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Preliminary Screening and Characterization of Novel Proteolytic Enzymes Produced by Extremophilic Bacteria Isolated from Tunisian and Algerian Biotopes

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Preliminary Screening and Characterization of Novel Proteolytic Enzymes Produced by Extremophilic Bacteria Isolated from Tunisian and Algerian Biotopes Submitted on: 15/03/2021 Revue Revised form accepted on: 14/06/2021 Nature et Technologie Corresponding author: [email protected] http://www.univ-chlef.dz/revuenatec ISSN : 1112-9778 – EISSN : 2437-0312 Preliminary screening and characterization of novel proteolytic enzymes produced by extremophilic bacteria isolated from Tunisian and Algerian biotopes Sondes MECHRI a, Khelifa BOUACEM b,c, Fawzi ALLALAb, Marwa KHALED a, Amel b b a, BOUANANE-DARENFED , Hocine HACÈNE , Bassem JAOUADI * a Laboratory of Microbial Biotechnology and Engineering Enzymes (LMBEE), Center of Biotechnology of Sfax (CBS), University of Sfax, Road of Sidi Mansour Km 6, P.O. Box 1177, Sfax 3018, Tunisia b Laboratory of Cellular and Molecular Biology (LCMB), Microbiology Team, Faculty of Biological Sciences (FSB), University of Sciences and Technology Houari Boumediene (USTHB), P.O. Box 32, El Alia, Bab Ezzouar, 16111 Algiers, Algeria c Department of Biochemistry & Microbiology, Faculty of Biological and Agricultural Sciences (FBAS), University Mouloud Mammeri of Tizi-Ouzou (UMMTO), P.O. Box 17, Tizi-Ouzou 15000, Algeria Abstract The current paper reports on the production and characterization of extracellular thermostable proteases produced from thermophilic bacteria namely Aeribacillus pallidus VP3, Anoxybacillus kamchatkensis M1V, Virgibacillus natechei FarDT, and Melghiribacillus thermohalophilus Nari2AT strains isolated from different extremophlic biotopes in Tunisia and Algeria. The key challenge of the use of the different proteases in industrial applications is their efficiency under unconventional conditions. An optimization via one-factor-at-a-time (OFAT) methodology has been used to obtain 3,000 U.mL-1; 4,600 U.mL-1; 15,800 U.mL-1; and 16,000 U.mL-1 of proteases activity from VP3, M1V, FarDT, and Nari2AT strains, respectively. Particularly, VP3 and FarDT proteases showed a high tolerance to several organic solvents. In addition, the protease produced from FarDT strain might be useful as potential candidate for application in detergency. The deproteinization of shrimp wastes by M1V and Nari2AT proteases was successful with a high rate of deproteinization of about 48% and 82%. The obtained proteolytic hydrolysate obtained by M1V strain showed high biological activities with half maximal inhibitory concentration (IC50) of 71.52 μg.mL-1, 107.67 μg.mL-1, and 133.24 μg.mL-1, respectively for the angiotensin, tyrosinase, and amylase inhibitory activities. Keywords: Extremophilic bacteria; Thermostable proteases; Biotechnologically applications. 1. Introduction temperature and pH, which could make them suitable for industries applications [2, 3]. Above all, proteases Microbial life does not seem to be restricted to are enzymes catalyses the hydrolysis of peptide and iso- specific environments. Over the past few decades, it has peptide bonds that join amino acids within proteins. become clear that microbial communities can be found Again, they can act near the ends of polypeptide chains in the most diverse conditions, including extremes of [4]. They are differentiated according to their substrate pressure, salinity, temperature, and pH [1]. These specificity as amino-peptidases, which are cleaves the microorganisms, called extremophiles, produce peptides at the N-terminus, and carboxypeptidases, biocatalysts mainly enzymes that are functional under which are degrades peptides at the C-terminus [5]. The extreme conditions. Accordingly, the unique properties unique catalytic activities of these enzymes make them of these biocatalysts have resulted in several novel an inexpensive choice for hydrolyzing peptide bonds for applications of enzymes in industrial and industrial uses [6]. biotechnological processes. Their ability to break down these compounds makes Biochemical properties of these enzymes them excellent for stain removal. Proteolytic enzymes demonstrated that they have a high optimum working catalyze the hydrolysis of proteinaceous material and This is an open-access document under the terms of the Creative Commons Attribution License CC-BY, which allows it to be shared, copied, reproduced, distributed, communicated, reused or adapted with the obligation to credit its author 62 Preliminary screening and characterization of novel proteolytic enzymes produced by extremophilic bacteria isolated from Tunisian and Algerian biotopes show the highest abundance amongst industrial relevant (Figure 1). The biotopes are saline lakes, hydrothermal biocatalysts [7, 8]. Proteolytic enzymes are found to hot springs, and petroleum reservoirs. The ATAM, have a large multiplicity in their applications, such as M1V, B5GN, HB14, and BA1 strains were isolated constituent in detergents [9], industrial processing of from Hammam Righa hot spring Ain Defla, in food [10], leather [11], peptide synthesis [12], and northwestern Algeria [16]. The heating system is pharmaceutical products [13]. Additionally, the use of characterized