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Improved anti-biofilm activity and long-lasting effects of novel serratiopeptidase immobilized on cellulose nanofibers

  • Biotechnological Products and Process Engineering
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Abstract

Today, enzymatic treatment is a progressive field in combating biofilm producing pathogens. In this regard, serratiopeptidase, a medicinally important metalloprotease, has been recently highlighted as an enzyme with proved anti-biofilm activity. In the present study, in order to increase the long-lasting effects of the enzyme, serratiopeptidase and the novel engineered forms with enhanced anti-biofilm activity were immobilized on the surface of cellulose nanofibers (CNFs) as a natural polymer with eminent properties. For this, recombinant serratiopeptidases including the native and previously designed enzymes were produced, purified and conjugated to the CNF by chemical and physical methods. Immobilization was confirmed using different scanning and microscopic methods. The enzyme activity was assessed using casein hydrolysis test. Enzyme release analysis was performed using dialysis tube method. Anti-biofilm activity of free and immobilized enzymes has been examined on Staphylococcus aureus and Pseudomonas aeruginosa strains. Finally, cytotoxicity of enzyme-conjugated CNFs was performed by MTT assay. The casein hydrolysis results confirmed fixation of all recombinant enzymes on CNFs by chemical method; however, inadequate fixation of these enzymes was found using cold atmospheric plasma (CAP). The AFM, FTIR, and SEM analysis confirmed appropriate conjugation of enzymes on the surface of CNFs. Immobilization of enzymes on CNFs improved the anti-biofilm activity of serratiopeptidase enzymes. Interestingly, the novel engineered serratiopeptidase (T344 [8-339ss]) exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms. In conclusion, incorporation of serratiopeptidases into CNFs improves their anti-biofilm activities without baring any cytotoxicity.

Key points

• Enzymes were successfully immobilized on cellulose nanofibers using chemical method.

• Immobilization of enzymes on CNFs improved their anti-biofilm activity.

• T344 [8-339ss] exhibited the highest anti-biofilm activity in both conjugated and non-conjugated forms.

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Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author upon a reasonable request.

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Acknowledgements

The authors wish to express their deep gratitude to all who provided support during the course of this research. Dr. Seyed Ali Nojoumi kindly revised the manuscript for proper language and grammar.

Funding

This project was financially supported by Pasteur Institute of Iran.

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Authors and Affiliations

  1. Department of Tissue Engineering and Applied Cell Science, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Maryam Rouhani & Sara Molasalehi

  2. Nano-Biotechnology Department, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran

    Maryam Rouhani, Vahideh Valizadeh, Haleh Bakhshandeh, Sara Ali Hosseinzadeh, Sara Molasalehi, Seyed Mohammad Atyabi & Dariush Norouzian

  3. QC Department, Osve Pharmaceutical Co., Tehran, Iran

    Haleh Bakhshandeh

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  1. Maryam Rouhani
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Contributions

MR, SAH, and SM contributed to the laboratory work, analysis of the data, and drafted the paper. HB, SMA, and DN offered critical suggestions for designing experimental assays and data analysis. V. V designed the work, supervised the study, and critically revised the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Vahideh Valizadeh.

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This article contained no studies incorporating human participants or animals to be performed by any of the authors. The local Ethics Review Committee approved the study protocol at the Pasteur Institute of Iran, Tehran, Iran (approval ID: IR.PII.REC.1398.040).

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The authors declare no competing interests.

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Rouhani, M., Valizadeh, V., Bakhshandeh, H. et al. Improved anti-biofilm activity and long-lasting effects of novel serratiopeptidase immobilized on cellulose nanofibers. Appl Microbiol Biotechnol 107, 6487–6496 (2023). https://doi.org/10.1007/s00253-023-12734-7

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  • DOI: https://doi.org/10.1007/s00253-023-12734-7

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