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
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.