Proteomic Analysis of HepG2 Cells Reveals FAT10 and BAG2 Signaling Pathways Affected by a Protease Inhibitor from Tinospora cordifolia (Willd.) Hook. f. and Thoms Stem. Extract Among the Different Plant and Microbial Samples Analyzed.

Objectives: Dysregulation of proteolysis underlies diseases like cancer. Protease inhibitors (PIs) regulate many biological functions and hence have potential anticancer properties. With this background, the current study aimed to identify the PI from natural sources such as plants and microbes against trypsin (a protease), which was assayed against casein, using an ultraviolet spectrophotometer-based methodology. Materials and Methods: PI extracted from a few plants and microbial samples were screened for their PI activity against trypsin. The PI from the most promising source in our study, Tinospora cordifolia (Willd.) Hook. f. and Thoms. stem, was partially purified using ammonium sulfate precipitation followed by dialysis. The PI activity of the partially purified inhibitor was analyzed against chymotrypsin and collagenase enzymes, and the cytotoxic effect of the PI was checked on HepG2 (liver carcinoma) cells by MTT- [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide]- assay. Liquid Chromatograography Mass Spectrometry -based proteomic studies were performed on HepG2 cells to understand the signaling pathways affected by the PIs in the liver cancer cell line. Results: Among the samples tested the PIs from T. cordifolia stem extract had the highest inhibitory activity (72.0%) against trypsin along with cytotoxicity to HepG2 cells. After partial purification by 80.0% ammonium sulfate precipitation, PI had increased inhibitory activity (83.0%) against trypsin and enhanced cytotoxicity (47.0%) to HepG2 cells. Proteomic analysis of the PI-treated HepG2 cells revealed that BAG2 and FAT10 signaling pathways were affected, which may have caused the inhibition of cancer cell proliferation. Conclusion: PI from T. cordifolia stem has promising anticancer potential and hence can be used for further purification and characterization studies toward cancer drug development.

Interactive effects of ammonium sulfate and lead on alfalfa in rare earth tailings: Physiological responses and toxicity thresholds.

Ion-adsorption rare earth ore contains significant levels of leaching agents and heavy metals, leading to substantial co-contamination. This presents significant challenges for ecological rehabilitation, yet there is limited understanding of the toxicity thresholds associated with the co-contamination of ammonium sulfate (AS) and lead (Pb) on pioneer plants. Here, we investigated the toxicity thresholds of various aspects of alfalfa, including growth, ultrastructural changes, metabolism, antioxidant system response, and Pb accumulation. The results indicated that the co-contamination of AS-Pb decreased the dry weight of shoot and root by 26 %-77 % and 18 %-92 %, respectively, leading to irregular root cell morphology and nucleus disintegration. The high concentration and combined exposures to AS and Pb induced oxidative stress on alfalfa, which stimulated the defense of the antioxidative system and resulted in an increase in proline levels and a decrease in soluble sugars. Structural equation modeling analysis and integrated biomarker response elucidated that the soluble sugars, proline, and POD were the key physiological indicators of alfalfa under stresses and indicated that co-exposure induced more severe oxidative stress in alfalfa. The toxicity thresholds under single exposure were 496 (EC5), 566 (EC10), 719 (EC25), 940 (EC50) mg kg-1 for AS and 505 (EC5), 539 (EC10), 605 (EC25), 678 (EC50) mg kg-1 for Pb. This study showed that AS-Pb pollution notably influenced plant growth performance and had negative impacts on the growth processes, metabolite levels, and the antioxidant system in plants. Our findings contribute to a theoretical foundation and research necessity for evaluating ecological risks in mining areas and assessing the suitability of ecological restoration strategies.

[Effects of Different Proportions of Ammonium Sulfate Replacing Urea on Soil Nutrients and Rhizosphere Microbial Communities].

