Search Results (140)

This study integrates microbial biotechnology, bioprocess optimization, and circular bioeconomy principles to valorize oilseed agro-industrial coproducts as low-cost inducing substrates for lipase production. Palm kernel cake (PKC), canola cake, andiroba cake, and brewer’s spent grain were evaluated as carbon sources for extracellular lipase production by the wild-type strain Trichoderma sp. LEPM-711 under submerged fermentation. Qualitative screening in olive oil/rhodamine B medium confirmed the strain’s lipolytic potential. Among the substrates tested, PKC showed the strongest induction, reaching 4.68 U mL⁻¹, and was therefore selected for nitrogen supplementation and statistical optimization. Peptone supplementation increased lipolytic activity to 8.41 U mL⁻¹ after 72 h. A Plackett–Burman design identified CaCl₂·2H₂O, CoCl₂·6H₂O, and peptone as significant medium components, while response surface methodology established the optimal operational conditions as pH 6.0, 10⁶ spores mL⁻¹, and 2% (w/v) PKC. Under these conditions, the crude enzymatic extract reached 18.31 U mL⁻¹ at pH 7.0 and 45 °C, corresponding to an approximately 3.9-fold increase over the initial PKC fermentation. These findings support the use of Trichoderma sp. LEPM-711 and oilseed coproducts as a sustainable platform for enzyme production and residue upgrading within applied natural sciences. Full article

Lipases are important enzymes in the esterase family that hydrolyze ester bonds in triglycerides, producing simpler molecules. This property makes them valuable in biotechnology and environmental cleanup. In this study, lipase-producing bacteria were isolated and characterized from petroleum-contaminated soil to establish a cost-effective platform for enzyme production and bioremediation. Among the recovered isolates, Serratia liquefaciens AB1 exhibited the highest lipolytic activity and was therefore selected for further investigation. The influence of various inducer oils and agro-industrial residues on enzyme production was systematically assessed. In addition, fermentation parameters were optimized through the evaluation of different carbon and nitrogen sources to enhance lipase yield. Waste frying oil was identified as the most effective inducer, while glucose and yeast extract supported optimal enzyme production. The enzyme lipase AB1 was fully purified using CM-Sephadex C-50 chromatography, Sephadex G-100 and further characterized by SDS-PAGE, kinetic studies, and stability assays. Purification of the enzyme resulted in a specific activity of 610.92 U/mg, corresponding to a 9.42-fold increase in purity with an overall recovery of 76%. The enzyme exhibited an apparent molecular mass of approximately 64 kDa. It demonstrated optimal catalytic activity at 60 °C and pH 8 and retained substantial stability at this temperature for up to 120 min. Kinetic analysis revealed a low Km value of 30 µM, indicating strong substrate affinity, along with a Vmax of 23.89 U/mL, reflecting a high catalytic efficiency under the tested conditions. Enzyme activity was enhanced by Ca²⁺, Na⁺, and Ba²⁺, but inhibited by Mn²⁺ and Hg²⁺. These findings demonstrate the favorable biochemical properties of the purified lipase and provide a basis for future investigations into its potential application as a biocatalyst for bioremediation. Full article

Six lactic acid bacteria isolated from São Jorge PDO cheese were characterised for technological, safety, antimicrobial, and probiotic properties. All isolates fermented a broad range of carbohydrates and lacked lipolytic activity, while SJC115 and SJC119 showed proteolysis. Safety profiling (γ-haemolysis, no DNase or gelatinase activity, and generally favourable antibiotic susceptibility) is promising, but tetracycline resistance warrants caution and genomic confirmation. L. paracasei and L. brevis isolates inhibited a wide range of foodborne pathogens (Listeria monocytogenes, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Bacillus spp.) and spoilage fungi (Penicillium and Aspergillus spp.). Notably, two isolates (SJC117 and SJC120) exhibited antibacterial activity in neutralized cell-free supernatants, indicating putative bacteriocin-like inhibitory substances (BLIS). The isolates survived intestinal conditions above the probiotic threshold, yet only SJC117 and SJC120 tolerated gastric acidity (pH 2.5, 1 h) with >5 log CFU/mL. Despite low hydrophobicity, strains showed good autoaggregation and pathogen coaggregation. All isolates produced exopolysaccharides (EPS) and angiotensin-converting enzyme (ACE) inhibitory peptides, whereas some exhibited moderate conjugated linoleic acid (CLA) production and glutamate decarboxylase (GAD) activity. L. paracasei SJC117 stood out by combining BLIS/antifungal activity, superior gastric tolerance, and an exceptional bioactive profile, making it a promising candidate for biopreservation and functional food applications that warrants further in vivo validation to confirm its efficacy and safety. Full article

