Search Results (35)

Global food waste management necessitates circular bioeconomy solutions to transform organic residues into high-value nutrients to address nutritional demands. This study investigated the valorization of two abundant waste streams, stale bread and sunflower oil through solid state fermentation using food-grade filamentous fungi. Three strains, Neurospora intermedia, Aspergillus oryzae and Rhizopus oryzae were evaluated for the bioconversion of stale bread. Oil supplementation levels of 10, 20 and 30% (g/100 g dry matter) using both fresh and spent sunflower oil were tested to assess changes in proximate composition, characterizing fungal growth dynamics and mycelial development. Furthermore, modifications in fatty acid profiles and hydrolytic enzyme activities were analyzed to determine species responses to oil source and concentration. The results demonstrated that N. intermedia achieved peak protein levels of 36% (g/100 g) alongside efficient starch catabolism, while 10% fresh oil supplementation induced a significant protein increase (26%) in A. oryzae. Regarding lipid accumulation, 10% spent oil supported higher fat content in R. oryzae (19%) compared to fresh oil (17%). PUFA/SFA ratio reached its maximum in A. oryzae with the highest of 5.91 ± 0.56 under 10% fresh oil. Enzymatic analysis identified A. oryzae as the most efficient lipase producer, reaching a maximum activity of approximately 0.10 U/g at 10% spent oil supplementation. Conversely, R. oryzae lipase activity peaked at 20% supplementation (0.08 U/g), reflecting its high capacity for lipid accumulation. These findings establish a potent bioprocess for upcycling mixed food wastes into enhanced functional ingredients for sustainable food and feed systems. Full article

Rhizopus oryzae lipase (ROL) is a key enzyme involved in olive oil spoilage and acts as a virulence factor in fungal infections. Natural lipophilic lipase inhibitors are crucial for mitigating economic losses resulting from lipid degradation in stored or decaying olive fruits. This study evaluated a series of enzymatically synthesized piperate esters with varying alkyl chain lengths (butyryl, C4; octyl, C8; dodecyl, C12) for their inhibitory effects on ROL activity. Octyl piperate (C8) demonstrated the highest potency, with IC₅₀ values of 0.05 mg/mL using methods B and C or 0.25 mg/mL using method A. Molecular docking indicated that C8 achieved the most favorable predicted binding energy (Gscore: –11.134 kcal/mol), primarily through hydrophobic interactions (Val329, Ala212, Phe209) and hydrogen bonds with oxyanion hole residues (Ser268, Thr206, Gln241). Molecular dynamics simulations confirmed that C8 maintained stable binding and stabilized the catalytic residues. In comparison, C4 exhibited weaker interactions, and the longer C12 chain induced conformational instability and steric hindrance. These results establish a parabolic structure–activity relationship, identifying the octyl chain (C8) as optimal for ROL inhibition among the tested derivatives. The rational design of lipophilic, biodegradable lipase inhibitors thus positions octyl piperate as a promising candidate for extending olive storage and shelf life, and as a scaffold for developing natural antifungal agents targeting virulent R. oryzae strains. 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

Commercial fungal lipases from Rhizopus oryzae, Rhizopus niveus, Aspergillus niger, Rhizomucor miehei, and Candida rugosa were immobilized via physical adsorption onto Accurel MP 1000, a hydrophobic polypropylene support. The effects of enzyme concentration, pH, temperature, and glutaraldehyde post-treatment were systematically evaluated. Immobilization generally enhanced enzyme stability, which was further improved in several cases by glutaraldehyde crosslinking. The immobilized preparations retained over 50% of their initial activity for 3–6 cycles, and 7–10 cycles following glutaraldehyde treatment. While soluble enzymes lost nearly all activity within three months at 5 °C and 25 °C and retained only 5–20% at −20 °C, the immobilized forms preserved 50–100% of their activity under all storage conditions tested. Immobilized lipases also exhibited improved thermal stability at 60 °C by general increments between 1.3 and 1.8 times compared to soluble lipases. Increased tolerance to pH fluctuations was observed in most immobilized enzymes, particularly from R. oryzae, R. niveus, R. miehei, and C. rugosa. Organic solvent tolerance of the immobilized enzymes showed highest stability in hexane (66–100% residual activity after 4 h incubation). Glutaraldehyde treatment affected solvent stability of immobilized lipases in enzyme and solvent dependent manner. These findings demonstrate the improved stability and applicability of the produced biocatalysts in varying reaction environments. Full article