In order to investigate the effects of ammonium sulfate, an industrial by-product, on soil nutrients and microbial community when applied in different proportions instead of using urea as nitrogen fertilizer, a pot corn experiment was conducted. A completely randomized block experimental design was used, with a total of five treatments:CK (no fertilization), U10S0 (100 % urea), U8S2 (80 % urea + 20 % ammonium sulfate), U6S4(60 % urea + 40 % ammonium sulfate), and U0S10 (100 % ammonium sulfate). The basic physical and chemical properties of soil and the dry weight of maize plants were determined by conventional methods, and microbial sequencing was performed using the Illumina NovaSeq platform. The experiment results showed that:① In each growth stage of maize, the pH of soil treated with fertilization (7.85-8.15) was decreased compared with that of CK (8.1-8.21), and the pH showed a decreasing trend with the increase in ammonium sulfate content. ② The soil available nitrogen content increased gradually with the increase in the ammonium sulfate ratio at each growth stage of maize. Compared with that in the CK and U10S0 treatments, the ratio in the U0S10 treatment increased 30.56 % to 63.68 % and 13.22 % to 38.43 %, respectively. The variation trend of organic carbon content was opposite to that of available nitrogen (U8S2 > U6S4 > U0S10), and the addition of ammonium sulfate was still higher than that of U10S0 at other growth stages except for the seedling stage. ③ The protease activity of all fertilization treatments was higher than that of the control, and the protease activity was gradually enhanced with the continuous growth of corn and the increase in the ammonium sulfate ratio. The protease activity of the U0S10 treatment was higher than that of the U10S0 treatment at each growth stage of corn, which increased by 10.54 %-100 %. Soil sucrase activity ranged from 0.04 to 0.24 mg·(g·24 h)-1, and those in the U0S10 treatments were significantly higher than those in the U10S0 and CK treatments at all growth stages, increasing by 20.32 % to 99.16 % and 24.31 % to 79.33 %, respectively. ④ The species abundance of bacteria and fungi in maize rhizosphere under all fertilization treatments were lower than those under the CK treatment, followed by those under the U10S0 treatment. The species diversity trend of the bacterial community in the three treatments with ammonium sulfate replacing urea were U8S2 > U0S10 > U6S4, and that of fungi were U6S4 > U8S2 > U0S10. ⑤ The maize dry weight of the U10S0 treatment and U0S10 treatment was the highest, which was 39.47 % and 36.16 % higher than that of the CK treatment, respectively, but the difference was not significant. The Pearson model showed that the species abundance and diversity of soil rhizosphere fungi and bacteria were affected by relevant environmental variables, among which pH value and soil available nitrogen content were the most important factors affecting microbial diversity. In conclusion, when corn planting in calcareous brown soil, replacing urea with a certain proportion of ammonium sulfate can improve soil nutrients more than urea alone, which affects the growth and rhizosphere microbial community of corn to a certain extent and has a greater yield.

Ammonium sulfate denatures transport medium less dependent on guanidinium isothiocyanate and enables SARS-CoV-2 RNA and antigen detection compatibility.

Introduction: Rapid identification of infected individuals through viral RNA or antigen detection followed by effective personal isolation is usually the most effective way to prevent the spread of a newly emerging virus. Large-scale detection involves mass specimen collection and transportation. For biosafety reasons, denaturing viral transport medium has been extensively used during the SARS-CoV-2 pandemic. However, the high concentrations of guanidinium isothiocyanate (GITC) in such media have raised issues around sufficient GITC supply and laboratory safety. Moreover, there is a lack of denaturing transport media compatible with SARS-CoV-2 RNA and antigen detection. Methods: Here, we tested whether supplementing media containing low concentrations of GITC with ammonium sulfate (AS) would affect the throat-swab detection of SARS-CoV-2 or a viral inactivation assay targeting coronavirus and other enveloped and non-enveloped viruses. The effect of adding AS to the media on RNA stability and its compatibility with SARS-CoV-2 antigen detection were also tested. Results and discussion: We found that adding AS to the denaturing transport media reduced the need for high levels of GITC, improved SARS-COV-2 RNA detection without compromising virus inactivation, and enabled the denaturing transport media compatible with SARS-CoV-2 antigen detection.

Enhancing erythromycin production in Saccharopolyspora erythraea through rational engineering and fermentation refinement: A Design-Build-Test-Learn approach.

