Comparative Studies of Structure and Regulatory Genes Controlling Pectinase Expression in Soft Rot Pectobacterium carotovorum NM-NRC;-Multistep Mutagenesis, Purification and Molecular Docking Studies For the Over-Production of Pectinase

Abd El-Aziz, Nagwa M.; El-gamal, Nora N.; Moharam, Maysa E.; Abdelhamid, Sayeda A.; Abdelaziz, Ahmed M.; El-Khonezy, Mohamed I.

doi:10.21608/ejchem.2024.298342.9881

Comparative Studies of Structure and Regulatory Genes Controlling Pectinase Expression in Soft Rot Pectobacterium carotovorum NM-NRC;-Multistep Mutagenesis, Purification and Molecular Docking Studies For the Over-Production of Pectinase

Document Type : Original Article

Authors

¹ Microbial Genetics Department, Biotechnology Research Institue, NRC

² Microbial Chemistry Department, Biotechnology Research Institue, NRC

³ Microbial Chemistry Department, NRC.

⁴ Microbial Biotechnology Department, Biotechnology Research Institue, NRC

⁵ Molecular biology department, Genetic Engineering and Biotechnology, and Biotechnology Research Division. National Research Centre, Dokki, Giza. Egypt.

⁶ Molecular Biology Department, Biotechnology Research Institute, National Research Centre

10.21608/ejchem.2024.298342.9881

Abstract

In this study, the pectinase activity investigated and assessed of 50 bacterial strains isolated from various rotting fruits and vegetables. Isolate No. 10, which measured 52.42 U/ml had the highest activity, was identified molecularly using the 16Sr DNA gene as Pectobacterium carotovorum subsp. carotovorum NM-NRC and deposited in the NCBI database with accession number OQ256290. Successive mutagenesis was performed utilizing ultraviolet, ethyl methansulfonate, and ethidium bromide for Pectobacterium carotovorum NM-NRC. A mutants assigned as Pectobacterium carotovorum E-36, displayed pectinase actions of 136.24 U/ml. To optimize the ideal conditions for pectinase expression, applied the Reaction Surface Strategy (RSM) to Pectobacterium carotovorum NM-NRC mutant E-36. The most significant pectinase-specific activity of 214.34 U/ml was under culture conditions of pH 9, 72 hours of hatching, and 2.5% lactose and 0.5% malt extract as carbon and nitrogen sources, respectively.Two pectinase isoenzymes (PecI and PecII) mutant E-36 have been isolated to homogeneity. Using SDS-PAGE electrophoresis, the primary isoenzyme PecII demonstrated molecular weight of 37 kDa. The 3D structure of the modeled polygalacturonase (PGase), pectate lyase (PL) and pectinesterase (PE) and regulatory gene (KdgR) proteins was validated using Ramachandran’s plot, which indicated that a high percentage were in the most favored region of the amino acid residues and docking studies revealed optimal binding affinities of the PGase, PL, PE and KdgR proteins with pectin substrate that was with high values of affinity score for the template strain Pectobacterium carotovorum strain PCC21, strain Pectobacterium NM-NRC and mutant Pectobacterium NM-NRC E-36; respectively.

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