Sondes Mechri

Bioprospecting for extremozymes from unique ecological niches is crucial for developing robust biocatalysts for green chemistry. Here, we report the de novo hybrid genome assembly of Penicillium crustosum CTM10622, isolated from the humid montane forest of El Feïdja National Park, Tunisia. Using Illumina NextSeq™ 500 and Nanopore PromethION 2 Solo, a highly contiguous 31.38 Mb assembly (N50 = 1.94 Mb; 98.3% BUSCOs) was achieved. This robust genomic foundation enabled the identification of an extensive hydrolase repertoire, leading to the discovery of a novel alkaline serine lipase, PCLIP, subsequently heterologously expressed in Pichia pastoris. Recombinant rPCLIP exhibited a high specific activity (15,000 U/mg at pH 10, 65 °C) and exceptional thermostability, with half-lives of 14 and 8 h at 80 and 90 °C, respectively. The enzyme’s identity as a serine lipase was confirmed by its complete inhibition by Orlistat or tetrahydrolipstatin (THL) (51 µM), PMSF (5 mM), and diisopropylfluorophosphate (DIFP) (2 mM). To determine its substrate specificity, advanced computational approaches, including convolutional neural network-based docking and explicitly solvated molecular dynamics, were employed to compare rPCLIP with its homologue PCrL, a recombinant serine alkaline lipase from Penicillium crustosum Thom P22. While rPCLIP showed optimal experimental activity toward short-chain glyceryl tributyrate, simulations revealed that long-chain trioctanoin acts as a ‘thermodynamic trap’ due to over-stabilization. Conversely, the rigid rPCrL favors tricaprylin, driven by a ‘hydrophobic engine’ effect where the solvated environment forces chain burial with minimal entropic penalty. The findings demonstrate that rPCLIP specificity is driven by a delicate interplay of geometric complementarity, Van der Waals enthalpy, and conformational entropy.

Hypersaline environments are unique ecosystems harboring specialized microbial communities with significant biotechnological potential. This study provides a comprehensive characterization of the taxonomic diversity and functional potential of two Tunisian salterns, Abbassia (Kerkennah) and Thyna (Sfax), using an integrated approach that combines 16S/18S rRNA gene amplicons (Illumina and full-length Nanopore) with shotgun metagenomics. Taxonomic profiling revealed a high species richness (S ≈ 1250 taxa); however, the Abbassia site was characterized by extreme taxonomic polarization, with over 95% of the community dominated by specialized halophilic Bacillota (Salinicoccus and Jeotgalicoccus). In contrast, Thyna exhibited a more even distribution dominated by Pseudomonadota and methanogenic Archaea. Beyond taxonomy, functional annotation via the HUMAnN 3.0 pipeline identified site-specific metabolic specializations. Abbassia was enriched in biosynthetic pathways and robust stress-response mechanisms, including ectoine biosynthesis and ppGpp-mediated stringent response, reflecting adaptation to stable hypersaline conditions. Conversely, Thyna’s microbiome prioritized energy extraction and nutrient recycling, with a high abundance of fermentation and glyoxylate cycle pathways. These findings demonstrate that environmental filtering shapes not only the microbial structure but also the metabolic landscape, highlighting the ecological plasticity of microbial life in extreme Tunisian salterns.

Pectins are high-value plant cell-wall polysaccharides with extensive applications in the food, pharmaceutical, textile, paper, and environmental sectors. Traditional extraction and processing methodologies rely heavily on harsh acids, high temperatures, and non-renewable solvents, generating substantial environmental and economic costs. This review consolidates recent advances across the entire Bacillus–pectinase value chain, from green pectin extraction and upstream substrate characterization, through process and statistical optimization of enzyme production, to industrial biocatalysis applications. We propose a practical roadmap for developing high-efficiency, low-environmental-footprint enzyme systems that support circular bioeconomy objectives. Critical evaluation of optimization strategies, including submerged versus solid-state fermentation, response surface methodology, artificial neural networks, and design of experiments, is supported by comparative data on strain performance, fermentation parameters, and industrial titers. Sector-specific case studies demonstrate the efficacy of Bacillus pectinases in fruit-juice clarification, textile bio-scouring, paper bio-bleaching, bio-based detergents, coffee and tea processing, oil extraction, animal feed enhancement, wastewater treatment, and plant-virus purification. Remaining challenges, including enzyme stability in complex matrices, techno-economic scale-up, and structure-guided protein engineering, are identified. Future directions are charted toward CRISPR-driven enzyme design and fully integrated circular-economy bioprocessing platforms.