The research presented in this manuscript aimed to conduct complex studies on epoxy composites filled with modified biowaste. This work discusses the entire process: first, the preparation and analysis of oak waste flour used as an additive to epoxy materials based on Epidian 6; then, obtaining and characterizing epoxy composites containing 5 wt.% of biowaste; and finally, the determination of the influence of wood filler and modification performed using selected ionic liquids (tetradecyltrihexylphosphonium bis(trifluoromethylsulfonyl)amide (IL-1), tetradecyltrihexylphosphonium bis(2,4,4-trimethylpentyl)phosphinate (IL-2), and 1-ethyl-3-methyl-imidazolium bis(trifluoromethylsulfonyl)imide) (IL-3)) on the susceptibility of epoxy composite to the enzymatic degradation. The enzymatic degradation was performed for four weeks using the lipase enzymes (Porcine Pancreas and Rhizopus Oryzae). The epoxy composition EP6-WF_IL-2, containing 5 wt.% of wood flour modified with IL-2, was characterized by the best mechanical parameters in terms of bending strength and flexural modulus (65.64 MPa and 1855.3 MPa, respectively). During enzymatic biodegradation, the lowest susceptibility to enzymatic degradation, regardless of the incubation conditions, was observed in samples of EP6-WF_IL-3 epoxy composition containing wood flour modified with methyltrioctylammonium bis(trifluoromethylsulfonyl)imide. Full article

Poly(ε-caprolactone) (PCL) is a synthetic plastic known for its excellent physicochemical properties and a wide range of applications in packaging, coatings, foaming, and agriculture. In medicine, its versatility allows it to function as a scaffold for drug delivery, sutures, implants, tissue engineering, and 3D printing. In addition to its biocompatibility, PCL’s most notable characteristic is its biodegradability. However, this property is affected by temperature, microbial activity, and environmental conditions, which means PCL can sometimes remain in nature for long periods. This review shows that various types of microorganisms can efficiently degrade PCL, including different strains of Pseudomonas spp., Streptomyces spp., Alcaligenes faecalis, and fungi like Aspergillus oryzae, Fusarium spp., Rhizopus delemar, and Thermomyces lanuginosus. These microorganisms produce enzymes such as lipases, esterases, and cutinases that break down PCL into smaller molecules that act as substrates. The review also examines the phylogenetic diversity of organisms capable of biodegrading PCL, the biochemical pathways involved in this process, and specific aspects of the genetic framework responsible for the expression of the enzymes that facilitate degradation. Targeted research on microbial PCL biodegradation and its practical applications could significantly aid in reducing and managing plastic waste on a global ecological scale. Full article

Triacylglycerols containing medium-chain fatty acids at the sn-1,3 positions and a long-chain fatty acid at the sn-2 position (MLM-TAG) are of nutritional interest. However, they are scarce in common food sources and are usually synthesized by chemical or enzymatic methods. In this work, the enzymatic synthesis of MLM-TAG was attempted using sn-2 monoacylglycerols (sn-2 MAG) from the ethanolysis of an arachidonic acid-rich fraction from ARASCO and fatty acid ethyl esters from the ethanolysis of coconut oil as substrates. The highest yield of sn-2 MAG (23.3 mol%) was obtained after 1 h of ethanolysis with Novozym 435 lipase at 25 °C, and the best profile of the ethanolysis products of coconut oil was obtained after 24 h of reaction catalyzed by the lipase from Thermomyces lanuginosus. Regarding the enzymatic synthesis of structured TAG, the lipase from Rhizopus oryzae gave better results than those from Thermomyces lanuginosus and Rhizomucor miehei, with the sn-2 position mainly esterified with arachidonic acid (34.8%) and the sn-1,3 positions mainly esterified with capric and lauric acids (35.1%). This work focuses on a simple process for the enzymatic production of structured TAG without prior purification of the sn-2 MAG. Full article

