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Pectinase from a Fish Gut Bacterium, Aeromonas guangheii (SS6): Production, Cloning and Characterization

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During the present research, 11 gut bacteria were isolated from the freshwater fish, Systomus sarana (General name: olive barb) and upon screening, the strains produced extracellular pectinase enzyme. Among them, the SS6 strain was found to produce a high quantity of 208.731 U/ml pectinase and through molecular characterization the SS6 strain was identified as Aeromonas guangheii. During the culture of SS6 strain, a set of parameters were optimized through the response surface methodology with a Box–Behnken design, for the production of the enzyme. The optimal conditions were found to be 2.11% of maltose, 2.20% of yeast extract, 6.5 of pH, and a temperature of 27.3 °C at 32-h incubation. Under the above conditions, the activity of pectinase production was enhanced to 371 U/ml. The purified pectinase’s molecular weight was determined to be ~ 50 kDa (by 10% 2-D PAGE). Totally, nine peptides were identified from the purified pectinase enzyme through the MALDI-TOF-MS analysis and MASCOT tool was used to get the mass spectrum of the peak at 2211 of peptide that indicated the reference pectinase protein. The referenced gene primer (pectate lyases) was PCR amplified and its nucleotide sequence was analyzed. The exo-pelA gene was cloned in pREST vector, which was found to be over expressed in Escherichia coli BL21. The ORF encoded for a mature protein comprising of 425 amino acids (1236 nucleotides) with a predicted molecular weight of ~ 48.7 kDa. The present findings underline the potential of the fish-gut microbes as a source of biotechnologically important enzymes.

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Acknowledgements

The authors are grateful to the authorities of the Periyar University for providing the necessary facilities to carry out this research work. Thanks are due to Dr. R. Rajaram, Associate Professor, Dept. of Marine Science, Bharathidasan University, for his help in plagiarism check. PP thanks the UGC, New Delhi, for the award of BSR Faculty Fellowship (Ref. No.F.18-1/2011 (BSR), 26.06.2018).

Funding

This research work was done without funding.

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Author notes

  1. Arul Dhayalan

    Present address: SRS of ICAR- National Dairy Research Institute, Adugodi, Bengaluru, Karnataka, 560030, India

  2. Natarajan Thillainathan

    Present address: Department of Biomedical Engineering, Central University of Rajasthan, Ajmer, Rajasthan, 305817, India

  3. Balasubramanian Velramar

    Present address: Amity Institute of Biotechnology, Amity University, Raipur, Chhattisgarh, 493225, India

  4. Palanisammi Athiyappagounder

    Present address: Veterinary College & Research Institute, Tamil Nadu Veterinary & Animal Science University, Tirunelveli, Tamil Nadu, 627358, India

  5. Dhanasundaram Sundaramoorthy

    Present address: Department of Marine Science, School of Marine Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India

Authors and Affiliations

  1. Department of Biotechnology, School of Biosciences, Periyar University, Salem, Tamil Nadu, 636011, India

    Arul Dhayalan, Natarajan Thillainathan, Balasubramanian Velramar & Perumal Pachiappan

  2. Department of Marine Science, School of Marine Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India

    Perumal Pachiappan

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Contributions

All authors brainstormed and came up with this study concept. NT provided the chemicals, equipment for the related microbiology tests and Response Surface Methodology (RSM-software) and related guidelines. BV provided the cloning vector, expression strain and related guidelines. PA assisted in review and editing for the manuscript. SD assisted in writing—review and editing, data curation of the manuscript. PP provided the full manuscript language correction, conceptualization, writing—review and editing—original, draft. AD provided the bacteria and carried out all the experiments, compiled and analyzed the resultant data and compiled this paper with insights from all the other authors.

Corresponding author

Correspondence to Perumal Pachiappan.

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Conflict of interest

All authors declare that they have no conflict of interest.

Ethical Approval

According to the Committee for the Purpose of Control and Supervision of Experiments on Animals-CPCSEA (under Ministry of Environment, Forests and Climate Change, Government of India), the edible fish which are used for laboratory experiments are exempted from obtaining Institutional Animal Ethics Committee (IAEC) approval.

Consent to Participate

The study animal that we have utilized for the present work is Systomus sarana (Hamilton, 1882) which is an edible fish, and hence the necessity did not arise to acquire the approval from IAEC. However, we have followed the OECD Guidelines for safe handling of experimental animals.

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Dhayalan, A., Thillainathan, N., Velramar, B. et al. Pectinase from a Fish Gut Bacterium, Aeromonas guangheii (SS6): Production, Cloning and Characterization. Protein J 41, 572–590 (2022). https://doi.org/10.1007/s10930-022-10077-2

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