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

Bhatti H. N., . and Amin F., . and Bilal M., . and Zhang S., . and Xiong M., . High-yield intracellular production of an exo-polygalacturonase enzyme via heterologous expression of Penicillium notatum gene in Saccharomyces cerevisiae. pp. 664-671. ISSN 22317546

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 30C in shake flask fermentation using minimal media without uracil in the presence of ampicillin (100 g/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.

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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 30C in shake flask fermentation using minimal media without uracil in the presence of ampicillin (100 g/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.

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Item Type:	Article
AGROVOC Term:	Polygalacturonase
AGROVOC Term:	Saccharomyces cerevisiae
AGROVOC Term:	Pectinolytic enzymes
AGROVOC Term:	Gel electrophoresis
AGROVOC Term:	analysis
AGROVOC Term:	Plasmids
AGROVOC Term:	Cloning
AGROVOC Term:	Gene sequence
AGROVOC Term:	Food industry
AGROVOC Term:	Yield increases
Depositing User:	Mr. AFANDI ABDUL MALEK
Last Modified:	24 Apr 2025 00:55
URI:	http://webagris.upm.edu.my/id/eprint/9992

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