High-yield intracellular production of an exo-polygalacturonase enzyme via heterologous expression of Penicillium notatum gene in Saccharomyces cerevisiae

Abstract

Exo-polygalacturonase (Exo-PG) is one of the most important members of the pectinolytic group of enzymes with immense applications in the food industry. The present work was undertaken to investigate the cloning, expression, and transformation of an Exo-PG gene in yeast Saccharomyces cerevisiae to achieve the high titre of Exo-PG from Penicillium notatum. For this, the Exo-PG gene from P. notatum was cloned into BamHI and XbaI digested pYES2 plasmid with GAL1 promoter, and heterologously expressed in S. cerevisiae. The recombinant yeast cells were cultivated at 30°C in shake flask fermentation using minimal media without uracil, in the presence of ampicillin (100 µg/mL), following the addition of 2.0% galactose as an expression inducer. Results revealed that the yeast was a good expression host, and successfully produced 6.67 U/mL of the recombinant enzyme into the culture media after 24 h of induction; under longer induction time, the activity was decreased. The secreted Exo-PG exhibited two strong bands with an approximate molecular weight of 20 - 25 kDa and 70 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, thus indicating a dimeric protein. In conclusion, the results demonstrated that the gene was successfully expressed, thus resulting in high-yield intracellular production of Exo-PG.

Isolation and Characterization of polygalacturonase producing thermophilic Aspergillus spp. isolated from decayed tomato fruits

“…As a result, the demand for enzyme time has risen dramatically (Cocok et al 2017;Li et al 2012;Ramesh et al 2020; Raveendran et al 2018). Pectinases are a class of pectinolytic enzymes that catalyse the depolymerisation and degradation of pectinaceous materials using hydrolases, lyases (depolymerisation reaction), or esterases (de-esteri cation reaction) (Zhang et al 2021). Pectinase can also hydrolyze the alpha-1, 4 glycosidic linkages that exist between galacturonic acid residues and sugar (Rahman et al 2019).…”