Lipolytic and esterase-like enzymes are crucial in the food industry for flavor production and ester hydrolysis. This study presents an exploratory, baseline feasibility approach to evaluate esterase-like enzyme production by Serratia marcescens 11E via a sustainable co-valorization matrix, where vegetable wax residues serve as structural solid support and cheese whey acts as the primary lipid nutritional source. Under fixed 48-h screening conditions, the recovered cell-free extract exhibited a distinct catalytic preference for short-chain esters, showing higher specific activity toward 4-nitrophenyl acetate (0.743 U/mg) than 4-nitrophenyl palmitate (0.125 U/mg), confirming a predominant carboxylesterase-like profile. Biochemical characterization revealed an initial optimal activity at pH 9.0 and 30 °C, along with a noteworthy bimodal catalytic behavior featuring a secondary activity peak at 70 °C. Size-exclusion chromatography resolved the extract into two distinct active elution pools (17.8 and 26.2 U/mg), which corresponded to candidate protein bands of 35–40 kDa on SDS-PAGE. Although definitive molecular identification remains subject to ongoing zymographic and proteomic characterization, these foundational findings demonstrate the potential of co-valorizing lipid- and carbohydrate-rich industrial wastes to produce resilient proteins with esterase-like activity at elevated temperatures. Full article

Several strategies can be employed for the identification of novel microbial lipases. Despite the increasing importance of metagenomics in bioprospecting, significant limitations in the expression of recombinant proteins, and lipases in particular, remain. Culture-based bioprospecting approaches are, therefore, still valuable. In this work, a collection of bacterial isolates, mainly of marine origin, was screened for lipase activity through a culture-based approach. Screening for lipolytic bacteria was performed in solid media containing olive oil emulsions and rhodamine B. Positive isolates were subsequently grown in liquid media, to confirm lipase production. Significant hydrolytic activity towards the triglyceride substrates tributyrin and triolein could be observed with the biomass produced, although no lipase activity could be detected in the culture supernatants. Six isolates presenting high activity were characterized as whole-cell biocatalysts, and all were found to be active at temperatures ranging between 25 and 65 °C, and at pH values between 6 and 10.5. Genomic analyses of two of these Gram-negative lipase-producing isolates revealed the presence of several hypothetical genes encoding for lipolytic enzymes, including outer cell-bound enzymes, predicted through the application of machine-learning tools. These natural isolates, containing cell-associated lipases, may therefore be of special interest for application as whole-cell biocatalysts. Full article

This study investigated the effect of ozonation of wheat flour (30 ppm O₃ for 30 min) on the microbiological status of flour, as well as the profile of volatile compounds, the mechanical properties, and the sensory characteristics of the resulting bakery products. Ozonation significantly reduced the microbial load of the flour, decreasing aerobic bacteria from 1.4 × 10⁵ to 1.7 × 10⁴ CFU·g⁻¹ and yeasts and moulds from 2.8 × 10³ to 1.3 × 10² CFU·g⁻¹, while lactic acid bacteria populations remained unchanged. HS-SPME-GC-MS analysis revealed that the ozonated flour contained six volatile compounds (compared to three in the control), predominantly nonanal (80.62%), an aldehyde formed via the ozonolysis of unsaturated fatty acids. Although these ozone-induced aldehydes were also detected in the final bakery products, their peak areas decreased substantially (to ≤3.3% of the flour values), suggesting thermal desorption during baking. Texture profile analysis demonstrated that products baked from ozonated flour exhibited increased hardness (Cycle 1: 68.06 N vs. 53.42 N; Cycle 2: 59.41 N vs. 47.52 N) and chewiness (427.95 mJ vs. 404.70 mJ) compared to controls. This textural degradation is likely due to ozone-induced modifications in enzyme activity (proteolytic, amylolytic, and lipolytic) and gluten protein cross-linking via disulphide bond formation. Furthermore, sensory evaluation using a five-point scale showed lower acceptability for the ozonated products (3.04 vs. 3.74), with panellists noting inferior taste, aroma, crumb colour, and flexibility. In conclusion, while ozonation effectively reduces the microbiological load of wheat flour, the applied high-dose treatment (30 ppm, 30 min) negatively impacts the sensory and textural quality of the bakery products, indicating that milder processing parameters are necessary to balance safety and quality. Full article

