Gene expression and molecular characterization of recombinant subtilisin from Bacillus subtilis with antibacterial, antioxidant and anticancer properties.

Autor: Shettar SS; Department of Biotechnology, KLE Technological University, Hubballi, Karnataka 580031, India., Bagewadi ZK; Department of Biotechnology, KLE Technological University, Hubballi, Karnataka 580031, India. Electronic address: zabinb@gmail.com., Yaraguppi DA; Department of Biotechnology, KLE Technological University, Hubballi, Karnataka 580031, India., Das S; Department of Chemistry, Indian Institute of Technology, Dharwad, Karnataka 580011, India., Mahanta N; Department of Chemistry, Indian Institute of Technology, Dharwad, Karnataka 580011, India., Singh SP; Department of Biosciences and Bioengineering, Indian Institute of Technology Dharwad, Karnataka 580011, India., Katti A; Department of Biotechnology, KLE Technological University, Hubballi, Karnataka 580031, India., Saikia D; Department of Biosciences and Bioengineering, Indian Institute of Technology Dharwad, Karnataka 580011, India.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2023 Sep 30; Vol. 249, pp. 125960. Date of Electronic Publication: 2023 Jul 28.
DOI: 10.1016/j.ijbiomac.2023.125960
Abstrakt: This study investigated the multifunctional attributes such as, antibacterial, antioxidant and anticancer potential of recombinant subtilisin. A codon-optimized subtilisin gene was synthesized from Bacillus subtilis and was successfully transformed into E. coli DH5α cells which was further induced for high level expression in E. coli BL21 (DE3). An affinity purified ~40 kDa recombinant subtilisin was obtained that revealed to be highly alkali-thermostable based on the thermodynamic parameters. The kinetic parameters were deduced that indicated higher affinity of N-Suc-F-A-A-F-pNA substrate towards subtilisin. Recombinant subtilisin demonstrated strong antibacterial activity against several pathogens and showed minimum inhibitory concentration of 0.06 μg/mL against B. licheniformis and also revealed high stability under the influence of several biochemical factors. It also displayed antioxidant potential in a dose dependent manner and exhibited cell cytotoxicity against A549 and MCF-7 cancerous cell lines with IC 50 of 5 μM and 12 μM respectively. The identity of recombinant subtilisin was established by MALDI-TOF mass spectrum depicting desired mass peaks and N-terminal sequence as MRSK by MALDI-TOF-MS. The deduced N- terminal amino acid sequence by Edman degradation revealed high sequence similarity with subtilisins from Bacillus strains. The structural and functional analysis of recombinant antibacterial subtilisin was elucidated by Raman, circular dichroism and nuclear magnetic resonance spectroscopy and thermogravimetric analysis. The results contribute to the development of highly efficient subtilisin with enhanced catalytic properties making it a promising candidate for therapeutic applications in healthcare industries.
Competing Interests: Declaration of competing interest All the authors confirm that they have no argument of interests.
(Copyright © 2023 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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