This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 10⁸ CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca²⁺/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca²⁺ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.

Biodiesel is one of the important biofuels as an alternative to petroleum-based diesel fuels. In the current study, enzymatic transesterification reaction was carried out for the production of biodiesel from waste cooking oil (WCO) and experimental conditions were optimized, in order to reach maximum biodiesel yield. Bacillus stearothermophilus and Staphylococcus aureus lipase enzymes were individually immobilized on CaCO₃ to be used as environmentally friendly catalysts for biodiesel production. The immobilized lipases exhibited better stability than free ones and were almost fully active after 60 days of storage at 4 °C. A significant biodiesel yield of 97.66 ± 0.57% was achieved without any pre-treatment and at 1:6 oil/methanol molar ratio, 1% of the enzyme mixture (a 1:1 ratio mixture of both lipase), 1% water content, after 24 h at 55 °C reaction temperature. The biocatalysts retained 93% of their initial activities after six cycles. The fuel and chemical properties such as the cloud point, viscosity at 40 °C and density at 15 °C of the produced biodiesel complied with international specifications (EN 14214) and, therefore, were comparable to those of other diesels/biodiesels. Interestingly, the resulting biodiesel revealed a linolenic methyl ester content of 0.55 ± 0.02% and an ester content of 97.7 ± 0.21% which is in good agreement with EN14214 requirements. Overall, using mixed CaCO₃-immobilized lipases to obtain an environmentally friendly biodiesel from WCO is a promising and effective alternative for biodiesel production catalysis.

A novel thermoactive phosphatidylcholine-specific phospholipase C (PC-PLC_Bs) was identified from Bacillus stearothermophilus isolated from a soil sample from an olive oil mill. Enhanced PLC_Bs production was observed after 10 h of incubation at 55 °C in a culture medium containing 1 mM of Zn²⁺ with an 8% inoculum size and 6 g/L glucose and 4/L yeast extract as the preferred carbon energy and nitrogen sources, respectively. PLC_Bs was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and anion exchange chromatography, resulting in a purification factor of 17.6 with 39% recovery. Interestingly, this enzyme showed a high specific activity of 8450 U/mg at pH 8–9 and 60 °C, using phosphatidylcholine PC as the substrate, in the presence of 9 mM sodium deoxycholate and 0.4 mM Zn²⁺. Remarkable stability at acidic and alkali pH and up to 65 °C was also observed. PLC_Bs displayed a substrate specificity order of phosphatidylcholine > phosphatidylethanolamine > phosphatidylserine > sphingomyelin > phosphatidylinositol > cardiolipin and was classified as a PC-PLC. In contrast to phospholipases C previously isolated from Bacillus strains, this PLC_Bs substrate specificity was correlated to its hemolytic and anti-bacterial potential against erythrocytes and Gram-positive bacterial membranes, which are rich in glycerophospholipids and cardiolipin. An evaluation of PLC_Bs soybean degumming process efficiency showed that the purified enzyme reduced the phosphorus content to 35 mg/kg and increased the amount of diacylglycerols released, indicating its ability to hydrolyze phospholipids in the crude soybean oil. Collectively, PLC_Bs could be considered as a potential catalyst for efficient industrial oil degumming, advancing the edible oil industry by reducing the oil gum volume through transforming non-hydratable phospholipids into their hydratable forms, as well as through generating diacylglycerols, which are miscible with triacylglycerols, thereby reducing losses.

Secreted phospholipases A₂ are snake-venom proteins with many biological activities, notably anti-tumor activity. Phospholipases from the same snake type but different geographical locations have shown similar biochemical and biological activities with minor differences in protein sequences. Thus, the discovery of a new phospholipase A₂ with unique characteristics identified in a previously studied venom could suggest the origins of these differences. Here, a new Group II secreted phospholipase A₂ (Cc-PLA₂-II) from the snake venom of Saudi Cerastes cerastes gasperetti was isolated and characterized. The purified enzyme had a molecular weight of 13.945 kDa and showed high specific activity on emulsified phosphatidylcholine of 1560 U/mg at pH 9.5 and 50 °C with strict calcium dependence. Interestingly, stability in extreme pH and high temperatures was observed after enzyme incubation at several pH levels and temperatures. Moreover, a significant dose-dependent cytotoxic anti-tumor effect against six human cancer cell lines was observed with concentrations of Cc-PLA₂ ranging from 2.5 to 8 µM. No cytotoxic effect on normal human umbilical-vein endothelial cells was noted. These results suggest that Cc-PLA₂-II potentially has angiogenic activity of besides cytotoxicity as part of its anti-tumor mechanism. This study justifies the inclusion of this enzyme in many applications for anticancer drug development.

This study highlights the potential of plant extracts as sustainable and cost-effective alternatives to traditional anti-inflammatory drugs, owing to their rich bioactive compounds. The chemical composition and biological activities of ethanolic extracts from Artemisia campestris, Haloxylon articulatum, and Retama raetam were investigated. Extraction yields ranged from 2.94% to 6.84%, with A. campestris showing the highest phenolic content (85.59 ± 2.4 mg GAE/g) and R. raetam having the highest flavonoid concentration (34.77 ± 3.09 mg CE/g). HPLC analysis identified therapeutic phenolic and flavonoid compounds, including sinapic, quinic, and caffeic acids in A. campestris, p-coumaric acid in H. articulatum, and salicylic acid in R. raetam. Antimicrobial tests revealed that Gram-positive bacteria like Staphylococcus aureus and Bacillus cereus were sensitive to the extracts, though Gram-negative strains were unaffected. Antifungal activity was limited, with only H. articulatum showing inhibition of Rhizoctonia solani. Strong antioxidant activities were noted, particularly in H. articulatum and R. raetam extracts (IC50 = 130 µg/mL). In anti-inflammatory assays, all extracts exhibited dose-dependent inhibition of enzymes linked to inflammation, including COX-1, COX-2, 5-LOX, and sPLA2. A. campestris demonstrated the most potent inhibition, reaching 100% inhibition of sPLA2 at 200 μg/mL, while A. campestris and R. raetam provided significant protection in human red blood cell membrane stabilization assays. These results suggest that these plant extracts have considerable biological potential, especially in enzyme inhibition related to inflammation, making them promising candidates for future therapeutic use.

Anaerobic digestion (AD) is an environmentally sustainable approach for managing invasive plants species, mitigating pollution, and generating renewable energy. However, the complex structure of these biomasses limits their biodegradability and necessitates pretreatment to enhance methane production. This study explored the biotransformation of two invasive species, Reynoutria japonica and Phragmites australis, harvested across diverse phenological stages. Bioprocess intensification was achieved through a single-stage process using a hydrolytic–methanogenic consortium under thermophilic conditions (55 °C, 25 days). The impact of harvest timing distinct plant fractions (shoot vs. root) on biogas production was meticulously evaluated. Results revealed progressive biogas production. Notably, winter-harvested shoot fractions exhibited the highest methane-rich biogas, achieving 551.12 ± 33.07 mL/g VS for Reynoutria and 401.42 ± 24.09 mL/g VS for Phragmites. The resulting digestate demonstrates a rich composition of essential macronutrients (N-P-K) vital for plant growth, highlighting its potential as a valuable biofertilizer. Significantly, complete inhibition of seed germination was observed, confirming the process’s efficacy in preventing the further propagation of invasive species. This research underscores that thermophilic anaerobic digestion, coupled with hydrolytic treatment, is a significant advancement in the valorization of invasive biomasses, contributing to both renewable energy production and ecological recovery.