Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation

Abstract

A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb(2+) and was not significantly affected by Hg(2+). Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca(2+). The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking.

Figure 1

Cation-exchange chromatography on CM-Sephadex A-50 column of the pectin lyase complex produced by Aspergillus giganteus. Solid line: absorbance at 280 nm; filled squares: PL activity; broken line: salt gradient.

Figure 2

Pure PLIII preparation analyzed by SDS-PAGE. Lanes: M: molecular mass standard proteins and PLIII: major pectin lyase after the Sephadex G-100 column step.

Figure 3

Influence of pH (a) and temperature (b) on PLIII activity and the thermal inactivation (c) of the purified PLIII from A. giganteus. The vertical bars indicate standard deviation (SD) of the mean calculated for three replicates.

Figure 4

Effect of Ca²⁺ ions on the purified PLIII activity. The vertical bars indicate standard deviation (SD) of the mean calculated for three replicates.

Figure 5

Degradation pattern of pectic substances by the purified PLIII from A. giganteus. Each lane corresponds to one substrate submitted to enzymatic degradation. Lane 1: digalacturonic acid, lane 2: polygalacturonic acid, lane 3: citrus pectin MD 72%, lane 4: citrus pectin MD 34%, lane 5; apple pectin MD 75%, and lanes 6, 7, and 8; citrus pectin MD 72% after 30 min, 2 h, and 24 h incubation, respectively. Standards: G1: monogalacturonic acid, G2: digalacturonic acid, and G3: trigalacturonic acid. The upper spots in lanes 2, 3, 6, 7, and 8 result from the alkaline hydrolysis of the substrate due to the reaction conditions also seen in the controls without enzyme addition (data not shown).