RESUMEN
Durante el periodo de poscosecha el principal problema de deterioro del lulo (Solanum quitoense Lam) es el ablandamiento que es generado principalmente por actividad de enzimas pécticas que atacan la red estructural de la pared celular. Esta investigación se basó en la búsqueda de las mejores condiciones de extracción y medida de actividad de las enzimas pectinesterasa, poligalacturonasa y pectato liasa; herramientas necesarias para estudiar posteriormente el rol de estas enzimas en el deterioro por ablandamiento sufrido por el fruto debido a diversos cambios metabólicos. Se encontró que las dos primeras enzimas pueden ser extraídas simultáneamente con buffer fosfatos 20 mM pH 7,0 + NaCl 0,06 M y 60 min de extracción, relación 1:2 (material vegetal: buffer de extracción), a su vez, pectato liasa se extrajo con buffer fosfatos 20 mM pH 7,0 + cisteína 20 mM y 30 min de extracción, relación 1:3. Para la cuantificación de la actividad pectinesterasa es necesario incubar 15 min a 42 °C 2.500 µL de extracto enzimático crudo (EE) en buffer fosfatos 20 mM pH 7,0 + NaCl 0,15 M y 1,6% de pectina cítrica como sustrato, con valores de Km aparente de 3,78% de PC y Vmax 17,95 µmolH+/min*mg prot. Para la cuantificación de la actividad poligalacturonasa es necesario incubar 15 min a 37 °C 30 µL (EE) en buffer acetatos 200 mM pH 4,5 + NaCl 0,25 M y 1,0% de APG como sustrato, con valores de Km aparente 0,141% de APG y Vmax 28,46 nKat/s*mg prot. Para la cuantificación de la actividad pectato liasa es necesario incubar 2 min a 17 °C 100 µL (EE) en buffer TRIS:HCl 50 Mm pH 8,5 + CaCl2 4 mM y 0,1% de APG como sustrato, con valores de Km aparente 0,0865% de APG y Vmax 82,75 µg/s*mg prot.
The main problem of post-harvest deterioration of lulo (Solanum quitoense Lam) is the softening is the main problem of post-harvest deteriorarion of Lulo, that is generated mainly by the activity of pectic enzymes, which attack the structural network of the cell wall. This research was based on finding the best conditions structural cell wall network for extraction and measurement of enzyme activity pectinesterase (PE), polygalacturonase (PG) and pectato liasa (PL); tools needed to study the further role of these enzymes in the deterioration of pectatelyase fruit softening, due to various metabolic changes. It was found that the first two enzymes can be extracted simultaneously with 20 mM phosphate buffer pH 7.0, 0.06 M NaCl and 60 minutes of extraction, ratio 1:2 (plant material: extraction buffer), pectatelyase extracted with 20 mM phosphate buffer pH 7.0, 20 mM cysteine and 30 minutes of extraction, ratio 1:3. For quantification of pectinesterase activity is necessary to incubate 15 minutes at 42 ° C, 2500 µL of crude enzyme extract (EE) in 20 mM phosphate buffer pH 7.0, to 0.15 M NaCl and 1.6% citrus pectin as (CP) substrate with apparent Km values of 3.78% CP and Vmax 17.95 mol H+/min * mg prot. For the quantification of pectinesterase activity is necessary to incubate 15 minutes to 42 °C 2500 µL of crude enzyme extract (EE) in 20 mM phosphate buffer pH 7.0, 0.15 M NaCl and 1.6% citrus pectin as substrate with apparent Km values of 3.78% CP and 17.95 µ Vmax mol H+/min*mg prot. For the quantification of polygalacturonase activity is necessary to incubate 15 minutes to 37 °C 30 µL (EE) in 200 mM Acetate buffer pH 4.5, 0.25 M NaCl and 1.0% of APG as substrate, with apparent Km values 0.141% of APG and Vmax 28.46 nKat/s*mg prot. For the quantification of the pectatelyase activity is necessary to incubate 2 minutes to 17 °C, 100 µL (EE) in buffer TRIS: HCl pH 8.5, 50 mM 4 mM CaCl2 and 0.1% PGA as substrate, with apparent Km values 0.0865% of APG and Vmax 82.75 µg/s*mg prot.
RESUMEN
Response surface methodology was used for optimization of polygalacturonase (PG) and pectinesterase (PE) production in submerged fermentation by A.niger. A Central Composite Experimental Design was applied, consisting of 22 experiments, including eight central points. Variables studied were: fermentation time (24 to 120 h), pH (3.5 to 6.5) and initial concentration of pectin (5 to 20 g/l). Maximum PE production was 220 U/l, after 74 h of culture, in a medium containing 20 g/l of pectin (pH 6.5). The optimal conditions for PG production were pH: 4.1, 20 g/l of pectin and 94 h of fermentation with a maximum value of 1032 U/l. Under these conditions, the PE production was low (15 U/l). A liquid extract with high PG activity and low PE activity could be suitable to be used in food processing in order to reduce the production of methanol.
A metodologia de superfície de resposta foi utilizada para a otimização da produção de poligalacturonasa (PG) e pectinesterasa (PE), por A. niger em fermentação submergida. Foi aplicado um Desenho Experimental Composto Central abrangendo 22 experiências, incluindo oito pontos centrais. As variáveis estudadas foram: tempo de fermentação (24 a 120 h), pH (3.5 a 6.5) e concentração inicial de pectina (5 a 20 g/l). A produção máxima de PE foi de 220 U/l, após 74h de cultivo, 20 g/l de pectina e pH 6.5. As condições ótimas para a produção de PG foram pH 4.1, 20 g/l de pectina e 94 h de fermentação, com um valor máximo de 1032 U/l. Sob estas condições, a produção de PE foi baixa (15 U/l). Um extrato líquido com alta atividade PG e baixa atividade PE poderia ser conveniente para ser utilizado no processamento e alimentos, visando reduzir a produção de metanol.