This work describes the immobilization and the characterization of purified Penicillium crustosum Thom P22 lipase (PCrL) using adsorption, encapsulation, and adsorption–encapsulation approaches. The maximum activity of the immobilized PCrL on CaCO₃ microspheres and sodium alginate beads was shifted from 37 to 45 °C, compared with that of the free enzyme. When sodium alginate was coupled with zeolite or chitosan, the immobilization yield reached 100% and the immobilized PCrL showed improved stability over a wide temperature range, retaining all of its initial activity after a one-hour incubation at 60 °C. The immobilization of PCrL significantly improves its catalytic performance in organic solvents, its pH tolerance value, and its thermal stability. Interestingly, 95% and almost 50% of PCrL’s initial activity was retained after 6 and 12 cycles, respectively. The characteristics of all PCrL forms were analyzed by X-ray diffraction and scanning electron microscopy combined with energy dispersive spectroscopy. The maximum conversion efficiency of oleic acid and methanol to methyl esters (biodiesel), by PCrL immobilized on CaCO₃, was 65% after a 12 h incubation at 40 °C, while free PCrL generated only 30% conversion, under the same conditions.

A novel extracellular lipase from a filamentous fungus Ascomycota strain, P22, was isolated from olive mill wastewater, then purified and characterized. This strain was identified as Penicillium crustosum Thom based on sequencing analyses. Penicilliumcrustosum Thom strain P22 lipase (PCrL) was purified 63-fold to homogeneity using ammonium sulfate precipitation and chromatography on a Q-Sepharose Fast Flow column, with a total yield of 34%. The purified PCrL had a molecular mass of 28 kDa, estimated by SDS-PAGE. The 20 NH₂-terminal amino-acid residues showed a high degree of homology with those of other Penicillium lipases. The specific activity of PCrL at pH 9 and 37 °C were found to be 5000 and 10,000 U/mg on olive oil and trioctanoin emulsions, respectively. PCrL exhibited clear regioselectivity toward the sn-1 position of the surface-coated triglycerides which were esterified with α-eleostearic acid at the sn-1/3 position. PCrL was completely inhibited by 53 µM of Orlistat, 5 mM of phenylmethylsulfonylfluoride, and 2 mM of diiodopropyl fluorophosphate, suggesting that it belonged to the serine lipase family. PCrL showed high activity and stability in the presence of water-immiscible organic solvents, surfactant, and oxidizing agents, and showed considerable compatibility with commercial laundry detergents. Washing performance analysis revealed that it could effectively remove oil stains. Hence, PCrL has several attractive properties that make it a promising potential candidate for detergent formulations.

The ability of microorganisms to grow at high temperature, alkaline pH, and high salinity makes them an attractive target for enzyme‐production with several industrial applications. One strain TN‐X30 has been selected as protease producer and identified as Streptomyces mutabilis after a phenotypic and molecular study. Its production of protease was improved using Taguchi L27 design. The strategy was carried out to identify the optimum levels and the interaction of the screened factors. Following this step, maximum protease activity (10,895 U/ml) was achieved after 6‐days of incubation. The TN‐X30 protease activity had an optimum of pH and temperature of 10 and 65°C, respectively. Thermodynamic parameters at 60°C were enthalpy 14.26 kJ/mol, entropy −220 J/mol/K, and Gibbs free energy 90.53 kJ/mol. TN‐X30 protease production displayed a 16‐fold increase reaching 175,000 U/ml in a 100‐L fermentor. Furthermore, the lyophilization in presence of sorbitol enhanced the stability of the TN‐X30 protease which remained active at 75% after 24‐months of storage. The lyophilized TN‐X30 protease exhibited exceptional stability indexes in presence of some known commercialized detergent components as NEODOL® 25‐7, Dehydol® LT 7, Na₂ CMC, Galaxy LAS, Galaxy LES 70, Galaxy 110, Galaxy CAPB Plus, and Sulfacid K. The lyophilized enzyme also displayed high stability with respect to both solid and liquid detergents. Finally, TN‐X30 protease exhibited remarkable destaining of blood, egg, and chocolate stained cloth pieces. These findings may promote TN‐X30 protease for use as bioadditive in detergent formulation, thereby reducing environmental chemical threat.