Pectinase activity

If we compare liquefaction to maceration, more activities are needed to liquefy the cell wall. Since 1991, new pectinases activities such as rhamnogalacturonase, pectin acetylesterase and xyloglucanases complex have been found to be important in the apple liquefaction by Henck Schols, Jean-Paul Vincken and Voragen [3]. The cellulose-xyloglucan complex accounts approximatively 57% of the apple cell-wall matrix. In a liquefaction process, an efficient enzymic degradation of this complex is crucial to increase the sugars extraction, to decrease the viscosity of the pulp then to be able to ultra-filtrate the juice without second depectinisation, at last to have negative alcohol tests required by some concentrate customers. 

The pectinase activity was found in void volume of the washing buffer while some contaminated protein was adsorbed to the ion exchanger (Figure 1. The activity was 100% recovered. Thoug i, the enzyme was not adsorbed to the DEAE-ceUulose, but it is useful since the some contaminated proteins coidd be separated. The enzyme was approx. 10 fold purified and its specific activity increased to 48.8 unit/mg protein (Table 1). 

When Rhizopus sp. 26R was cultivated in the solid substrates without addition of rice bran but composed of only wheat bran and rice husk at the ratio of 18 2. The pectinase activity from the culture was approx. 25-35 unit/ml within 2 days and the production remained constant for 4 days (Figure 3). One gram of raw starch from cassava tuber, 1 g of pectin or 0.5 g of yeast extract was added to the solid substrates in order to induce higher activity of the enzsrme. The results showed that either 1 g raw cassava starch or 1 g pectin that was added to the 20 g solid substrates increased the enzyme activity to 1.7 and 2.4 times, respectively (Figure 3). The production of pectinase in soHd substrates with wheat bran and rice husk could be enhanced with the addition of raw cassava starch and pectin. 

Figure 3 Pectinase activity in 20 g of solid substrates composting of wheat bran, rice bran and rice husk (18 0 2) with the addition of 1 g raw starch from cassava tuber, 1 g pectin or 0.5 g yeast extract.

Figure 5 Pectinase activity in solid substrates of wheat bran, rice bran and rice husk at different ratios of substrates. Initial moisture content 66 % Incubation temperature 32°C Initial pH 5.7...

Figure 6,7 and 8 showed the results of the pectinase activity when produced in the solid substrates containing wheat bran, rice bran and rice husk in the ratio of 6 12 2. The highest activity obtained when the strain was grown on the solid substrates with 58 % initial moisture content, pH adjusted to 5.7 and incubation temperature was at 32°C. Under these conditions, the highest activity of the enzyme that could be obtained from Rhizopus sp. 26R was ca. 700 units of enzyme activity per gram of solid substrates. 

Screening of cells for pectinase activity was done on MP-5 medium [5] after precipitation of polygalacturonic acid with 1% Cetrimide (Sigma). Liquid culture experiments were done under the self-induced anaerobic conditions [6]. 

Endo pectinase activity was found to be very high for the strain FP-180 at a initial pH of 2.5 and 3.5 producing about 3 to 5 fold respectively in relation to A. niger. at 37°C. (Fig.2C). At other pH values the level of endo-pectinase secreted diminished as pH increased, and was practically negligible at 6.5 pHi showing a clear relation between pHi and enzyme production or secretion. Endo pectinase produced by A. n/gerwas very low as was the case of exo activity. However low production vras accentuated at very acidic pH values (i.e., 2.5 and 3.5). There was not a clear correlation in the production of this activity with the initial pH. 

Figure 2. Specific Exo (A and B)and Endo (C) pectinase activities produced by Aspergillus FP-180 (open bars) and Aspergillus niger N402 (dashed bars) at different pH values growing on 1% pectin at 30 C (A) and 37 C (B, C).

Figure 3. SDS-PAGE and in situ pectinase activity on pectin and polygalacturonic acid-agarose overlays of culture filtrates of Aspergillus niger N-402 (upper panel) and Aspergillus FP-180 (lower panel) at 2.5, 3.5, 5.5 and 6.5 pHi (Lanes a, b, c, and d, respectively). Electrophoresis on 10% acrylamide slab gel (14 X 8 cm) in the presence of SDS was according to Laemmli (6), run at 30 mA constant current for 2 hours. Crude cell-free samples were concentrated by lyophilization, dialyzed, boiled with sample buffer by 60 sec. and applied to each well. Polyacrylamide gel and overlays were incubated overnight with 0.17 acetate buffer at room temperature.

Hadj-Taieb, N., Ayadi, M., Trigui, S., Bouabdallah, F., and Gargouri, A., Hyperproduction of pectinase activities by a fully constitutive mutant (CT1) of Penicillium occitanis. Enzyme Microbial Technol 2002, 30 (5), 662-666. 

Pectinol [Rohm Haas]. TM for formulated enzyme concentrate of fungal origin, with varying degrees of pectinase activity, that hydrolyzes pectic substances. 

A parallel situation may exist with pectinase activity on... 

The commercial liquefaction enzyme typically used (Rhoapect 7016) contained predominantly cellulase and hemicellulase activity but with some pectinase activity. The use of more pectinase and less liquefaction enzyme represented a more favorable treatment economically and was actually more effective in reducing viscosity (Table II). Also, as predicted, the steady state flux of the membranes was higher when pectinase predominated. 

A comparison of the total polysaccharides in juice from healthy grapes and those affected by rot shows that the latter contain glucane but no homogalacturonan, due to the intense pectinase activity of the fungus (Table 3.6). 

Bhattacharya and Shah [26] studied the influence of environment-friendly enzyme treatment of flax fabrics using BGLU enzyme with hemicellulase and pectinase activities. Under optimal conditions of enzymalysis, weight loss was in the region of 12%, similar to that obtained by conventional caustic soda treatment (10 16%). In addition, the fabrics had improved absorbency, whiteness, and dyeability with tolerable loss of tensile strength. 

In the samples of fruit bodies of basidiomycetes level of activity of the proteolytic, cellulolytic and pectolytic enzymes is very high. Xylotrophic mushrooms obtain a high amylolytic activity. In saprotrophic fungi the amylases activity is absent. Mycorrhizal saprotrophs have a high pectinase activity as compared with the Litter saprotrophs... 

High levels of rot in the harvest increase juice turbidity and make clarification difficult, due to the protective colloidal effect of glucan produced by Botrytis. A low concentration of rot (less than 5%) tends to facilitate juice clarification, due to a pectinase activity in contaminated grapes that is nearly 100 times higher than in healthy grapes. 

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