Search Results (30)

Background: Membrane proteins are preferentially solubilized with sodium dodecyl sulfate (SDS), which necessitates a purification protocol to deplete the surfactant prior to mass spectrometry analysis. However, maintaining solubility of intact membrane proteins is challenged in an SDS-free environment. SDS precipitation with potassium salts (KCl) offers a potentially viable workflow to deplete SDS and permit proteoform analysis. The purpose of this study is to devise a robust detergent-based protocol applicable for processing and analysis of intact membrane-associated proteoforms. Methods: The precipitation conditions impacting SDS removal from spinach chloroplasts and liver membrane proteome preparations were evaluated, capitalizing on optimization of pH (highly basic), addition of MS-compatible solubilizing additives (urea) and adjustment of the KCl to SDS ratio to maximize recovery and purity. Results: Characterization of the SDS-solubilized, KCl-precipitated spinach membrane preparation revealed multiple charge envelope MS spectra displaying high signal to noise, free of SDS adducts. Precipitation at pH 12 or with urea improved protein recovery and purity. Bottom-up analysis identified 1826 distinct liver protein groups from four independent SDS precipitation conditions. While precipitation at pH 8 without urea revealed a greater number of protein identifications by mass spectrometry, precipitation under highly basic conditions (pH 12) with urea provided higher membrane protein recovery and achieved the greatest number (732 of 1056) and largest percentage (69.3%) of membrane proteins identified in the SDS removal workflow. Conclusion: This workflow provides new opportunities for MS-based proteoform analysis by capitalizing on the benefits of SDS for protein extraction while maintaining high solubility and purity of intact proteins though KCl precipitation of the surfactant. Full article

The bioprocessing of agricultural wastes to produce microbial enzymes has become significant due to its benefits in reducing enzyme production costs and improving waste management. In this study, various substrates, including spent coffee grounds, coffee husks, coffee pulp, rice husks, rice bran, pomelo albedo, pomelo flavedo, orange peel, banana peel, sugarcane bagasse, and starch, were used as organic nutrient sources for α-amylase biosynthesis by B. licheniformis TKU004. Among the tested substrates, pomelo albedo (3%, w/v) was the most suitable carbon source for amylase production, with a productivity of 80.645 U/mL. The purification process resulted in a 60 kDa amylase. The protein identification of B. licheniformis TKU004 amylase revealed a coverage rate of 39% with α-amylase from Bacillus subtilis 168. B. licheniformis TKU004 amylase exhibited optimal activity at 60 °C and pH = 7 and showed a high compatibility with EDTA (Ethylenediaminetetraacetic acid). HPLC (high-performance liquid chromatography) analysis demonstrated that B. licheniformis TKU004 amylase is an α-amylase with the final products of maltobiose, maltose, and glucose. Due to its important properties, such as tolerance to EDTA, B. licheniformis TKU 004 amylase may be valuable for industrial applications, especially in detergents and food processing. Full article

Tick-Borne Encephalitis Virus (TBEV) is impacting public health in the Eurasian region, with increasing case numbers. There is, therefore, a need to expand research efforts and the corresponding infrastructure capacity. Since TBEV is classified as a risk group 3 organism in Switzerland, handling infectious material containing the virus is restricted to biosafety level 3 laboratories. In some instances, downstream analyses may need to be performed outside of the containment facility. It is, therefore, essential to validate effective inactivation protocols compatible with the safe and accurate processing of samples. This study evaluated UV irradiation, chemical treatment with detergents, and mechanical filtration as candidate methods to inactivate TBEV infectious samples, including culture supernatants and tissue homogenates, while preserving their compatibility for different assays. Among the methods tested, 45 s of UV irradiation or Triton-X100 at concentrations between 0.05% and 0.1% effectively inactivated TBEV while mostly preserving the integrity of the processed samples for immuno- or enzymatic assays. These findings establish safe and reliable procedures for advancing TBEV research beyond high-containment settings. Full article

Obesity, a complex condition that involves genetic, environmental, and behavioral factors, is a non-infectious pandemic that affects over 650 million adults worldwide with a rapidly growing prevalence. A major contributor is the consumption of high-fat diets, an increasingly common feature of modern diets. Maternal obesity results in an increased risk of offspring developing obesity and related health problems; however, the impact of maternal diet on the adipose tissue composition of offspring has not been evaluated. Here, we designed a generational diet-induced obesity study in female C57BL/6 mice that included maternal cohorts and their female offspring fed either a control diet (10% fat) or a high-fat diet (45% fat) and examined the visceral adipose proteome. Solubilizing proteins from adipose tissue is challenging due to the need for high concentrations of detergents; however, the use of a detergent-compatible sample preparation strategy based on suspension trapping (S-Trap) enabled label-free quantitative bottom-up analysis of the adipose proteome. We identified differentially expressed proteins related to lipid metabolism, inflammatory disease, immune response, and cancer, providing valuable molecular-level insight into how maternal obesity impacts the health of offspring. Data are available via ProteomeXchange with the identifier PXD042092. Full article

