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

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.

Figure 1. Detailed profile for the hydrolysis of pectin during the first 40 min of the reaction.

The hydrolysis was performed at 40°C, with an initial pectin concentration of 0.5% (w/v), using the pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation. The reaction was undertaken in triplicate. The mean values for each time were plotted, with the error bars representing the standard error of the mean. The curve represents the best fitting fifth-order polynomial.

Figure 2. Reaction profiles obtained over the first 40 min at different temperatures.

The hydrolysis was performed using 0.5% (m/v) pectin, with the pectinase complex produced by Aspergillus niger CH4 in solid-state fermentation. Symbols: (•) 30°C, (▴) 40°C and (+) 50°C.

Figure 3. 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.

Figure 4. 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).

Figure 5. Short hydrolysis profiles (40 min), obtained using different pectinase complexes and either polygalacturonic acid (A) or citric pectin (B).

The hydrolysis was performed at 40°C, with an initial substrate concentration of 0.5% (w/v). The pectinase complexes used were the extracts of Aspergillus niger (•) and Aspergillus oryzae (+), produced in solid-state fermentation, and the commercially available Pectinex Ultra SPL (Novozymes) (□) and Pectinase P4716 (Sigma-Aldrich) (⋄).

Figure 6. Extended hydrolysis profiles (24 h), obtained using different pectinase complexes and either polygalacturonic acid (A) or citric pectin (B).

The hydrolysis was performed at 40°C, with an initial substrate concentration of 0.5% (w/v). The pectinase complexes used were the extracts of Aspergillus niger (•) and Aspergillus oryzae (+), produced in solid-state fermentation, and the commercially available Pectinex Ultra SPL (Novozymes) (□) and Pectinase P4716 (Sigma-Aldrich) (⋄).