila and mezcal industry, generating vast amounts of agroindustrial solid waste. This type of lignocellulosic biomass is considered to be agroindustrial residue, which can be used to produce enzymes, giving it added value. However, the structure of lignocellulosic biomass makes it highly recalcitrant, and results in relatively low yield when used in its native form. The aim of this study was to investigate an effective pre-treatment method for the production of commercially important hydrolytic enzymes. In this work, the physical and chemical modification of Agave durangensis leaves was analysed using ultrasound and high temperature as pre-treatments, and production of enzymes was evaluated. The pre-treatments resulted in modification of the lignocellulosic structure and composition; the ultrasound pre-treatment improved the production of inulinase by 4â¯U/mg and cellulase by 0.297â¯U/mg, and thermal pre-treatment improved ß-fructofuranosidase by 30â¯U/mg.
Assuntos
Agave , beta-Frutofuranosidase , Celulase , Hidrólise , Folhas de PlantaRESUMO
ABSTRACT The present study evaluated the purification of inulinase by changing the ionic strength of the medium by addition of NaCl and CaCl2 followed by precipitation with n-propyl alcohol or iso-propyl alcohol. The effects of the concentration of alcohols and the rate of addition of alcohols in the crude extract on the purification yield and purification factor were evaluated. Precipitation caused an activation of enzyme and allowed purification factors up to 2.4-fold for both alcohols. The purification factor was affected positively by the modification of the ionic strength of the medium to 0.5 mol.L-1 NaCl before precipitation with the alcohol (n-propyl or iso-propyl). A purification factor of 4.8-fold and an enzyme yield of 78.1 % could be achieved by the addition of 0.5 mol.L-1 of NaCl to the crude extract, followed by the precipitation with 50 % (v/v) of n-propyl alcohol, added at a flow rate of 19.9 mL/min.
Assuntos
Concentração Osmolar , Precipitação Química , Álcoois/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/química , Valores de Referência , Sais/química , Solventes/química , Kluyveromyces/isolamento & purificação , Kluyveromyces/química , Cloreto de Cálcio/química , Cloreto de Sódio/química , Reprodutibilidade dos Testes , Meios de Cultura/químicaRESUMO
Thirty fungal species grown on Cichorium intybus L. root extract as a sole carbon source, were screened for the production of exo-inulinase activities. The thermophile Thielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 gave the highest production levels of inulinases I & II at 50 and 24 ºC respectively. Yeast extract and peptone were the best nitrogen sources for highest production of inulinases I & II at five and seven days of incubation respectively. The two inulinases I & II were purified to homogeneity by gel-filtration and ion-exchange chromatography with 66.0 and 42.0 fold of purification respectively. The optimum temperatures of purified inulinases I & II were 75 and 50 ºC respectively. Inulinase I was more thermostable than the other one. The optimum pH for activity was found to be 4.5 and 5.5 for inulinases I & II respectively. A comparatively lower Michaelis-Menten constant (2.15 mg/ml) and higher maximum initial velocity (115 µmol/min/mg of protein) for inulinase I on inulin demonstrated the exoinulinase's greater affinity for inulin substrate. These findings are significant for its potential industrial application. The molecular mass of the inulinases I & II were estimated to be 72 & 78 kDa respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
RESUMO
Thirty fungal species grown on Cichorium intybus L. root extract as a sole carbon source, were screened for the production of exo-inulinase activities. The thermophile Thielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 gave the highest production levels of inulinases I & II at 50 and 24 ºC respectively. Yeast extract and peptone were the best nitrogen sources for highest production of inulinases I & II at five and seven days of incubation respectively. The two inulinases I & II were purified to homogeneity by gel-filtration and ion-exchange chromatography with 66.0 and 42.0 fold of purification respectively. The optimum temperatures of purified inulinases I & II were 75 and 50 ºC respectively. Inulinase I was more thermostable than the other one. The optimum pH for activity was found to be 4.5 and 5.5 for inulinases I & II respectively. A comparatively lower Michaelis-Menten constant (2.15 mg/ml) and higher maximum initial velocity (115 µmol/min/mg of protein) for inulinase I on inulin demonstrated the exoinulinase's greater affinity for inulin substrate. These findings are significant for its potential industrial application. The molecular mass of the inulinases I & II were estimated to be 72 & 78 kDa respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
RESUMO
Thirty fungal species grown on Cichorium intybus L. root extract as a sole carbon source, were screened for the production of exo-inulinase activities. The thermophile Thielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 gave the highest production levels of inulinases I & II at 50 and 24 ºC respectively. Yeast extract and peptone were the best nitrogen sources for highest production of inulinases I & II at five and seven days of incubation respectively. The two inulinases I & II were purified to homogeneity by gel-filtration and ion-exchange chromatography with 66.0 and 42.0 fold of purification respectively. The optimum temperatures of purified inulinases I & II were 75 and 50 ºC respectively. Inulinase I was more thermostable than the other one. The optimum pH for activity was found to be 4.5 and 5.5 for inulinases I & II respectively. A comparatively lower Michaelis-Menten constant (2.15 mg/ml) and higher maximum initial velocity (115 µmol/min/mg of protein) for inulinase I on inulin demonstrated the exoinulinase's greater affinity for inulin substrate. These findings are significant for its potential industrial application. The molecular mass of the inulinases I & II were estimated to be 72 & 78 kDa respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.