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

BioMed Research International

Bouacem

Amziane

et al. 2019

Self Cite

A new peptidase designated as SAPV produced from a moderately halophilic Virgibacillus natechei sp. nov., strain FarDT was investigated by purification to homogeneity followed by biochemical and molecular characterization purposes. Through optimization, it was determined that the optimum peptidase activity was 16,000 U/mL. It was achieved after 36 h incubation at 35°C in the optimized enzyme liquid medium (ELM) at pH 7.4 that contains only white shrimp shell by-product (60 g/L) as sole energy and carbon sources. The SAPV enzyme is a monomer protein with a molecular mass of 31 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance liquid chromatography (HPLC) gel filtration chromatography. The sequence of its NH2-terminal amino-acid residues showed homology with those of Bacillus peptidases S8/S53 superfamily. The SAPV showed optimal activity at pH 9 and 60°C. Irreversible inhibition of enzyme activity by diiodopropyl fluorophosphates (DFP) and phenylmethanesulfonyl fluoride (PMSF) confirmed its belonging to the serine peptidases. Considering its interesting biochemical characterization, the sapV gene was cloned, sequenced, and heterologously overexpressed in the extracellular fraction of E. coli BL21(DE3)pLysS. The biochemical properties of the recombinant peptidase (rSAPV) were similar to those of the native one. The highest sequence identity value (97.66%) of SAPV was obtained with peptidase S8 from Virgibacillus massiliensis DSM 28587, with 9 amino-acid residues of difference. Interestingly, rSAPV showed an outstanding and high resistance to several organic solvents than SPVP from Aeribacillus pallidus VP3 and Thermolysin type X. Furthermore, rSAPV exhibited an excellent detergent stability and compatibility than Alcalase 2.4 L FG and Bioprotease N100L. Considering all these remarkable properties, rSAPV has attracted the interest of industrialists.

Section: Resultsmentioning

confidence: 99%

Identification of a New Serine Alkaline Peptidase from the Moderately Halophilic Virgibacillus natechei sp. nov., Strain FarD^T and its Application as Bioadditive for Peptide Synthesis and Laundry Detergent Formulations

BioMed Research International

Bouacem

Amziane

et al. 2019

Self Cite

“…A maximum protease activity of 2,000 U/mL was obtained with 25 g/L shrimp waste. e VP3 and C250R strains also produce large amounts of proteases activity in the order of 600 and 200 U/mL (Mechri et al [13,19], submitted for publication), respectively. Bacillus licheniformis RP1 was shown to produce proteases (1,400 U/ mL) when grown in media containing shrimp wastes powder as carbon and nitrogen source, indicating that this bacteria could obtain its needs directly from shrimp by-product [28].…”

Section: Production Of Proteases In Economical Mediummentioning

confidence: 99%

“…Recently, the application of proteases and lactic acid-producing bacteria, to extract chitin from crustacean by-products, has gained an increasing interest. Actually, this method has the advantage of being relatively simple and less expensive and using a chemical-free process [12,13]. Hence, in comparison with chemical and enzymatic methods, it has been found that microbial fermentation methods are the most effective for chitin extraction, especially, in terms of final product quality and environment safety [14].…”

Section: Introductionmentioning

confidence: 99%

Statistical Experimental Design Optimization of Microbial Proteases Production under Co-Culture Conditions for Chitin Recovery from Speckled ShrimpMetapenaeus monocerosBy-Product

