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In accordance with the framework of the Circular Blue Bioeconomy in the Mediterranean region, the objective of this study was to evaluate the biotransformation of blue swimming crab (Portunus segnis) residues obtained from the port of Sfax by an extracellular chitinase produced by Nocardiopsis halophila strain TN-X8 isolated from Chott El Jerid (Tozeur, Tunisia). From the analysis of multiple extremophilic Actinomycetota, it was determined that strain TN-X8 exclusively utilized 60 g/L of raw blue swimming crab as its carbon and energy source, achieving a chitinase activity of approximately 950 U/mL following a 6-day incubation period at 40 °C. Pure chitinase, designated as ChiA-Nh30, was obtained after heat treatment, followed by ammonium sulfate fractionation and Sephacryl® S-200 column chromatography. The maximum ChiA-Nh30 activity was observed at pH 3 and 75 °C. Interestingly, compared with cyclohexamidine, ChiA-Nh30 showed a good antifungal effect against four pathogenic fungi. Furthermore, when using colloidal chitin as substrate, ChiA-Nh30 demonstrated a higher degree of catalytic efficiency than the commercially available Chitodextrinase®. In addition, ChiA-Nh30 could be immobilized by applying encapsulation and encapsulation-adsorption techniques. The kaolin and charcoal used acted as excellent binders, resulting in improved ChiA-Nh30 stability. For the immobilized ChiA-Nh30, the yield of N-acetyl-D-glucosamine monomers released from 20% (w/v) blue swimming crab residues increased by 3.1 (kaolin) and 2.65 (charcoal) times, respectively., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Bacillus amyloliquefaciens is a food spoilage spore-forming bacterium. Its spores are useful for multiple biotechnological applications. Nevertheless, few reports are available regarding the achievement of a high cell density and good sporulation effectiveness under fermentation conditions. Therefore, the current study was designed to optimize a low-cost fermentation medium allowing the highest sporulation yield by B. amyloliquefaciens strain BS13. Our data revealed that tryptone and starch were the best carbon and energy sources. In addition, two nitrogen sources namely, corn steep liquor (CSL) and yeast extract (YE), allowed a significant enhancement of spore production and they were both retained for further optimization. A combination of CaCl 2 , MgSO 4 , and MnSO 4 showed a positive impact on spores' production. The composition of the optimized medium was (in g/L); tryptone 3, starch 15, CSL 13.5, YE 1.5, CaCl 2 0.1, MgSO 4 ·7H 2 O 0.012, and MnSO 4 ·7H 2 O 0.0012. Such medium was further validated in a 400-L fermentor. The spore yield by B. amyloliquefaciens strain BS13 was enhanced from 3.0 × 10 10 spores/mL under flask culture conditions to 6.2 × 10 10 spores/mL when cultures were performed on large scale. Therefore, strain BS13 spore preparation could be proposed as a promising probiotic and a biocontrol agent useful for plants, animals, and humans., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

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 blue swimming crab Portunus segnis bio-waste. Proteases production was optimized 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 (P spp ). The biological value of P spp and its obtained derivatives were evidenced via accredited protocols. The recovered protein hydrolysate (P Hyd ) was found to be active towards radical scavenging power and against angiotensin I-converting enzyme (ACE). The blue crab chitin (BC) extraction efficiency was achieved with a yield of 32%. Afterwards, chitosan was prepared through chitin N-deacetylation with a yield of 52%, leading to an acetylation degree (AD) of 19% and solubility of 90%. In addition, chitosan is found to be active against the growth of all pathogenic bacteria tested., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This paper reports the isolation and identification of an acido-thermostable chitinase (ChiA-Ba43) which was purified, from the culture liquid of Bacillus altitudinis strain KA15, and characterized. Purification of ChiA-Ba43 produced a 69.6-fold increase in the specific activity (120,000 U/mg) of the chitinase, with a yield of 51% using colloidal chitin as substrate. ChiA-Ba43 was found to be a monomeric protein with a molecular mass of 43,190.05 Da as determined by MALDI-TOF/MS. N-terminal sequence of the first 27 amino-acids (aa) of ChiA-Ba43 displayed homology to chitinases from other Bacillus species. Interestingly, ChiA-Ba43 exhibited optimum pH and temperature of 4-5.5 and 85 °C, respectively. Thin-layer chromatography (TLC) showed that the final hydrolyzed products of the enzyme from chitin-oligosaccharides and colloidal chitin are a mixture of (GlcNAc) 2 , (GlcNAc) 3 , (GlcNAc) 4 , and (GlcNAc) 5 , which indicates that ChiA-Ba43 possesses an endo-acting function. More interestingly, compared to ChiA-Mt45, ChiA-Hh59, Chitodextrinase®, N-acetyl-β-glucosaminidase®, and ChiA-65, ChiA-Ba43 demonstrated a high level of catalytic efficiency and outstanding tolerance towards various organic solvents. The chiA-Ba43 gene (1332 bp) encoding ChiA-Ba43 (409 aa) was cloned, sequenced, and expressed in Escherichia coli strain HB101. The biochemical properties of the recombinant chitinase (rChiA-Ba43) were equivalent to those of the natively expressed enzyme. These properties make ChiA-Ba43 an ideal candidate for industrial bioconversion of chitinous waste., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The efficiency of the proteolytic strain Anoxybacillus kamchatkensis M1V in the fermentation of speckled shrimp by-product was investigated for the recovery of a deproteinized bioactive hydrolysate. The biological activities of the resulting hydrolysate were also examined by applying several antioxidant and enzyme inhibitory assays. The strain M1V was found to produce high level of protease activity (2000 U/mL) when grown in media containing only shrimp powder at 25 g/L. The crude protease displayed a significant deproteinization capabiliy, with the best efficiency (48%) being recorded for an enzyme to substrate (E/S) ratio of 30 U/mg. Following the deproteinization, chitin was recovered and the authenticity was confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis. On the other hand, the obtained hydrolysate showed a significant enzymatic inhibitory potential against acetylcholinesterase, tyrosinase, amylase, and angiotensin I convertase, and a strong antioxidant activity. Graphical Abstract.

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., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Fadoua Jabeur et al.)

The present study investigates the purification and biochemical characterization of a novel extracellular serine alkaline protease, subtilisin (called SAPN) from Melghiribacillus thermohalophilus Nari2A T . 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 NH 2 -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.