Molecular Cloning and Characterization of an Acidic Polygalacturonase from Grapes and Its Potential in Industry
| Autor: | Zahra Nazir, Mahjabeen Saleem, Riffat Chohan, Malik Siddique Mahmood, Roquyya Gul, Hafiz Muzzammel Rehman |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
chemistry.chemical_classification
food.ingredient Pectin Chemistry food and beverages lac operon Molecular cloning medicine.disease_cause Genes Plant Polymerase Chain Reaction Hydrolysis Enzyme food Polygalacturonase Fiber cell Biochemistry Genetics medicine Microscopy Electron Scanning Electrophoresis Polyacrylamide Gel Vitis Pectinase Cloning Molecular Molecular Biology Escherichia coli |
| Zdroj: | Critical reviews in eukaryotic gene expression. 30(5) |
| ISSN: | 1045-4403 |
| Popis: | Pectinase enzymes from different plants have many possible biotechnological applications in various industries. Considering industrial significance of pectinolytic enzymes, the polygalacturonase (PG) gene from grapes was cloned into Escherichia coli DH5α using pTZ57R/T vector. Homologous sequences established a close link to Vitis vinifera. Conserve domain analysis predicted PLN02218 domain belongs to the cl31843 superfamily, showing its function as polygalacturonase. After confirmation by PCR and restriction analysis, the PG gene was expressed in E. coli BL21 and induced by IPTG. Expression level was assessed by 12% SDS-PAGE, which showed a 47 kDa protein. High expression level in the soluble fraction indicated that the protein is intracellular or transmembrane. Maximum expression was obtained with 1 mM IPTG and 6 hour induction time, and autoinduction with lactose increased production of the recombinant enzyme. Zymogram analysis revealed that the induced protein was an active enzyme. Expressed PG showed pectinolytic effect on the physiochemical properties of lemon juice. Natural biopolymers are greatly needed because synthetic fibers can negatively affect health. Pectin hydrolysis of banana pseudostem by the PG enzyme produced better quality fiber. Morphological studies by scanning electron microscopy revealed pectin degradation within the fiber cell architecture, showing the effectiveness of PG treatment on banana pseudostem. |
| Databáze: | OpenAIRE |
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