Along with the development of technology and the increasing consumption of polymeric materials, which have become an integral part of man’s everyday life, problems related to their disposal are arising. The presented research concentrates on the studies on the enzymatic degradation of selected epoxy-polyurethane materials filled with 2 or 5 wt.% of waste unmodified or chemically modified through mercerization wood flour. Composites, subjected to the degradation process, contained up to 60% of raw materials of natural origin. The enzymatic degradation was carried out for 28 days, in three environmental conditions, differing in the type of applied buffer, pH, process temperature, the amount, and the type of applied enzyme. In this study, the influence of two lipases was tested (specifically: lipase of microbiological origin—Rhizopus Oryzae Lipase, and one of animal origin—Porcine Pancreas Lipase). There were seven compositions tested, based on the polyaddition product of epoxidized soybean oil with bisphenol A, differing in the amount of filler and the type of modification to which wood flour was subjected before the application in the polymer composite. After enzymatic degradation, the greatest progress of biodegradation was observed at T = 30 °C, in a complex phosphate buffer with pH = 6.8, in the presence of the Porcine Pancreas enzyme. Under these conditions, a slightly smaller effect was also observed in the presence of the Rhizopus Oryzae enzyme. At the same time, the compositions containing mercerized wood flour turned out to be the most susceptible to biodegradation with the above-mentioned enzymes. After conducting the process in the full 4-week cycle numerous changes were noticed within the tested sample, such as (1) 7.0 %wt. of the overall weight loss of samples, (2) reducing the value of the static contact angle (e.g., from 116.7° before degradation to 27.2° at the end of the study), and (3) morphological appearance of the sample (sample’s surface had suffered erosion noticed as smoothest roughnesses and numerous empty holes throughout its entire volume), concerning sample’s condition before enzymatic degradation. Full article

Metal–organic frameworks (MOFs) are regarded as excellent carriers for immobilized enzymes due to their substantial specific surface area, high porosity, and easily tunable pore size. Nevertheless, the use of UIO−66 material is significantly limited in immobilized enzymes due to the absence of active functional groups on its surface. This study comprised the synthesis of UIO−66 and subsequent modification of the proline (Pro) on UIO−66 through post-synthetic modification. UIO−66 and UIO−66/Pro crystals were employed as matrices to immobilize Rhizopus oryzae lipase (ROL). The contact angle demonstrated that the introduction of Pro onto UIO−66 resulted in favorable conformational changes in the structure of ROL. The immobilized enzyme ROL@UIO−66/Pro, produced via the covalent-bonding method, exhibited greater activity (0.064715 U/mg (about 1.73 times that of the free enzyme)) and stability in the ester hydrolysis reaction. The immobilized enzymes ROL@UIO−66 (131.193 mM) and ROL@UIO−66/Pro (121.367 mM), which were synthesized using the covalent-bonding approach, exhibited a lower K_m and higher substrate affinity compared to the immobilized enzyme ROL@UIO−66/Pro (24.033 mM) produced via the adsorption method. This lays a solid foundation for the industrialization of immobilized enzymes. Full article

This study assessed the application of whole lipolytic cells in the pretreatment of slaughterhouse wastewater to reduce its lipid content. The fungal biomass of Rhizopus oryzae was evaluated in the hydrolysis of slaughterhouse wastewater containing high lipid concentrations, focusing on the biomass’s concentration and the effect of using an emulsifier and surfactant. The use of the whole-cells lipase of Rhizopus oryzae grown in a residual vegetable oil medium proved effective in the hydrolysis of slaughterhouse wastewater, generating concentrations of free fatty acids (FFA) ranging from 40.36 to 90.14 mM. The action of lipase in the hydrolysis of slaughterhouse residues indicated its effectiveness in pretreating lipid-rich liquid residues, potentially boosting the microbiota of this anaerobic treatment. The results showed that lipase activity without surfactant exhibited a similar performance to that of Triton X-100 in the hydrolysis of liquid residues. However, the combination of lipase and surfactant could represent a promising strategy to optimize free fatty acid production from slaughterhouse residues, strengthening anaerobic treatment processes and potentially enhancing the overall efficiency of waste management systems. Full article

