Identification of a novel protease from the thermophilic Anoxybacillus kamchatkensis M1V and its application as laundry detergent additive

  • Sondes Mechri
  • Khelifa Bouacem
  • Nadia Zaraî Jaouadi
  • Hatem Rekik
  • Mouna Ben Elhoul
  • Maroua Omrane Benmrad
  • Hocine Hacene
  • Samir Bejar
  • Amel Bouanane-Darenfed
  • Bassem JaouadiEmail author
Original Paper


A thermostable extracellular alkaline protease (called SAPA) was produced (4600 U/mL) by Anoxybacillus kamchatkensis M1V, purified to homogeneity, and biochemically characterized. SAPA is a monomer with a molecular mass of 28 kDa estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion using high performance liquid chromatography (HPLC). The sequence of its NH2-terminal amino-acid residues showed high homology with those of Bacillus proteases. The SAPA irreversible inhibition by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine proteases family. Optimal activity of SAPA was at pH 11 and 70 °C. The sapA gene was cloned and expressed in the extracellular fraction of E. coli. The highest sequence identity value (95%) of SAPA was obtained with peptidase S8 from Bacillus subtilis WT 168, but with 16 amino-acids of difference. The biochemical characteristics of the purified recombinant extracellular enzyme (called rSAPA) were analogous to those of native SAPA. Interestingly, rSAPA exhibit a degree of hydrolysis that were 1.24 and 2.6 than SAPB from Bacillus pumilus CBS and subtilisin A from Bacillus licheniformis, respectively. Furthermore, rSAPA showed a high detergent compatibility and an outstanding stain removal capacity compared to commercial enzymes: savinase™ 16L, type EX and alcalase™ Ultra 2.5 L.

Graphic Abstract


  • A new extracellular Anoxybacillus kamchatkensis protease was purified (SAPA) and characterized.

  • SAPA was a serine protease and a monomer with a molecular mass of ∼28 kDa.

  • Optimum pH and temperature values for activity were pH 11 and 70 °C, respectively.

  • The sapA gene encoding SAPA was cloned, sequenced, and expressed in E. coli.

  • rSAPA is a potential candidate for future application as laundry detergent additive.


Anoxybacillus kamchatkensis Hot spring Protease Expression Detergent 



The authors would like to thank Mrs. N. Masmoudi, Mrs. N. Kchaou, and Mr. K. Walha (Analysis Unit-CBS) for their technical help. They would also like to express their gratitude to Pr. Ali Gargouri, Pr. H. Belghith, and Dr. W. Saibi (CBS) for their helpful suggestions and discussions; and Pr. W. Hariz from the English Department at the Sfax Faculty of Sciences, University of Sfax, Tunisia for language editing services and constructive proofreading.

Author contributions

All authors cited, have made intellectual, substantial, and direct participation to the work, and approved it for publication.


This study was supported by the Ministry of Higher Education and Scientific Research in Tunisian under the contract program LMBEE-CBS/code: LR15CBS06_2015-2019.

Compliance with ethical standards

Conflict of interest

Authors Nadia Zaraî Jaouadi, Hatem Rekik, Mouna Ben Elhoul, Samir Bejar, and Bassem Jaouadi were employed by company (Biotech ECOZYM Start-up, Business Incubator at the CBS). All other authors declare no competing interests.

Ethical approval

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

Supplementary material

792_2019_1123_MOESM1_ESM.docx (2.9 mb)
Supplementary file1 (DOCX 2986 kb)


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Copyright information

© Springer Japan KK, part of Springer Nature 2019

Authors and Affiliations

  • Sondes Mechri
    • 1
  • Khelifa Bouacem
    • 1
    • 3
  • Nadia Zaraî Jaouadi
    • 1
    • 2
  • Hatem Rekik
    • 1
    • 2
  • Mouna Ben Elhoul
    • 1
    • 2
  • Maroua Omrane Benmrad
    • 1
  • Hocine Hacene
    • 3
  • Samir Bejar
    • 1
    • 2
  • Amel Bouanane-Darenfed
    • 3
  • Bassem Jaouadi
    • 1
    • 2
    Email author
  1. 1.Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Centre of Biotechnology of Sfax (CBS)University of SfaxSfaxTunisia
  2. 2.Biotech ECOZYM Start-Up, Business Incubator, Centre of Biotechnology of Sfax (CBS)University of SfaxSfaxTunisia
  3. 3.Laboratory of Cellular and Molecular Biology (LCMB), Microbiology Team, Faculty of Biological SciencesUniversity of Sciences and Technology of Houari Boumediene (USTHB)Bab EzzouarAlgeria

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