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Pectinase activity determination: an early deceleration in the release of reducing sugars throws a spanner in the works!

Biz A, Farias FC, Motter FA, de Paula DH, Richard P, Krieger N, Mitchell DA - PLoS ONE (2014)

Bottom Line: In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction.In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude.In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.

ABSTRACT
Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.

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Residual activity curves for the extract incubated over 60 min at different temperatures.The pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation was used. Residual activities were obtained at 50°C, in a 10-min assay, using 0.5% pectin (w/v).
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pone-0109529-g004: Residual activity curves for the extract incubated over 60 min at different temperatures.The pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation was used. Residual activities were obtained at 50°C, in a 10-min assay, using 0.5% pectin (w/v).

Mentions: Increasing temperatures not only increase the intrinsic rate of enzyme-catalyzed reactions, but also increase the rate of enzyme denaturation. In order to investigate the degree to which denaturation might be occurring in the profiles in Figure 2, the extract was incubated at various temperatures (Figure 4). The pectinase activity was stable over 60 min at 30°C and 40°C. At 50°C, the residual activity fell significantly, reaching only 25% at 60 min. At 55°C, denaturation was significantly faster, with only 6% residual activity at 10 min. These results suggest that thermal denaturation is not the cause of the early deceleration in Figure 1, since that reaction was carried out at 40°C, but that some denaturation probably occurred in the reactions carried out at higher temperatures in Figures 2 and 3.


Pectinase activity determination: an early deceleration in the release of reducing sugars throws a spanner in the works!

Biz A, Farias FC, Motter FA, de Paula DH, Richard P, Krieger N, Mitchell DA - PLoS ONE (2014)

Residual activity curves for the extract incubated over 60 min at different temperatures.The pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation was used. Residual activities were obtained at 50°C, in a 10-min assay, using 0.5% pectin (w/v).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206274&req=5

pone-0109529-g004: Residual activity curves for the extract incubated over 60 min at different temperatures.The pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation was used. Residual activities were obtained at 50°C, in a 10-min assay, using 0.5% pectin (w/v).
Mentions: Increasing temperatures not only increase the intrinsic rate of enzyme-catalyzed reactions, but also increase the rate of enzyme denaturation. In order to investigate the degree to which denaturation might be occurring in the profiles in Figure 2, the extract was incubated at various temperatures (Figure 4). The pectinase activity was stable over 60 min at 30°C and 40°C. At 50°C, the residual activity fell significantly, reaching only 25% at 60 min. At 55°C, denaturation was significantly faster, with only 6% residual activity at 10 min. These results suggest that thermal denaturation is not the cause of the early deceleration in Figure 1, since that reaction was carried out at 40°C, but that some denaturation probably occurred in the reactions carried out at higher temperatures in Figures 2 and 3.

Bottom Line: In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction.In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude.In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.

ABSTRACT
Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.

Show MeSH