Biomimetic ligands are synthetic compounds that mimic the structure and binding properties of natural biological ligands. The first uses of textile dyes as pseudo-affinity ligands paved the way for the rational design and de novo synthesis of low-cost, non-toxic and highly stable triazine-scaffolded affinity ligands. A novel method to assess and enhance protein stability, employing triazine-based biomimetic ligands and using cutinase from Fusarium solani pisi as a protein model, has been previously reported. This innovative approach combined the concepts of molecular modeling and solid-phase combinatorial chemistry to design, synthesize and screen biomimetic compounds able to bind cutinase through complementary affinity-like interactions while maintaining its biological functionality. The screening of a 36-member biased combinatorial library enabled the identification of promising lead ligands. The immobilization/adsorption of cutinase onto a particular lead (ligand 3′/11) led to a noteworthy enhancement in thermal stability within the temperature range of 60–80 °C. In the present study, similar triazine-based compounds, sourced from the same combinatorial library and mimicking dipeptides of diverse amino acids, were selected and studied to determine their effectiveness in binding and/or improving the thermal stability of several lipases, enzymes which are closely related in function to cutinases. Three ligands with different compositions were screened for their potential thermostabilizing effect on different lipolytic enzymes at 60 °C. An entirely distinct enzyme, invertase from Saccharomyces cerevisiae, was also assessed for binding to the same ligands and functioned as a ‘control’ for the experiments with lipases. The high binding yield of ligand 3′/11 [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid] to cutinase was confirmed, and the same ligand was tested for its ability to bind lipases from Aspergillus oryzae (AOL), Candida rugosa (CRL), Chromobacterium viscosum (CVL), Rhizomucor miehei (RML) and Rhizopus niveus (RNL). The enzymes CRL, CVL, RNL and invertase showed significant adsorption yields to ligand 3′/11—32, 29, 36 and 94%, respectively, and the thermal stability at 60 °C of free and adsorbed enzymes was studied. CVL and RNL were also stabilized by adsorption to ligand 3′/11. In the case of CRL and invertase, which bound but were not stabilized by ligand (3′/11), other ligands from the original combinatorial library were tested. Between the two alternative ligands, one was effective at stabilizing C. rugosa lipase, while none stabilized invertase. Full article

Lipases have valuable potential for industrial use, particularly those mostly active against water-insoluble substrates, such as triglycerides composed of long-carbon chain fatty acids. However, in most cases, engineered variants often need to be constructed to achieve optimal performance for such substrates. Protein engineering techniques have been reported as strategies for improving lipase characteristics by introducing specific mutations in the cap domain of esterases or in the lid domain of lipases or through lid domain swapping. Here, we improved the lipase activity of a lipase (WP_075743487.1, or Lip_MRD) retrieved from the Marine Metagenomics MarRef Database and assigned to the Actinoalloteichus genus. The improvement was achieved through site-directed mutagenesis and by substituting its lid domain (FRGTEITQIKDWLTDA) with that of Rhizopus delemar lipase (previously R. oryzae; UniProt accession number, I1BGQ3) (FRGTNSFRSAITDIVF). The results demonstrated that the redesigned mutants gain activity against bulkier triglycerides, such as glyceryl tridecanoate and tridodecanoate, olive oil, coconut oil, and palm oil. Residue W89 (Lip_MRD numbering) appears to be key to the increase in lipase activity, an increase that was also achieved with lid swapping. This study reinforces the importance of the lid domains and their amino acid compositions in determining the substrate specificity of lipases, but the generalization of the lid domain swapping between lipases or the introduction of specific mutations in the lid domain to improve lipase activity may require further investigation. Full article

