Isolation of a bacterial strain from the gut of the fish, Systomus sarana, identification of the isolated strain, optimized production of its protease, the enzyme purification, and partial structural characterization

Arul Dhayalan; Balasubramanian Velramar; Balasubramani Govindasamy; Karthik Raja Ramalingam; Aiswarya Dilipkumar; Perumal Pachiappan

doi:10.1186/s43141-022-00299-3

Isolation of a bacterial strain from the gut of the fish, Systomus sarana, identification of the isolated strain, optimized production of its protease, the enzyme purification, and partial structural characterization

J Genet Eng Biotechnol. 2022 Feb 10;20(1):24. doi: 10.1186/s43141-022-00299-3.

Authors

Arul Dhayalan^{1

2}, Balasubramanian Velramar^{1

3}, Balasubramani Govindasamy^{1

4}, Karthik Raja Ramalingam^{1

5}, Aiswarya Dilipkumar^{1

6}, Perumal Pachiappan^{7

8}

Affiliations

¹ Department of Biotechnology, School of Biosciences, Periyar University, Salem, 636011, Tamil Nadu, India.
² ICAR- National Dairy Research Institute, SRS, Adugodi, Bengaluru, 560030, Karnataka, India.
³ Amity Institute of Biotechnology, Amity University, Raipur, 493225, Chhattisgarh, India.
⁴ ICAR- Central Institute of Brackishwater Aquaculture, Chennai, 600028, Tamil Nadu, India.
⁵ Department of Microbiology, Alagappa University, Karaikudi, 630003, Tamil Nadu, India.
⁶ 1/145, New Mariyaman Kovil Street, Bominayakanpatti post, Pagalpatti, Salem, 636304, Tamil Nadu, India.
⁷ Department of Biotechnology, School of Biosciences, Periyar University, Salem, 636011, Tamil Nadu, India. perumalarticles@gmail.com.
⁸ Department of Marine Science, School of Marine Sciences, Bharathidasan University, Tiruchirappalli, 620024, Tamil Nadu, India. perumalarticles@gmail.com.

Abstract

Background: The present study focuses on the isolation of Bacillus thuringiensis bacterium from the gut of fresh water fish, Systomus sarana, the innovative optimization of culture parameters to produce maximum protease enzyme, by the isolated bacterium, and the elucidation of peptide profile of the protease. And the experimental data and results were authenticated through the response surface method (RSM) and Box-Behnken design (BBD) model.

Results: During the RSM optimization, the interaction of the highest concentrations (%) of 2.2 maltose, 2.2 beef extract, and 7.0 pH, at 37 °C incubation, yielded a maximum protease enzyme of 245 U/ml by the fish gut-isolated, B. thuringiensis. The spectral analysis of the obtained enzyme revealed the presence of major functional groups at the range of 610-3852 cm^-1 viz., alkynes (-C≡C-H: C-H stretch), misc (P-H phosphine sharp), α, β-unsaturated aldehydes, and through PAGE analysis, its molecular weight was determined as 27 kDa. The enzyme's MALDI-TOF/MS analysis revealed the presence of 15 peptides from which the R.YHTVCDPR.L peptide has been found to be a major one.

Conclusions: The fish gut-isolated bacterium, B. thuringiensis, SS4 exhibited the potential for high protease production under the innovatively optimized culture conditions, and the obtained result provides scope for applications in food and pharmaceutical industries.

Keywords: Bacillus thuringiensis; Enzyme assay; Homology modeling; Peptide fingerprinting; Response surface methodology.