Search Results (40)

Vans (light commercial vehicles) account for only about 11% of the European light-duty vehicle fleet. However, they are mostly used in urban delivery and service operations where frequent stop-and-go driving increases tyre abrasion. Furthermore, their annual mileage is on average more than 70% higher than that of passenger cars. For these reasons, vans are estimated to generate tyre wear emissions that are at least 2.5 times higher than those of passenger cars on a per-vehicle basis, and therefore make a disproportionate contribution to microplastic pollution in cities. The Euro 7 pollutant emission standards introduce, for the first time, regulatory limits on tyre abrasion for passenger car tyres (C1 class) from 2028 and for light-commercial-vehicle tyres (C2 class) from 2030, building on United Nations (UN) tyre testing procedures developed under UN Regulation 117. While two candidate test methods (a real-world method and a laboratory method) have been agreed on for C1 tyres, no equivalent standard exists yet for C2 tyres, and very few experimental data have been published so far. In this study, we adapt the C1 real-world-based method to winter C2 tyres (snow three-peak mountain snowflake, 3PMSF) fitted to vans, and we discuss the practical and regulatory challenges encountered. The resulting abrasion rate and abrasion level indices provide first experimental emission factors for C2 tyres and can inform the ongoing development of regulatory test procedures and limit values for van tyres. We also develop an experimental and analytical framework to relate abrasion measurements to tyre service life (mileage potential). Full article

The intestine is considered a habitat for both bacteria and parasites. In this study, many fecal bacterial isolates and the protozoan Blastocystis sp. were recovered from stool samples of individuals with gastrointestinal conditions. Isolated bacteria were tested for extracellular protease production, and the most potent producer was identified by 16SrDNA gene sequencing as P. aeruginosa FZM498. The enzyme was extracted and purified to electrophoretic homogeneity by the DEAE-Sepharose ion-exchanger and SDS-PAGE revealed a major band at 42.15 KDa. It exhibited maximal activity at 35 °C with thermostability at 60 °C (T_1/2 = 200.04 min). It was most active at pH 8.0 and stable at 5.0–9.5. Enzymatic activity was greatly stimulated in the presence of Fe²⁺ ions, but was repressed by Zn²⁺ and Hg²⁺ ions. Inhibition by PMSF, TLCK, aprotinin, benzamidine, and SBTI protease reagents suggests a serine protease family. The V_max and K_m dynamic constants against azocasein were 36.232 U/mL and 0.0072 mM, respectively. It exhibited the lowest K_m value against the synthetic substrate D-Val-Leu-Lys-pNA among all substrates, indicating a plasmin-like activity. Interestingly, when tested against Blastocystis sp., cysts appeared progressively shrunken, ruptured, and mycelial-like, indicating complete structural collapse with leakage of intracellular contents. The importance of this research is that it is the first study to test the anti-Blastocystis activity of an extracted bacterial serine protease from the gut. This could be a promising, eco-friendly, natural alternative as an anti-Blastocystis agent. The objective of this study was to isolate, purify, and biochemically characterize an extracellular serine protease produced by gut-associated bacteria, as well as to assess its in vitro anti-Blastocystis efficacy as a potential natural and ecologically friendly antiparasitic therapy. Full article

Chloroplast-to-nucleus ROS retrograde signaling is essential for acclimation of the photosynthetic apparatus to environmental stresses. One of the key mechanisms is the regulation of the photosystem II antenna size depending on light conditions and other environmental factors. However, the molecular components linking chloroplast redox status to nuclear gene regulation remain poorly defined. Here, we demonstrate that H₂O₂, generated in chloroplasts, in particular with involvement of the plastoquinone pool components, enhances the protease activity in the chloroplast envelope. As it is known, protease activity leads to the processing of the chloroplast envelope-bound transcription factor PTM, enabling its relocation to the nucleus, where it induces ABI4 expression. ABI4, in turn, represses transcription of lhcb genes, resulting in downsizing of the PS II antenna. Gene expression analysis confirms the coordinated upregulation of ABI4, and PTM, as well as metallo-ASP and serine SPPA1 envelope proteases in high light. We further show that H₂O₂ at physiologically relevant concentrations specifically stimulates the serine protease activity, since this activation is inhibited by PMSF. Our findings indicate a link between redox changes in the plastoquinone pool and the H₂O₂ level in chloroplasts with protease-mediated signaling cascades. Therefore, the obtained data reveal the connection between chloroplast and nuclear control of photosynthetic light harvesting, highlighting a signaling strategy for the photosystem II antenna size regulation in higher plants. Full article

