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
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.
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.
KeywordsMelghiribacillus 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.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Annamalai N, Rajeswari MV, Sahu SK, Balasubramanian T (2014b) Purification and characterization of solvent stable, alkaline protease from Bacillus firmus CAS 7 by microbial conversion of marine wastes and molecular mechanism underlying solvent stability. Process Biochem 49:1012–1019CrossRefGoogle Scholar
- Arbia W, Arbia L, Adour L, Amrane A (2013) Chitin extraction from crustacean shells using biological methods: a review. Food Technol Biotech 51:12–25Google Scholar
- Balti R, Bougatef A, Sila A, Guillochon D, Dhulster P, Nedjar Arroume N (2015) Nine novel angiotensin I-converting enzyme (ACE) inhibitory peptides from cuttlefish (Sepia officinalis) muscle protein hydrolysates and antihypertensive effect of the potent active peptide in spontaneously hypertensive rats. Food Chem 170:519–525CrossRefGoogle Scholar
- Benkiar A, Zaraî Jaouadi N, Badis A, Rebzani F, Boulkour Touioui S, Rekik H, Naili B, Ferradji FZ, Bejar S, Jaouadi B (2013) Biochemical and molecular characterization of a thermo- and detergent-stable alkaline serine keratinolytic protease from Bacillus circulans strain DZ100 for detergent formulations and feather-biodegradation process. Inter Biodeter Biodegrad 83:129–138CrossRefGoogle Scholar
- Bouacem K, Bouanane Darenfed A, Laribi-Habchi H, Ben Elhoul M, Hmida Sayari A, Hacene H, Ollivier B, Fardeau ML, Jaouadi B, Bejar S (2015) Biochemical characterization of a detergent-stable serine alkaline protease from Caldicoprobacter guelmensis. Int J Biol Macromol 81:299–307CrossRefGoogle Scholar
- Hamiche S, Mechri S, Khelouia L, Annane R, El Hattab M, Badis A, Jaouadi B (2019) Purification and biochemical characterization of two keratinases from Bacillus amyloliquefaciens S13 isolated from marine brown alga Zonaria tournefortii with potential keratin-biodegradation and hide-unhairing activities. Int J Biol Macromol 122:758–769CrossRefGoogle Scholar
- Mechri S, Ben Elhoul Berrouina M, Omrane Benmrad M, Zaraî Jaouadi N, Rekik H, Moujehed E, Chebbi A, Sayadi S, Chamkha M, Bejar S, Jaouadi B (2017a) Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest. Int J Biol Macromol 94:221–232CrossRefGoogle Scholar
- Mechri S, Kriaa M, Ben Elhoul Berrouina M, Omrane Benmrad M, Zaraî Jaouadi N, Rekik H, Bouacem K, Bouanane Darenfed A, Chebbi A, Sayadi S, Chamkha M, Bejar S, Jaouadi B (2017b) Optimized production and characterization of a detergent-stable protease from Lysinibacillus fusiformis C250R. Int J Biol Macromol 101:383–397CrossRefGoogle Scholar
- Mohamed S, Bouacem K, Mechri S, Addou NA, Laribi-Habchi H, Fardeau ML, Jaouadi B, Bouanane-Darenfed A, Hacène H (2019) Purification and biochemical characterization of a novel acido-halotolerant and thermostable endochitinase from Melghiribacillus thermohalophilus strain Nari2AT. Carbohydr Res 473:46–56CrossRefGoogle Scholar
- Omrane Benmrad M, Moujehed E, Ben Elhoul M, Zaraî Jaouadi N, Mechri S, Rekik H, Kourdali S, El Hattab M, Badis A, Sayadi S, Bejar S, Jaouadi B (2016) A novel organic solvent-and detergent-stable serine alkaline protease from Trametes cingulata strain CTM10101. Int J Biol Macromol 91:961–972CrossRefGoogle Scholar
- Omrane Benmrad M, Moujehed E, Ben Elhoul M, Mechri S, Bejar S, Zouari R, Baffoun A, Jaouadi B (2018) Production, purification, and biochemical characterization of serine alkaline protease from Penicillium chrysogenium strain X5 used as excellent bio-additive for textile processing. Int J Biol Macromol 119:1002–1016CrossRefGoogle Scholar
- Rani K, Rana R, Datt S (2012) Review on latest overview of proteases. Int J Curr Life Sci 2:12–18Google Scholar
- Zaraî Jaouadi N, Rekik H, Badis A, Trabelsi S, Belhoul M, Yahiaoui AB, Ben Aicha H, Toumi A, Bejar S, Jaouadi B (2013) Biochemical and molecular characterization of a serine keratinase from Brevibacillus brevis US575 with promising keratin-biodegradation and hide-dehairing activities. PLoS One 8:e76722CrossRefGoogle Scholar