A novel alkaline serine protease, derived from the Staphylococcus aureus strain ALA1 previously isolated from dromedary milk, was subjected to purification and characterization. Optimal protease production occurred under specific culture conditions. The purified protease, designated S. aureus Pr with a molecular mass of 23,662 Da and an N-terminal sequence, showed an approximately 89% similar identity with those of other Staphylococcus strains. It exhibited its highest enzymatic activity at a pH of 10.0 and 60 °C in the presence of 3 mM Ca²⁺. Remarkable thermostability was observed at temperatures up to 70 °C and within a pH range of 6.0 to 10.0 for 2 h. The presence of Ca²⁺ or Mg²⁺ and Zn²⁺ significantly enhanced both enzymatic activity and thermal stability. Additionally, notable stability was demonstrated in the presence of reducing and chaotropic agents as well as in surfactants, oxidizing agents, and organic solvents commonly found in detergent compositions. This highlights the enzyme’s potential as a versatile biocatalyst, especially in detergents. Its stability and compatibility with laundry detergents matched Alcalase 2.5 L, type Dx, and the Stearothermophilus protease, used as controls. Collectively, this study investigated the potential utilization of S. aureus Pr in industrial detergents as an excellent candidate for incorporation as an additive in detergent formulations. Full article

The objective was to characterize the pastures by grazing cycle, as well as to evaluate the performance of buffaloes in intensive rotational grazing in a silvopastoral system in the eastern Amazon supplemented with agro-industry co-products in order to characterize the grazing cycles, the composition of the fractions, and the carcass yield. Fifteen non-castrated, crossbred water buffaloes (Murrah × Mediterranean) were used. All animals used in the study were clinically healthy and weighed approximately 458 kg. The animals were grazed in a single group, and supplementation (1% of live weight—LW/day) was divided into three treatments: control (control—conventional ingredients); Cocos nucifera coconut cake (Cocos nucifera) (coconut cake—70%); and palm kernel cake (Guinean elaeis) (palm kernel cake—70% palm kernel cake). The chemical composition of the forage is different in each part of the plant, with higher protein values in the leaves (11.40%) and higher acid detergent fiber (ADF) values in the stems (50.03%). Among the ingredients of the supplement, corn has the highest percentage of indigestible protein (35.57%), most of the protein in palm kernel cake is B3 (49.11%), and in Coco, B2 (51.52%). Mombasa grass has a higher percentage of B3 and B2 proteins; the indigestible fraction is lower in the leaves (17.16%). The leaf/stem ratio also varied between grazing cycles, being better in the second cycle (2.11%) and with an overall average of 1.71. Supplement consumption varied between cycles and was higher in the control treatment, with an overall mean of 4.74. There was no difference in daily weight gain and carcass yield, with an average of 1 kg/day and 49.23%, respectively. Therefore, it can be concluded that including supplements based on by-products from the coconut and palm oil agro-industries promotes performance and carcass yields compatible with conventional supplements. Their use reduces production costs, optimizes the utilization of forage mass, enhances the sustainability of the production chain, and, therefore, is recommended. Full article

Membrane proteins are underrepresented during proteome characterizations, primarily owing to their lower solubility. Sodium dodecyl sulfate (SDS) is favored to enhance protein solubility but interferes with downstream analysis by mass spectrometry. Here, we present an improved workflow for SDS depletion using transmembrane electrophoresis (TME) while retaining a higher recovery of membrane proteins. Though higher levels of organic solvent lower proteome solubility, we found that the inclusion of 40% methanol provided optimal solubility of membrane proteins, with 86% recovery relative to extraction with SDS. Incorporating 40% methanol during the electrophoretic depletion of SDS by TME also maximized membrane protein recovery. We further report that methanol accelerates the rate of detergent removal, allowing TME to deplete SDS below 100 ppm in under 3 min. This is attributed to a three-fold elevation in the critical micelle concentration (CMC) of SDS in the presence of methanol, combined with a reduction in the SDS to protein binding ratio in methanol (0.3 g SDS/g protein). MS analysis of membrane proteins isolated from the methanol-assisted workflow revealed enhanced proteome detection, particularly for proteins whose pI contributed a minimal net charge and therefore possessed reduced solubility in a purely aqueous solvent. This protocol presents a robust approach for the preparation of membrane proteins by maximizing their solubility in MS-compatible solvents, offering a tool to advance membrane proteome characterization. Full article

