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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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Reaction profiles obtained over the first 40 min with different initial pectin concentrations.The hydrolysis was performed at 45°C, with the pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation. Symbols: Initial pectin concentrations of (▴) 0.25% m/v, (+) 0.5% m/v and (•) 1% m/v.
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pone-0109529-g003: Reaction profiles obtained over the first 40 min with different initial pectin concentrations.The hydrolysis was performed at 45°C, with the pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation. Symbols: Initial pectin concentrations of (▴) 0.25% m/v, (+) 0.5% m/v and (•) 1% m/v.

Mentions: Reaction profiles were then followed, at 45°C, with different initial pectin concentrations (Figure 3). The initial reaction rate was highest with 1% (w/v) pectin. However, this concentration led to a viscous solution, which made pipetting difficult, and undissolved pectin was visible suspended in the reaction medium. These problems are probably responsible for the greater dispersion of data points in the 1% (w/v) profile in Figure 3, compared to the profiles obtained at lower initial pectin concentrations. Again, in all the conditions, a significant deceleration occurred over the first 10 min.


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)

Reaction profiles obtained over the first 40 min with different initial pectin concentrations.The hydrolysis was performed at 45°C, with the pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation. Symbols: Initial pectin concentrations of (▴) 0.25% m/v, (+) 0.5% m/v and (•) 1% m/v.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0109529-g003: Reaction profiles obtained over the first 40 min with different initial pectin concentrations.The hydrolysis was performed at 45°C, with the pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation. Symbols: Initial pectin concentrations of (▴) 0.25% m/v, (+) 0.5% m/v and (•) 1% m/v.
Mentions: Reaction profiles were then followed, at 45°C, with different initial pectin concentrations (Figure 3). The initial reaction rate was highest with 1% (w/v) pectin. However, this concentration led to a viscous solution, which made pipetting difficult, and undissolved pectin was visible suspended in the reaction medium. These problems are probably responsible for the greater dispersion of data points in the 1% (w/v) profile in Figure 3, compared to the profiles obtained at lower initial pectin concentrations. Again, in all the conditions, a significant deceleration occurred over the first 10 min.

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