Intramuscular fat (IMF) content determines marbling levels and influences the sensory and edible qualities of livestock meat. Its deposition is influenced by the animal’s age and gut microbial community. This study assessed age-related differences in IMF deposition and shifts in gut microbiota between yearlings (1-year-old) and mature (4-year-old) grazing Tan sheep. Then correlations among these factors were examined to investigate the potential role of gut bacteria in IMF deposition. The results demonstrated that mature sheep exhibited higher IMF content in shoulder and rump muscles (p < 0.05), elevated serum lipid levels (p < 0.001), and increased lipolytic enzyme abundances in the liver and pancreas (p < 0.05), compared with yearlings. In contrast, the concentrations of acetate and propionate in ruminal and colonic contents were lower in mature sheep (p < 0.05), despite a higher abundance of lipolytic and synthetic enzymes in colonic content (p < 0.05). Gut microbial diversity differed between age groups, particularly in the rumen and colon, with clear shifts in specific bacterial taxa. Correlation analyses revealed that the abundance of Copromorpha and RUG420 in the colon were positively correlated with IMF content in shoulder and rump muscles, and serum lipid levels (including free fatty acids, FFA; low-density lipoprotein, LDL; high-density lipoprotein, HDL; and very-low-density lipoprotein, VLDL), but negatively correlated with propionate content (|r| > 0.45, FDR < 0.05). Conversely, the abundance of Cryptobacteroides in the colon was negatively correlated with IMF content in shoulder muscle (r < −0.6, FDR < 0.05), and with the levels of triglyceride (TG), LDL, HDL, and VLDL, while showing positive correlations with acetate and propionate contents (r > 0.45, FDR < 0.05). These findings highlight the potential role of specific colon bacteria (Copromorpha, RUG420, and UBA5905) in IMF deposition, identifying them as candidate bacteria for further investigation regarding their effects on meat quality. Full article

The thermophilic bacterium Thermus thermophilus represents a crucial genetic reservoir for exploring thermostable enzymes as valuable biocatalysts for industrial and biotechnology applications. Here, we identify, clone, and characterize Ces1-ET, Est1-ET, and Plp1-ET, three lipolytic enzymes obtained from T. thermophilus strain ET-1 isolated from El Tatio Geothermal Field in Northern Chile. To enable recombinant expression, we constructed the pTGT-1 expression system, a versatile bifunctional shuttle vector compatible with both Escherichia coli and T. thermophilus. The three thermoenzymes Ces1-ET, Est1-ET, and Plp1-ET, were successfully cloned, expressed, and purified using the pTGT-1 system, with a molecular mass of 25 kDa, 36 kDa, and 28 kDa, respectively. The recombinant purified enzymes displayed optimal temperatures at 60 °C, 80 °C, and 70 °C and optimal pH of 7.5, 9.0, and 8.0 for Ces1-ET, Est1-ET, and Plp1-ET, respectively. Functional biochemical assays revealed a broad tolerance to surfactants, detergents, divalent cations, and high salinity, relevant properties for their application in an industrial setting. These thermostable esterases expand the repertoire of thermozymes from Thermus spp., introducing pTGT-1 as an innovative tool for thermophilic protein expression and highlighting T. thermophilus strain ET-1 from El Tatio Geothermal Field as a valuable source of thermostable enzymes for industrial and biotechnology applications. Full article

Moulds of the Rhizopus oryzae species exhibit high biotechnological potential due to their significant metabolic activity, which is influenced by cultivation conditions. The study aimed to evaluate the ability of R. oryzae DSM 2199 to synthesize extracellular lipolytic and proteolytic enzymes in solid-state fermentation (SSF) using rapeseed cake as a substrate. The effectiveness of the SSF method in stimulating the synthesis of hydrolytic enzymes by R. oryzae was confirmed. The effect of an additional carbon and nitrogen source with three different dilution variants of the solid substrate on lipase and protease activity was analyzed. No significant correlation was found between enzyme activity and the applied diluents. The extracellular enzyme solution obtained from R. oryzae in SSF was lyophilized. The freeze-dried raw preparation exhibited high lipolytic activity (111.59 U/g) compared to its low proteolytic activity (0.013 U/g). Demonstrated hydrolytic activity made the biocatalyst useful for the hydrolysis and esterification reactions. Full article