In this study, thirty-seven alkaline protease-producing bacteria were recovered from different regions of Saudi Arabia. The proteolytic strain with the highest productivity was identified as Bacillus sp. Mar64. Maximum productivity of Mar64P alkaline protease was reached at 60 h, pH 9.0, and 45 °C using 1% tyrosine and 0.5% maltose as nitrogen and carbon supplies, respectively. Specific activity was intensified to 8.5-fold with a recovery of 12.4% and SDS—PAGE revealed one band at 28 kDa after enzyme purification. Mar64P was maximally active at 55 °C and pH 11.0 with thermal stability up to 70 °C and pH stability at 7.0–12.0 for 1 h. It was inhibited by EDTA and unaffected by PMSF, therefore tentatively classified as metalloprotease-type. Storage efficacy was effective for up to eight weeks and it was durable in presence of organic solvents (20%, v/v) such as acetonitrile, acetone, and isopropanol upto to 15 days. The enzyme was compatible with dry detergents at both low and high temperature, in addition, was successful in removing various stains such as blood, egg yolk, chocolate, tea, coffee, and sweat. Furthermore, it was successful in removing skin hairs and hydrolyzing gelatin of waste X-ray films. Collectively, due to these unique properties, Mar64P could be considered an environmentally friendly candidate in both detergent and leather industries. Full article

Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R² = 0.85–0.90) and plasma (SPEED R² = 0.84; SDC R² = 0.76, S-Trap R² = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired. Full article

Lichenysin, an amphiphilic biosurfactant with structural and physicochemical properties similar to surfactin, is produced by Bacillus licheniformis. Its low toxicity, good environmental compatibility, solubilization, foaming, emulsification and detergent activities have led to a wide range of applications in agricultural biocontrol, enhanced oil recovery, foaming agents for cosmetics and detergents for household cleaning products. However, despite the extraordinary surface-active properties and potential applications of lichenysin, the number of wild bacteria found so far is relatively low. Low titers and high costs are the main limiting factors for widespread industrial applications. In this study, a factorial design was used to optimize the composition of the medium for the production of lichenysin by Bacillus licheniformis Ali5. Firstly, the solutions of carbon, nitrogen, amino acids, inorganic salts and trace elements in the medium were evaluated in flasks using a single-factor optimization method. Meanwhile, the operating conditions were optimized in the same way. Afterwards, a partial factorial design was used to investigate the effect of six variables (five medium compositions and inoculum size) on lichenysin production. Based on the results obtained, the concentrations of sucrose and ammonium nitrate and the inoculum size were considered to be important for lichenysin production. Subsequently, a full factorial design was used to optimize these three variables. The optimized medium composition were sucrose 19.8 g/L, NH₄NO₃ 3.9 g/L, K₂HPO₄·3H₂O 4.0 g/L, MgSO₄·7H₂O 0.6 g/L, FeSO₄·7H₂O 0.1 g/L, CaCl₂ 0.01 g/L, NaCl 3.0, trace elements 1.2 mL/L. Finally, the titer of lichenysin after fed-batch fermentation reached 1425.85 mg/L, which was approximately 5.5 times higher than the titer of lichenysin from the original medium. Consequently, the method was further demonstrated to be suitable for lichenysin production. Full article