In acidic environments, acid phosphatases (EC 3.1.3.2) play a crucial role in hydrolyzing phosphate ester linkages. Two isoforms of acid phosphatases, namely AP-I and AP-II, were purified to homogeneity from the seeds of Erythrina indica using a combination of gel filtration and affinity chromatography techniques. The purification process involved multiple steps to ensure the enzymes were free from other seed components, thereby facilitating detailed characterization. We report in this study the active site characterization of acid phosphatase form AP-I. The active site of purified AP-I was characterized in detail through chemical modification studies, which revealed the presence of one residue each of carboxylate, tryptophan, and serine amino acid. Substrate protection experiments using p-nitrophenyl phosphate effectively prevented the modification of all three residues, suggesting their essential role in the enzyme’s active site. These experiments provided strong evidence that these residues are directly involved in the catalytic process. Kinetic studies of the partially inactivated enzyme, achieved through the use of the specific modifying agents dicyclohexylcarbodiimide (DCCD) for carboxylate, N-bromosuccinimide (NBS) for tryptophan, and phenylmethylsulfonyl fluoride (PMSF) for serine further confirmed the involvement of these residues in the catalytic mechanism. The results demonstrated that the inactivation of any of these residues significantly impaired the enzyme’s activity, highlighting their critical roles in the catalytic process. The results provide a comprehensive understanding of the active site architecture and the catalytic mechanism of AP-I function. Full article

Tyre wear is a major contributor to global microplastic pollution, affecting air, soil, water, and wildlife as well as human health. In the European Union (EU), the latest Euro 7 regulation foresees the introduction of tyre abrasion limits covering all tyre categories, referring to two testing methods (convoy on road or laboratory drum) developed by the United Nations (UN) Economic Commission for Europe (UNECE) World Forum for Harmonization of Vehicle Regulations (WP.29). In this study, we applied the convoy method adopted by the UNECE Working Group on Noise and Tyres (GRBP) as part of the UN Regulation 117 on tyre performance parameters. The method has been developed by the Task Force on Tyre Abrasion (TFTA) of the UNECE and involves vehicles driving on public roads for about 8000 km. Candidate and reference tyres are fitted in a convoy of up to four vehicles, and an abrasion index for each candidate tyre is determined as a ratio of the abrasion of the candidate and reference tyres. In our tests, in addition to the abrasion rate, we measured the tread depth reduction and defined a service life index (i.e., total mileage potential) without the need of a different methodology. The results from six summer and nine winter C1 class passenger car tyres of various sizes showed a wide range of abrasion rates and service life values. We also compared our results with values reported in the literature and on websites. The conclusions of this study are expected to support the ongoing discussion on limit setting for C1 tyres and the definition of a service life index. Full article

Phthalic acid esters (PAEs), ubiquitously employed as a plasticizer, have been classified as priority environmental pollutants because of their persistence, bioaccumulation, and endocrine-disrupting properties. As a characterized PAE-degrading strain of marine origin, Mycolicibacterium phocaicum RL-HY01 utilizes di-(2-ethylhexyl) phthalate (DEHP) as its sole carbon and energy source. Genome sequencing and RT-qPCR analysis revealed a previously uncharacterized hydrolase gene (dehpH) in strain RL-HY01, which catalyzes ester bond cleavage in PAEs. Subsequently, recombinant expression of the cloned dehpH gene from strain RL-HY01 was established in Escherichia coli BL21(DE3). The purified recombinant DehpH exhibited optimal activity at 30 °C and pH 8.0. Its activity was enhanced by Co²⁺ and tolerant to most metal ions but strongly inhibited by EDTA, SDS, and PMSF. Organic solvents (Tween-80, Triton X-100, methanol, ethanol, isopropanol, acetone, acetonitrile, ethyl acetate, and n-hexane) showed minimal impact. Substrate specificity assay indicated that DehpH could efficiently degrade the short and long side-chain PAEs but failed to hydrolyze the cyclic side-chain PAE (DCHP). The kinetics parameters for the hydrolysis of DEHP were determined under the optimized conditions, and DehpH had a V_max of 0.047 ± 0.002 μmol/L/min, K_m of 462 ± 50 μmol/L, and k_cat of 3.07 s⁻¹. Computational prediction through structural modeling and docking identified the active site, with mutagenesis studies confirming Ser228, Asp324, and His354 as functionally indispensable residues forming the catalytic triad. The identification and characterization of DehpH provided novel insights into the mechanism of DEHP biodegradation and might promote the application of the target enzyme. Full article