with a temperature of 68°C, a salinity of these proteases is highly efficient, by allowing the 150 g.L-1 and a pH of 6.9. The VP3 strain is isolated recovery of chitin and bioactive peptides from from the production water of the Litayem oil-field crustacean’s bio-wastes [14, 15]. This was supported by managed by Thyna Petroleum Services1, Sfax, Tunisia. cloning amino-acid sequences inspection and The formation water was withdrawn from the oil- homology-modeling of the genes encoding bearing horizons from depths of 1,300 m, with a extremozymes, which is endowed with a number of temperature of 78°C, a salinity of 100 g.L-1, and a pH characteristics that are highly valued. Thus, these 7.6, after passing through a pipeline of about 20 km. observations inspired us to explore other enzymes from The FarDT strain is isolated from sediments of a saline the extremophile strains. lake located in Taghit, 93 km from Bechar, southwest of For particular interest, North Africa countries by Algeria. The temperature at the sampling site was 39°C their geographical position present a great diversity of and pH was 6.8. The Nari2AT strain is isolated from the extreme biotopes occupied by an important soil of Chott Melghir, an Algerian Salt Lake located at microbiological richness. Mainly, Tunisia and Algeria north-east of Biskra city, Algeria. The temperature at possess many extreme biotopes such as saline lakes, the sampling site was 39°C and the pH was 6.8. The soil hydrothermal hot springs, and petroleum reservoirs. is characterized by alkaline pH of 8.4 and a salinity of Furthermore, the family of Bacillaceae comprises the 112 g.L-1. The C2SS100 and C2SS10 strains are rod-shaped bacteria which form endospores, having a isolated from the production water of Sercina petroleum wide diversity of physiological characteristics. Their reservoir, located near the Kerkennah Island, Tunisia. ability to form hardy spores enables them to be widely The production water is of about 30 km with a distributed in nature, from Arctic environments to hot temperature of 25°C, a salinity of 71 g.L-1, and a pH of springs and desert sands and from fresh water to salt or 7.8. The positive strains were screened on skimmed marine sediments. Very few reports are available on milk agar plates (SMAP) as earlier reported [17]. enzymes production from Aeribacillus, Anoxybacillus, Virgibacillus, and Melghiribacillus genera. 2.2. Identification of microorganisms, DNA Keeping these points in view, the present sequencing, and phylogenetic analysis contribution was undertaken the screening of bacteria T T isolated from different extreme ecological niches in The VP3, M1V, FarD , and Nari2A strains were Tunisian and Algeria and hyperproducing of proteolytic identified previously based on both catabolic and enzymes. molecular approaches [18-21]. For example, the species Additionally, the optimization through one-factor- level identification of strain M1V was performed using at-a-time (OFAT) methodology and biotechnological microbiological characteristics and 16S rRNA (rDNA) application of these proteases, having expected gene sequence analysis. The colony morphologies were proprieties for their potential uses as bio-additives with determined using cultures grown aerobically on nutrient various laundry detergents and deproteinization of agar (NA). Cell morphology and motility were crustacean’s by-products are also investigated. examined microscopically in exponentially growing liquid cultures after 18 to 24 h of incubation at 60°C by means of conventional tests. Acid production from 2. Materials and Methods carbohydrates and hydrolyses of some polymers were 2.1. Microorganisms determined using analytical profile index (API) strip tests API 20E and API 50 CHB (bioMérieux, SA, Different strains were isolated from different 1 extreme ecological niches in Tunisia and Algeria http://www.made-in-tunisia.net/vitrine/contact.php?tc1=lKmYnqyS MECHRI et al. 63 Marcy-l’Etoile, France) as recommended by the The preculture of M1V and VP3 strains was carried manufacturer. The temperature range for growth was out in a 1 L Erlenmeyer flask containing 100 mL of LB determined by incubating the isolate at 30 to 80°C. The liquid medium at pH 7.4, then incubated at 45°C effect of NaCl on bacterial growth was studied in the overnight. While, the preculture of FarDT strain was presence of 1 to 5% (w/v) NaCl. The pH dependence of carried out on Sehgal and Gibbons medium (SG) growth was tested in the pH range of 6 to 10. All the containing the following (in g.L-1): casamino acids, 7.5; physiological tests were determined in NA medium, yeast extract, 10; sodium glutamate, 1; trisodium citrate, with the exception of the temperature growth at 80°C 3; MgSO4·7 H2O, 20; KCl, 2; FeSO4·7 H2O, 0.036; and -1 which was performed in Nutriment broth (NB). The MnCl2·4 H2O, 0.00036 supplemented with 100 g.L of polymerase chain reaction (PCR) amplification of the NaCl at pH 7 then incubated at 35°C overnight. 16S rRNA gene was carried out with two universal For Nari2AT strain, the preculture was carried out on primers, one forward and the other reverse, designed International Streptomyces Project 2 medium (ISP2) from the conserved zones within the rRNA operon of E. containing the following (in g.L-1): glucose, 4; yeast coli [22].
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