Soybean (Glycine max Merr.), with a high nutritional value, is an important oil crop and a good protein feed crop. Due to the shortage of high-protein feed and the high import pressure on soybean, scarce high-protein feed is the main research target for improving feed quality. High-quality soybean (Qihuang 34) and high-yield silage maize (Zea mays L.) (Jingling silage 386) varieties were used as the experimental materials in this study. The silage quality and the microbial community of the mixed silage of soybean and maize were analyzed, and the compatible intercropping ratios of maize–soybean mixed silage were evaluated. This experiment designed five strip intercropping systems (SIS) in a randomized block. The intercropping row ratios of maize and soybean were 0:1 (pure soybean, S), 1:0 (pure maize, M), 1:1 (MS1), 1:3 (MS2), and 1:5 (MS3). Dry matter yield and fresh matter yield were improved in the treatments of MS1 and MS2. In the mixed silage systems of maize and soybean, with an increase in soybean proportion, crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF) contents gradually increased, but the contents of water-soluble carbohydrates (WSC) and dry matter (DM) reduced to different degrees (p < 0.05). Moreover, the soybean silage alone had a poor fermentation performance, as indicated by high pH, high acetic acid (AA), propionic acid (PA), butyric acid (BA), and ammonia-N (NH₃-N) concentrations, and low lactic acid (LA) concentration. By contrast, the mixed silage materials were conducive to reducing the pH, PA, BA, and NH₃-N, and increasing the LA content. The relative abundance of Lactobacillus and Weissella in the MS were higher, and the abundance of undesirable bacteria were lower than in the S. The MS2 materials had the lowest pH, the highest LA concentration, and the highest relative abundance of Lactobacillus and Weissella among the three mixed silage groups. Therefore, the mixed silage in the SIS modified the microbial communities and improved the feed fermentation quality while increasing yields. The better intercropping ratio of maize–soybean mixed silage was 1:3. These results could provide a theoretical basis for the wide application and popularization of soybean as a high-protein silage forage source. Full article

Gel-free bottom-up shotgun proteomics is the principal methodological platform for the state-of-the-art proteome research. This methodology assumes quantitative isolation of the total protein fraction from a complex biological sample, its limited proteolysis with site-specific proteases, analysis of the resulted peptides with nanoscaled reversed-phase high-performance liquid chromatography-(tandem) mass spectrometry (nanoRP-HPLC-MS and MS/MS), protein identification by sequence database search and peptide-based quantitative analysis. The most critical steps of this workflow are protein reconstitution and digestion; therefore, detergents and chaotropic agents are strongly mandatory to ensure complete solubilization of complex protein isolates and to achieve accessibility of all protease cleavage sites. However, detergents are incompatible with both RP separation and electrospray ionization (ESI). Therefore, to make LC-MS analysis possible, several strategies were implemented in the shotgun proteomics workflow. These techniques rely either on enzymatic digestion in centrifugal filters with subsequent evacuation of the detergent, or employment of MS-compatible surfactants, which can be degraded upon the digestion. In this review we comprehensively address all currently available strategies for the detergent-assisted proteolysis in respect of their relative efficiency when applied to different biological matrices. We critically discuss the current progress and the further perspectives of these technologies in the context of its advances and gaps. Full article

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. Full article

Cellulose fibers isolated from oil palm empty fruit bunches (OPEFB) have been studied as a potential reinforcement for polyvinyl alcohol (PVA) biocomposite. Analysis of variance (ANOVA) showed that all three parameters—hydrolysis temperature, time and acid concentration, as well as their interactions—significantly affected the yield of cellulose. Moving Least Squares (MLS) and Multivariable Power Least Squares (MPLS) models demonstrated good fitness. The model also proved that acid concentration was the dominant parameter, supported by the Fourier transform infrared spectroscopy (FTIR) analysis. Hydrolysis using 54% acid at 35 °C and 15 min achieved the highest cellulose yield of 80.72%. Cellulose-reinforced PVA biocomposite films demonstrated better mechanical strength, elongation at break, moisture barrier properties, thermal stability and poorer light transmission rate compared to neat PVA due to the high aspect ratio, crystallinity and good compatibility of cellulose fibers. These findings suggested the potential of cellulose fibers-reinforced PVA biocomposite film as water-soluble detergent capsules. Full article

Sugar amides, such as aldonamides, are interesting, sugar-based molecules used in various fields, from detergency to medicine. Nevertheless, their valorization, especially as alternatives to petroleum-based substances, can be slowed down by their synthetic pathway, which is generally not in accordance with green chemistry principles, and is also not economically competitive. We propose herein a fast procedure for the synthesis of aldonamide-derived glycoconjugates with mechanochemistry. The conditions were first optimized with galactonolactone, used as a model lactone, and dodecylamine. After only 5 min of grinding of stoechiometric amounts of amine and lactone, in the presence of water used as a Liquid Assisted Grinding (LAG) agent, the corresponding galactonamide was isolated with a high yield (90%) after a simple aqueous work-up. The optimized conditions were then applied to a wide variety of amines and sugar lactones, showing the versatility of the methodology. Gluco- and ribono-lactone exhibited similarly excellent reactivity, showing that the procedure is not sugar-dependent. Furthermore, the procedure was shown to be compatible with various functional groups such as alkene, alkyne, thiol, ester and hydroxyl. Full article