, Volume 23, Issue 5, pp 529–547 | Cite as

Purification and biochemical characterization of a novel thermostable and halotolerant subtilisin SAPN, a serine protease from Melghiribacillus thermohalophilus Nari2AT for chitin extraction from crab and shrimp shell by-products

  • Sondes Mechri
  • Khelifa Bouacem
  • Fadoua Jabeur
  • Sara Mohamed
  • Nariman Ammara Addou
  • Ahlam Dab
  • Aicha Bouraoui
  • Amel Bouanane-Darenfed
  • Samir Bejar
  • Hocine Hacène
  • Laura Baciou
  • Florence Lederer
  • Bassem JaouadiEmail author
Original Paper


The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2AT. The highest yield of protease (395 IU/g) with white shrimp shell by-product (40 g/L) as a unique source of nutriments in the growth medium was achieved after 52 h at 55 °C. The monomeric enzyme of about 30 kDa was purified to homogeneity by ammonium sulfate fractionation, heat treatment, followed by sequential column chromatographies. The optimum pH and temperature values for subtilisin activity were pH 10 and 75 °C, respectively, and half lives of 9 and 5 h at 80 and 90 °C, respectively. The sequence of the 25 NH2-terminal residues pertaining of SAPN exhibited a high homology with those of Bacillus subtilisins. The inhibition by DFP and PMSF indicates that this enzyme belongs to the serine proteases family. SAPN was found to be effective in the deproteinization (DDP %) of blue swimming crab (Portunus segnis) and white shrimp (Metapenaeus monoceros) by-products, with a degree of 65 and 82%, respectively. The commercial and the two chitins obtained in this work showed a similar peak pattern in Fourier-Transform Infrared (FTIR) analysis, suggesting that SAPN is suitable for the bio-production of chitin from shell by-products.

Article Highlights

  • The purification of subtilisin (SAPN) from M. thermohalophilus Nari2A was carried out.

  • The molecular weight and the NH2-terminal sequence of the subtilisin were determined.

  • Optimum pH and temperature values for activity were pH 10 and 75 °C respectively.

  • SAPN was found to be effective in the deproteinization of crab and shrimp by-products.

  • SAPN may be used as candidate for chitin extraction from crustacean by-products.

Graphic abstract


Melghiribacillus thermohalophilus Subtilisin Portunus segnis Metapenaeus monoceros Chitin 



The authors would like to express their gratitude to Mr. K. Walha, Mrs. N. Kchaou, and Mrs. N. Masmoudi (Analysis Unit-CBS) for their technical assistance. We would also like to thank Dr. W. Saibi and Pr. H. Belghith (CBS) and Mr. F. Allala (LCBM, FSB-USTHB) for their constructive discussions and suggestions. Special thanks are also due to Pr. W. Hariz from the English Department at the Faculty of Sciences of Sfax (FSS), University of Sfax (Tunisia) for constructive proofreading and language polishing services.


This study was supported by the Tunisian Ministry of Higher Education and Scientific Research under the Contract Program LMBEE-CBS/code: LR15CBS06_2015-2019 and the Ph.D. Student Fellowship of the Doctoral Institute of Fundamental Sciences of the Sfax University represented by the FSS, University of Sfax/Code: ED08FSSf01.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


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Copyright information

© Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  • Sondes Mechri
    • 1
  • Khelifa Bouacem
    • 1
    • 2
  • Fadoua Jabeur
    • 1
  • Sara Mohamed
    • 2
  • Nariman Ammara Addou
    • 2
  • Ahlam Dab
    • 1
  • Aicha Bouraoui
    • 4
  • Amel Bouanane-Darenfed
    • 2
  • Samir Bejar
    • 1
  • Hocine Hacène
    • 2
  • Laura Baciou
    • 4
  • Florence Lederer
    • 4
  • Bassem Jaouadi
    • 1
    • 3
    Email author
  1. 1.Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS)University of SfaxSfaxTunisia
  2. 2.Laboratory of Cellular and Molecular Biology (LCMB), Microbiology Team, Faculty of Biological SciencesUniversity of Sciences and Technology of Houari Boumediene (USTHB)AlgiersAlgeria
  3. 3.Biotech ECOZYM Start-up, Business Incubator, Centre of Biotechnology of Sfax (CBS)University of SfaxSfaxTunisia
  4. 4.Laboratoire de Chimie Physique (LCP), CNRS UMR 8000, Faculté des Sciences, Université Paris-Sud, Université Paris-SaclayOrsay CedexFrance

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