Cold-active lipolytic enzymes enable low-temperature biocatalysis, but remain underexplored in Antarctic actinomycetes. Here, we report the discovery and first-step characterization of a CALB-like cold-active lipolytic enzyme (PanLip) from Pseudonocardia antarctica. Sequence and structure analyses revealed a canonical α/β-hydrolase fold with a conserved Ser–Asp–His triad and short helical elements around the pocket reminiscent of CALB’s α5/α10 lid. Mature PanLip was expressed primarily as inclusion bodies in E. coli; an N-terminally truncation (PanLipΔN) improved solubility and PanLipΔN was purified by Ni–NTA. Far-UV CD confirmed a folded α/β architecture. PanLipΔN favored short-chain substrates (p-NPA, k_cat/K_M = 2.4 × 10⁵ M⁻¹·s⁻¹) but also showed measurable hydrolytic activity toward natural triglycerides, consistently with a lipase-family esterase. The enzyme showed an activity optimum near 25 °C and pH 8.0. The enzyme tolerated low salt (maximal at 0.1 M NaCl), mild glycerol, and selected organic solvents (notably n-hexane), but was inhibited by high salt, Triton X-100, and SDS. AlphaFold predicted high local confidence for the catalytic core; DALI placed PanLip closest to fungal lipases (AFLB/CALB). Temperature-series MD and CABS-flex indicated enhanced surface breathing and flexible segments adjacent to the active site—including a region topologically matching CALB α10—supporting a flexibility-assisted access mechanism at low temperature. Structure-based MSAs did not support a cold adaptation role for the reported VDLPGRS motif. Taken together, these findings position PanLip as a promising cold-active catalyst with CALB-like access control and potential for low-temperature biocatalysis. Full article

Obesity is linked to metabolic dysfunction-associated steatotic liver disease (MASLD), but the molecular mechanisms and effective treatments remain unclear. This study investigated whether ST32db, an inducer of activating transcription factor 3 (ATF3), affects lipid metabolism in MASLD. An in vitro model was established involving the treatment of HepG2 cells with 1 mM oleic acid (OA) with or without 20 µM ST32db. In an in vivo model, C57BL/6 mice were fed a high-fat diet (HFD) for 18 weeks to induce obesity and treated or not with ST32db (1 mg kg⁻¹). ST32db significantly decreased intracellular lipid accumulation in OA-treated HepG2 cells. In these cells, ST32db remarkably decreased mRNA and protein levels of adipogenesis- and lipogenesis-related genes and increased mRNA levels of adipose triglyceride lipase (ATGL), a lipolytic enzyme. In HFD-fed mice, the ST32db treatment significantly decreased the liver weight, serum triglycerides, and fat vacuole and triglyceride accumulation in the liver. Livers from these mice also showed significantly decreased CCAAT/enhancer-binding protein β mRNA and protein levels, increased ATF3 mRNA and protein and ATGL mRNA levels, and increased levels of phosphorylated AMP-activated protein kinase (AMPK) and protein kinase A (PKA). These findings suggest that ST32db may exert protective effects against MASLD through activating hepatic AMPK and PKA pathways. Full article

Endophytic fungi were isolated from ginger (Zingiber officinale) tubers and identified through molecular characterization of ITS and 28S rRNA regions. Nine species were obtained, belonging to the genera Aspergillus, Penicillium, Plectosphaerella, and Pseudogymnoascus. Several isolates, particularly Penicillium melinii, Aspergillus ustus, and Plectosphaerella cucumerina, exhibited strong antagonistic activity against Botrytis cinerea (up to 98.6% growth inhibition), while moderate effects were observed against Colletotrichum acutatum, Staphylococcus aureus and Klebsiella pneumoniae. All isolates produced at least one extracellular enzyme, with lipolytic and cellulolytic enzymes being the most frequently observed, and showed measurable antioxidant activity (EC₅₀ values ranging from 21.7 to 673.6 µg/mL). P. melinii and P. cucumerina demonstrated the highest radical scavenging capacities. These findings reveal the multifunctional potential of ginger-associated endophytic fungi as sustainable sources of bioactive compounds, with promising applications in biocontrol, food preservation, and industrial biotechnology. Full article