Collagenases are enzymes with broad biotechnological applications in medicine. This study describes the production and characterization of a collagenase from Aspergillus serratalhadensis URM 7866, isolated from the Caatinga biome. Solid-state fermentations were conducted using wheat bran under varying conditions of pH (6, 7, 8), moisture content (50%, 60%, 70%), and substrate concentration (2.5 g, 5 g, 10 g). The optimal condition—10 g of wheat bran at pH 8 and 70% moisture—yielded the highest collagenolytic activity (177.96 U/mL) and a specific activity of 50.55 U/mg. The enzyme was purified via multiple chromatography, with pre-purification and final purification factors of 18.09 and 20.21, respectively, reaching a specific activity of 1021.86 U/mg. The enzyme showed optimal activity at 50 °C and pH 8, with stability from 20 to 40 °C and pH 7–9. PMSF caused >80% inhibition; EDTA caused ~34% inhibition. Activity increased with Na⁺ and Ca²⁺ and was inhibited by Zn²⁺. The enzyme retained full activity in anionic and non-ionic surfactants (1–10%). FTIR confirmed characteristic amide bands, and kinetic analysis revealed a Km of 1.72 mg/mL and Vmax of 6.89 mg/mL/min. These findings support its potential for alkaline and surfactant-rich industrial processes. Full article

In the search for new alternatives for controlling parasitic agents, proteases from Beauveria bassiana stand out. The aim of this study was to evaluate in silico and in vitro the ovicidal potential of B. bassiana proteases on Eurytrema pancreaticum Janson, 1889 (Dicrocoeliidae) eggs. Beauveria bassiana Bals. -Criv., 1835 (Cordycipitaceae) (IP 361) was cultivated for enzymatic production. Proteins were precipitated with acetone (1:4 ratio), and specific activity was determined. Protease profiles were assessed via zymography, and inhibition by phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA) was tested. Three-dimensional models of the proteases were generated. Eurytrema pancreaticum eggs were used for the in vitro anthelmintic evaluation of the proteases. The results showed that precipitation significantly concentrated proteolytic activity (p < 0.01) compared to the crude extract. However, no chitinase activity was detected. The proteolytic profile of the precipitate revealed five bands with molecular weights from 25.6 to 66.9 kDa. In the in vitro tests, the proteases significantly (p < 0.01) reduced the number of intact E. pancreaticum eggs by 53% compared to the control with denatured enzymes. These findings highlight the ovicidal potential of B. bassiana proteases, though further studies are needed to confirm their application in parasite control. Full article

Whey, a large-scale dairy industry by-product, can be converted into whey protein concentrate (WPC), providing a cost-effective nutrient-rich substrate for microbial fermentation. We investigated protease production by Aspergillus oryzae using WPC as the sole substrate in submerged fermentation. Following fermentation, the protease was purified sequentially from the crude extract by salting-out, which yielded a substantial purification factor (~39), and subsequent ion-exchange chromatography. The non-adsorbed chromatographic fraction showed the highest protease activity (92.6 U/mL) and revealed one main protein band ~45 kDa via SDS-PAGE. Enzyme characterization demonstrated activity across neutral-to-alkaline conditions, optimal at pH 9.0 and 37 °C, with stability maintained between 30 °C and 37 °C. The enzyme was classified as a serine protease based on strong inhibition by PMSF and SDS; its activity was also inhibited by Zn²⁺, Mg²⁺, and K⁺, but enhanced by Ca²⁺. This work validates WPC as an efficient substrate for protease production by A. oryzae and presents a promising strategy for valorizing industrial by-products through sustainable biotechnology. Full article