Polylactic acid (PLA) is the main biobased plastic manufactured on an industrial scale. This polymer is synthetized by chemical methods, and there is a strong demand for the implementation of clean technologies. This work focuses on the microbial fermentation of agro-industrial waste rich in starch for the production of lactic acid (LA) in a consolidated bioprocess, followed by the enzymatic synthesis of PLA. Lactic acid bacteria (LAB) and the fungus Rhizopus oryzae were evaluated as natural LA producers in pure cultures or in fungal–lactobacteria co-cultures formed by an LAB and a fungus selected for its metabolic capacity to degrade starch and to form consortia with LAB. Microbial interaction was analyzed by scanning electron microscopy and biofilm production was quantified. The results show that the fungus Talaromyces amestolkiae and Lactiplantibacillus plantarum M9MG6-B2 establish a cooperative relationship to exploit the sugars from polysaccharides provided as carbon sources. Addition of the quorum sensing molecule dodecanol induced LA metabolism of the consortium and resulted in improved cooperation, producing 99% of the maximum theoretical yield of LA production from glucose and 65% from starch. Finally, l-PLA oligomers (up to 19-LA units) and polymers (greater than 5 kDa) were synthetized by LA polycondensation and enzymatic ring-opening polymerization catalyzed by the non-commercial lipase OPEr, naturally produced by the fungus Ophiostoma piceae. Full article

In this study, the performance characteristics of hemp shives impregnated with linseed oil and tung tree oil (HS)- and corn starch (CS)-based biocomposites containing flame retardants were evaluated before and after treatment with the mixture of bacterium Pseudomonas putida and fungus Rhizopus oryzae. Enzymatic activities and physical-mechanical properties such as water absorption, thickness swelling, compressive strength, and thermal conductivity were tested to evaluate the suitability of selected composites for thermal insulation purposes. In addition, electron microscopy was used to investigate the impact of microorganisms on the microstructure of the material. It was determined that the type of oil used for impregnation significantly affects the properties of biocomposites after 6 months of incubation with mixture of bacterium P. putida and fungus Rh. oryzae. Biocomposites impregnated with linseed oil and after treatment with a mixture of microorganisms had cellulase activity of 25 U/mL, endo β-1-4-glucanase activity of 26 U/mL, lipase activity of 101 U/mL, only a 10% decrease in compressive strength, 50% higher short-term water absorption, unchanged swelling in thickness, and slightly decreased thermal conductivity compared to control biocomposites. At the same time, biocomposites with tung tree oil had a much more pronounced deterioration of the properties tested, cellulase activity of 28 U/mL, endo β-1-4-glucanase activity of 37 U/mL, lipase activity of 91 U/mL, two times lower compressive strength and two times higher short-term water absorption, 2.5 times greater thickness swelling, and a slightly increased thermal conductivity. We conclude that linseed oil provides better protection against the action of microorganisms compared to impregnation with tung tree oil. Full article

Rhizopus oryzae is widely distributed in nature and can be isolated from different substrates such as decomposing vegetables, fruits and various soils. It is generally classified as GRAS filamentous fungi and commonly used in the production of oriental traditional food such as tempeh or peka. This microorganism has great industrial potential due to the capability to synthesize enzymes (glucoamylases, cellulases and lipases) and organic acids (lactic acid, fumaric acid). The most studied enzymes of the fungi are lipases (ROL). Therefore, the aim of the study was the selection of growth medium content and initial pH rate, which would provide high lipase synthesis yield in 5 days shaken cultures. Two fractions of lipases were investigated in order to obtain lipase biocatalysts: extracellular enzymes present in supernatant and cell-bound lipases in biomass. The used nutrient-rich media were: YPG (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose), YPO (10 g/L yeast extract, 20 g/L peptone, 20 g/L olive oil), YMG (3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 20 g/L glucose), YMO (3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 20 g/L olive oil). The mineral media were: SMG (10 g/L peptone, 14 g/L KH₂PO₄, 2.4 g/L K₂HPO₄, 0.4 g/L MgSO₄, 20 g/L glucose) and SMO (10 g/L peptone, 14 g/L KH₂PO₄, 2.4 g/L K₂HPO₄, 0.4 g/L MgSO₄, 20 g/L olive oil). Fungi biomass and supernatant were separated and used to measure lipase activity by a spectrophotometric method based on the hydrolysis of p-nitrophenyl laurate. The results showed that the highest lipase activity after 5 days of cultivation was reached in YPO medium for biomass (from 7- to 60-fold higher results depending on the compared variant of culture media) and YMG for supernatant (from 3- to 6.5-fold higher results depending on the used variant of culture media). The addition of citric acid resulted in a two times increase of the activity of produced lipases after 5 days of cultivation. Full article