The study investigated the effects of chloride salts (control: 100% NaCl, salt mixture I: NaCl/KCl (50/50), salt mixture II: NaCl/KCl/CaCl₂ (40/40/20), salt mixture III: NaCl/KCl/CaCl₂/MgCl₂ (30/40/20/10)) on enzymatic activity, lipid oxidation, and volatile compounds in reduced-sodium salt pastırma, a Turkish dry-cured meat product. Lipid oxidation and instrumental color values were not affected by different salt mixtures. Salt mixtures II and III decreased pH value (p < 0.05). However, the mean pH value did not fall below 5.5 in any sample. The salt mixture treatment had significant effect on water activity, cathepsin B, and cathepsin B + L. In contrast, a_w value was under 0.90 in all treatments. The highest mean values for cathepsin B and B + L were determined in the control group with 11.69 ± 2.73 and 85.82 ± 12.65 U g⁻¹ × 10⁻³ dry matter, respectively. The closest correlation for lipolytic enzyme activities was determined by the mixture II and III groups, while a closer correlation was observed between salt mixtures I and III in terms of proteolytic enzyme activities. With regard to volatile compounds, there was a closer relationship between the control and salt mixture I. As a result, it can be concluded that salt mixture I in reduced-sodium salt pastırma showed closer results to the control group. Full article

Protein purification is often performed for various applications. However, enzyme purification processes typically involve multiple steps that reduce yield and increase production costs. To overcome these challenges, we developed a novel three-step process to purify a lipase from whole sardine viscera (WSV), leveraging protein properties and the structural affinity of lipases for dye ligands. A crude extract of the viscera (CEV) was obtained by grinding the whole viscera in 50 mM phosphate buffer (pH 7.0, Solution B) followed by centrifugation (6000× g; 30 min, 0 °C). Lipolytic activity (3.3 U/mg) was recorded only in the supernatant. The purification process began with ammonium sulfate fractionation (30–50% saturation), increasing lipolytic activity in the precipitate (PF30-50) to 32.9 U/mg. PF30-50 was then ultrafiltered using a 30 KDa MWCO membrane, where 5% of semi-purified lipases (SPLSV) was retained with an activity of 156.5 U/mg (UF30). Finally, the SPLSV was injected into a column packed with dye ligand affinity adsorbent, pre-equilibrated with 1.0 M ammonium sulfate in buffer A. The WSV lipase was eluted using a step gradient to progressively reduce salt concentration. SDS-PAGE analysis revealed a single band of purified lipase from sardine viscera (PLSV) corresponding to a molecular weight of 123.4 kDa, with a specific activity of 266.4 U/mg. The combination of ammonium sulfate precipitation, ultrafiltration, and dye-ligand affinity chromatography provides a scalable and reproducible approach with potential industrial relevance, particularly in biocatalysis and waste valorization contexts. Full article

Background: Enzymes secreted by the digestive glands are excreted from the body with urine, sweat and feces, and they are also removed from the blood due to their participation in the enzymatic provision of the secretion entering the gastrointestinal tract. Objective: The aim of this work was to analyze the activity of pepsinogen, amylase and lipase in the coprofiltrate of pregnant women in each trimester of pregnancy and in the postpartum period, taking into account the timing and type of delivery (term, premature, late delivery or cesarean section). Methods: Data from studies of non-pregnant (n = 45) and pregnant (n = 193) women were analyzed. The materials for preparation coprofiltrate were collected during delivery. Pepsinogen activity was determined by proteolytic activity at pH = 1.5–2.0 using the tyrosine spectrophotometric method, while amylase activity was determined by the amyloclastic method of Karavey, and lipolytic activity was determined by a unified kinetic method using olive oil as a substrate. Outcomes: A small amount of pepsinogen was excreted in the coprofiltrate, and while the level of its excretion increased after childbirth, it remained below the control values. At the same time, an increase in the amylolytic activity of the coprofiltrate was observed in all groups of pregnant women examined from the first to the third trimester of pregnancy. In pregnant women, multidirectional changes in lipase activity were observed depending on the timing and type of delivery. Conclusions: At the end of pregnancy, amylolytic activity increased in all women, and pepsinase activity decreased compared to the indicators of non-pregnant women. No reliable differences were found in the lipolytic activity of the coprofiltrate in pregnant women at the end of pregnancy and the indicators of non-pregnant women. Full article