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Background: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. Objectives: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. Methods: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. Results: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 μM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. Conclusion: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

In this study, a heterologously expressed exo-polygalacturonase from Sporothrix schenckii1099-18 was purified and then its two magnetic immobilized counterparts were prepared in yolk–shell-structured mesoporous silica. The exo-polygalacturonase was initially precipitated on porous calcium carbonate in the presence of Fe3O4magnetic nanoparticles. Then, a silica layer was created on the surface of the precipitated particles. The obtained immobilized exo-polygalacturonase (silica@Fe3O4/SsExo-PG) was further treated with glutaraldehyde (silica@Glu- Fe3O4/SsExo-PG). Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy techniques were used to confirm the immobilization of polygalacturonase. The optimum pH and temperature of all the exo-polygalacturonase preparations were determined to be 4.0 and 60 °C, respectively which are also optimum conditions for apple juice clarification. The silica@ Fe3O4/SsExo-PG and silica@Glu- Fe3O4/SsExo-PG showed 3- and 3.7-fold much stability, respectively than the free exo-polygalacturonase at 60 °C. The free and immobilized SsExo-PG preparations were used for the clarification of apple juice and the result showed that the turbidity of apple juice was reduced by 82% after 1 h treatment time. After ten consecutive uses, silica@Fe3O4/SsExo-PG and silica@Glu-Fe3O4/SsExo-PG retained 80% and 90% of their initial activities, respectively. Altogether, biochemical, thermal-stability, and apple juice clarification characteristics of the immobilized SsExo-PG suggest a high potential for biodegradation of pectin.