Jabeur

BioMed Research International

Kriaa

et al. 2020

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This study was designed with the aim to produce microbial proteases in presence of speckled shrimp by-product. For this reason, three strains belonging to Bacillus genus, namely, Aeribacillus pallidus VP3, Lysinibacillus fusiformis C250R, and Anoxybacillus kamchatkensis M1V were studied under co-culture procedure. A Taguchi L27 experimental design was applied to optimize the co-culture parameters. The experimental design was built with 9 factors (by-product powder concentration, the pH of the medium, the temperature, the sucrose concentration, the agitation speed, the inoculum sizes of VP3, M1V, and C250R strains, and the culture volume) at three different levels. The obtained results showed that a total protease activity of 8,182 U/mL could be achieved after 24 h of incubation in presence of 20 g/L shrimp by-product and 10 g/L sucrose, at an initial pH of 7, a 40°C temperature and absorbance, at 600 nm, of inoculum sizes of 0.1, 0.3, and 0.1 for VP3, M1V, and C250R strains, respectively. The agitation was set at 200 rpm, and the final volume was 25 mL. Taguchi’s design allowed the identification of temperature, the inoculum size for strain VP3, the inoculum size for strain M1V, and the final culture volume as the most influencing variables. A Box–Behnken design with 27 experiments was carried out for the optimization of these four selected factors. Following such design, the highest protease production reached was 11,300 U/mL. This yield was obtained in a final culture volume of 15 mL containing 20 g/L shrimp by-product powder and 10 g/L sucrose and inoculated with VP3, C250R, and M1V strains at 0.05, 0.1, and 0.2, respectively. The flasks were incubated at 45°C for 24 h with shaking at 200 rpm. The efficiency of chitin extraction by co-cultivation was investigated under the latter conditions. The chitin yield from shells by-product was 16.7%. Fourier-Transform Infrared (FTIR) analysis of the obtained chitin displayed characteristic profiles similar to that of the commercial α-chitin.

“…In the same way, the proteolytic hydrolysis of crustacean's bio-wastes by proteases has been used for the recovery of chitin. For example, Alcalase® from Novozymes Biopharma DK A/S (Bagsvaerd, Denmark) and serine alkaline protease (SAPN) from Melghiribacillus thermohalophilus Nari2A T were proposed for the recovery of chitin from Portunus segnis and Metapenaeus monoceros, respectively (Mechri et al 2019b;Mechri et al 2020b). A comprehensive overview of recent literature shows that, several studies have been concentrated on an in-depth investigation of the bioactive compounds derived from Portunus segnis swimming blue crab products, using enzymatic and chemical biotechnological procedures (Hamdi et al 2020;Hamdi et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…This process allows healthier solubilization of proteins in the form of protein and peptide hydrolysates. Indeed, marine by-products could be valued by transformation into homogeneous and ne our which is mainly intended for biotechnological applications as proteases production (Jabeur et al 2020;Mechri et al 2019a;Mechri et al 2019b). Again, the recovery of chitin requires recourse to strong acids and bases, which remains a double-edged sword because, although it allows the recovery of pure chitin, it can cause depolymerization of the chitin and seriously pollute the environment.…”

Section: Introductionmentioning

confidence: 99%

Trends in valorization of the invasive crab Portunus segnis for cleaner production of chitin, chitosan, and protein hydrolysate

Jabeur

Mensi

et al. 2021

Preprint

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The diversity of marine biomasses is a set of exploitable and renewable resources with application in several sectors. In this context, a co-culture based on three protease-producing bacterial isolates namely; Aeribacillus pallidus VP3, Lysinibacillus fusiformis C250R , and Anoxybacillus kamchatkensis M1V strains, was carried out in a medium based on the invasive blue crab Portunus segnis bio-waste. Optimization of proteases production was performed using a central composite design (CCD). The highest level of proteases production obtained was 8,809 U/mL in a medium comprising 75 g/L of Portunus segnis by-product powder (Pspp ). The recovered protein hydrolysate (P Hyd ) was found to be active towards radical scavenging power, and against angiotensin I-converting enzyme (ACE). The extraction efficiency of the blue crab chitin (BC) was achieved with a yield of 32%. Afterward, chitosan was prepared through chitin N -deacetylation with a yield of 52%, leading to an acetylation degree (AD) of 19% and solubility of 90%. The spectrum of chitin and chitosan were depicted by Fourier-transform infrared spectroscopy (FTIR). The biological value of Pspp and its obtained derivates were evidenced via accredited protocols. These data constitute a roadmap towards a circular and sustainable bio-economic strategy for a clean transformation of a recalcitrant waste to bio-based products.