The application of slow-release fertilizers is essential for improving fertilizer utilization efficiency and promoting sustainable agricultural development. Unlike traditional single organic polymer-coated or inorganic-coated fertilizers, this study utilized biodegradable modified polyvinyl alcohol (PVA) as a binder and cheap, readily available phosphogypsum–bentonite as an inorganic coating material to develop a novel slow-release potassium magnesium sulfate fertilizer (SRPMSF). This study initially examined the influence of SA dosage on PVA properties. XRD, FTIR, TGA, and water resistance analyses revealed that sodium alginate exhibits good compatibility with polyvinyl alcohol, enhancing its heat and water resistance. Ultimately, PVA–SA-2 (1.2% sodium alginate) was chosen as the optimal binder for SRPMSF production. Furthermore, this study investigated the impact of bentonite on the physical and slow-release properties of the SRPMSF by varying the phosphogypsum-to-bentonite ratio. This experiment included five treatment methods: the treatments consist of SRPMSF-1 (0 g bentonite), SRPMSF-2 (phosphogypsum/bentonite ratio of 4:1), SRPMSF-3 (3:2), SRPMSF-4 (2:3), and SRPMSF-5 (1:4). A control group (PMSF) was also included. The results indicated that, as the bentonite content increased, both the particle size and compressive strength of the coated slow-release fertilizer increased, with the SRPMSF particle sizes ranging from 3.00 to 4.50 mm. The compressive strength of the SRPMSF ranged from 20.85 to 43.78 N, meeting the requirements for industrial production. The soil column leaching method was employed to assess the nutrient release rate of the fertilizers. The experimental results indicated that, compared to the PMSF, the SRPMSF effectively regulated nutrient release. Pot experiments demonstrated that the SRPMSF significantly enhanced garlic seedling growth compared to the PMSF. In conclusion, a new type of slow-release fertilizer with good slow-release performance is prepared in this paper, which can improve the utilization rate of fertilizer and reduce the economic loss and is conducive to the sustainable development of agriculture. Full article

A novel protease was isolated from the fruiting bodies of the straw mushroom Volvariella volvacea. The protease was purified 13.48-fold using a series of techniques, including ammonium sulfate precipitation, ultrafiltration, diethylaminoethyl fast-flow (DEAE FF) ion-exchange chromatography, and Superdex 75 gel filtration chromatography, resulting in a specific enzyme activity of 286.82 U/mg toward casein as a substrate. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the purified protease had a molecular weight of 24 kDa. The enzyme exhibited optimal activity at pH 7 and 50 °C, showing sensitivity to alkaline conditions and instability at elevated temperatures. The presence of Ca²⁺ significantly enhanced enzyme activity, whereas Ni²⁺ and Cu²⁺ exerted strong inhibitory effects, with other metal ions showing weak inhibition. β-mercaptoethanol, Tween-80, and Triton X-100 had more pronounced inhibitory effects, whereas PMSF, EDTA, and CTAB had weaker inhibitory effects. The Michaelis constant (Km) and maximum velocity (Vm) of the protease were determined to be 1.34 g/L and 3.45 μg/(mL·min), respectively. The protease exhibited a greater degree of enzymatic degradation of soybean-isolate protein (7.58%) compared to trypsin (5.24%), with the enzyme product containing a high percentage of medicinal amino acids (73.54%), particularly phenylalanine (Phe) and arginine (Arg), suggesting their presence at the enzyme’s active site. These findings suggest that the protease from V. volvacea holds promising potential for applications in the food industry, particularly in protein hydrolysate production and flavor enhancement. Full article

A novel caseinolytic protease (ClpP) of the S14 family from Cobetia amphilecti KMM 296 (CamClpP), comprising 206 amino acids, with a calculated molecular weight of 22.66 kDa and a pI of 4.88, was expressed in Escherichia coli cells to verify the functional annotation of the encoding gene that has low identity with known structures. The proteolytic activity of the purified recombinant enzyme was found to be 2824 U/mg, using 1% casein as a substrate. Enzyme activity was maximal at pH 5.6 and 7.4 in phosphate buffer and was maintained over a wide pH range of 4-10. The optimum temperature for protease activity was 45 °C. The enzyme in its optimal state required the presence of either NaCl or KCl at concentrations of 0.3 and 0.2 M, respectively. The addition of the metal ions Mg²⁺, Ca²⁺, Ni²⁺, Mn²⁺, Li⁺, and Zn²⁺ at 2 mM resulted in a significant inhibition of the protease activity. However, the presence of Co²⁺ led to a marked activation of the enzyme in the absence of ATP. The enzyme activity was inhibited by ethanol, isopropanol, glycerol, SDS, EGTA, and EDTA. The presence of Triton X-100, acetone, DTT, and PMSF resulted in a significant increase in the CamClpP protease activity. The protease CamClpP effectively and preferentially degrades high-polymer wheat and rye flour proteins. This new proteolytic enzyme with unique properties is of great ecological and biotechnological importance. Full article

The large-scale production of soybean proteins results in the generation of a significant volume of wastewater, containing a substantial amount of valuable β-amylase. The β-amylase enzyme was purified from the soybean whey wastewater using a three-step process, including alcohol precipitation, ion-exchange chromatography, and gel filtration chromatography. The specific activity of the purified β-amylase was 29,700 U/mg, with an enzyme activity recovery of 17.3% and purification fold of 16.5. The β-amylase had a molecular mass of around 56 kDa and an isoelectric point (pI) value of 4.8. The β-amylase exhibited optimal activity at 55 °C and reasonable stability between 30 °C and 40 °C. The enzyme demonstrated an optimum pH of 6.0 and relative stability in the pH range of 5.0–8.0. Furthermore, the β-amylase activity was stimulated by PMSF, Tween-20, Tween-40, Tween-60, Tween-80, and Triton X-100. In terms of substrate preference, the enzyme hydrolyzed potato starch worked most effectively, followed by amylose, amylopectin, soluble starch, maltose, and pullulan. The purified β-amylase showed K_m and V_max values of 3.62 μM and 1.04 μM/ (g protein min), respectively. The purification process was simple and yielded high purification and recovery. The β-amylase from soybean whey wastewater has potential applications in the food and beverage industries. Full article

Lipases and carboxylesterases are enzymes of biotechnological interest both for their reactions and their specificity. They have wide-ranging applications in the food, pharmaceuticals, biodiesel synthesis, and bioremediation industries. For that reason, the strain Neobacillus thermocopriae C255 was isolated from ash from Popocatepetl volcano and studied as a new source of lipolytic enzymes. It was identified using 16S ribosomal RNA and flagellar protein FliF sequence homology, yielding 100% identity. From the sequencing of its genome, an enzyme with lipolytic activity, classified as a monoacylglycerol lipase, and named Mgl-C255, was cloned in E. coli BL21, and then expressed, biochemically characterized, and tested via transesterification reactions with alcohols and monosaccharides. Based on its sequence and structure, it was placed within family V, having a catalytic triad of S90-D207-H237. Biochemical characterization showed its highest activity at 40 °C, pH 7.5 to 8.5, with C-2 length substrate preference. No metal ions or inhibitors influenced lipolytic activity, except for PMSF, SDS, Cu⁻², and Hg⁻². Mgl-C255 retained about 50% of its activity in non-polar solvents and showed synthetic activity in organic solvents, making it a good candidate for studying its catalytic potential and selectivity. Full article

Background/Objectives: Heterodera avenae is a highly significant plant-parasitic nematode, causing severe economic losses to global crop production each year. Trichoderma species have been found to parasitize nematodes and control them by producing enzymes that degrade eggshells. The T. longibrachiatum T6 (T6) strain has been demonstrated the parasitic and lethal effects on H. avenae cysts and eggs, associated with the increased serine protease activity and trypsin-like serine protease gene (PRA1) expression. Methods: Our present study aimed to purify the recombinant PRA1 protease through a prokaryotic expression system and identify its nematicidal activity. Results: The recombinant PRA1 protease was identified as S1 family trypsin-like serine protease, with a molecular weight of 43.16 kDa. The purified soluble protease exhibited the optimal activity at 35 °C and pH 8.0, and also demonstrating higher hydrolytic ability toward casein and skimmed milk. Meanwhile, the Ca²⁺ and Mg²⁺ enhanced its activity, while the inhibitor PMSF significantly reduced it. The contents of H. avenae eggs leaked out after treatment with the recombinant PRA1 protease, with egg hatching inhibition and relative hatching inhibition rates at 70.60% and 66.58%, respectively. In contrast, there was no sign of content dissolution, and embryos developed normally in the control group. Conclusions: Our present study revealed that the PRA1 protease of T6 strain has a lethal effect on H. avenae eggs, which providing a theoretical basis for developing biocontrol agents to control nematodes. Full article