Industrial production of bioactive compounds from actinobacteria, such as erythromycin and its derivatives, faces challenges in achieving optimal yields. To this end, the Design-Build-Test-Learn (DBTL) framework, a systematic metabolic engineering approach, was employed to enhance erythromycin production in Saccharopolyspora erythraea (S. erythraea) E3 strain. A genetically modified strain, S. erythraea E3-CymRP21-dcas9-sucC (S. erythraea CS), was developed by suppressing the sucC gene using an inducible promoter and dcas9 protein. The strain exhibited improved erythromycin synthesis, attributed to enhanced precursor synthesis and increased NADPH availability. Transcriptomic and metabolomic analyses revealed altered central carbon metabolism, amino acid metabolism, energy metabolism, and co-factor/vitamin metabolism in CS. Augmented amino acid metabolism led to nitrogen depletion, potentially causing cellular autolysis during later fermentation stages. By refining the fermentation process through ammonium sulfate supplementation, erythromycin yield reached 1125.66 mg L-1, a 43.5% increase. The results demonstrate the power of the DBTL methodology in optimizing erythromycin production, shedding light on its potential for revolutionizing antibiotic manufacturing in response to the global challenge of antibiotic resistance.

UCST-Type Thermoresponsive Sol-Gel Transition Triblock Copolymer Containing Zwitterionic Polymer Blocks.

Thermoresponsive sol-gel transition polymers are of significant interest because of their fascinating biomedical applications, including as drug reservoirs for drug delivery systems and scaffolds for tissue engineering. Although extensive research has been conducted on lower critical solution temperature (LCST)-type sol-gel transition polymers, there have been few reports on upper critical solution temperature (UCST)-type sol-gel transition polymers. In this study, we designed an ABA-type triblock copolymer composed of a poly(ethylene glycol) (PEG) block and zwitterionic polymer blocks that exhibit UCST-type thermoresponsive phase transitions. A sulfobetaine (SB) monomer with both ammonium and sulfonate (-SO3) groups in its side chain or a sulfabetaine (SaB) monomer with both ammonium and sulfate (-OSO3) groups in its side chain was polymerized from both ends of the PEG block via reversible addition-fragmentation chain-transfer (RAFT) polymerization to obtain PSB-PEG-PSB and PSaB-PEG-PSaB triblock copolymers, respectively. Although an aqueous solution containing the PSB-PEG-PSB triblock copolymer showed an increase in viscosity upon cooling, it did not undergo a sol-to-gel transition. In contrast, a sol-to-gel transition was observed when a phosphate-buffered saline containing PSaB-PEG-PSaB was cooled from 80 °C to 25 °C. The PSaB blocks with -OSO3 groups exhibited a stronger dipole-dipole interaction than conventional SB with -SO3 groups, leading to intermolecular association and the formation of a gel network composed of PSaB assemblies bridged with PEG. The fascinating UCST-type thermoresponsive sol-gel transition properties of the PSaB-PEG-PSaB triblock copolymer suggest that it can provide a useful platform for designing smart biomaterials, such as drug delivery reservoirs and cell culture scaffolds.

Evaporating crystallization effect of ammonium sulfate at atmospheric pressure under the action of ultrasound.

Ultrasound enhanced evaporating crystallization has been proposed to solve the problems of low crystallization yield and uneven particle size in the evaporating crystallization process of ammonium sulfate solution at atmospheric pressure. The effects of key operating parameters, including the ultrasound power, stirring speed, pH value, and ultrasound time, on the yield of ammonium sulfate product and the duration of solid-liquid transformation time are studied. The results show that the ultrasound crystallization can increase the ammonium sulfate yield by 52.9 %, reduce the solid-liquid transformation time of ammonium sulfate by 10 %, and obtain ammonium sulfate products with higher crystallinity and more uniform particle size. Ultrasound promotes the crystallization of ammonium sulfate by enhancing the transfer of heat in the solution and reducing the supersolubility of the ammonium sulfate solution from 937.5 g/L to 833.33 g/L. This study provides experimental justification for the use of ultrasound in atmospheric evaporative crystallization.

Influence of the Nitrogen Fertilization on the Yield, Biometric Characteristics and Chemical Composition of Stevia rebaudiana Bertoni Grown in Poland.

Stevia rebaudiana Bertoni is a plant native to South America that has gathered much interest in recent decades thanks to diterpene glycosides, called steviosides, which it produces. These compounds are characterised by their sweetness, which is 250-300 times higher than saccharose, and they contain almost no caloric value. Stevia is currently also grown outside the South American continent, in various countries characterised by warm weather. This research aimed to determine whether it is viable to grow Stevia rebaudiana plants in Poland, a country characterised by a cooler climate than the native regions for stevia plants. Additionally, the impact of adding various dosages and forms of nitrogen fertiliser was analysed. It was determined that Stevia rebaudiana grown in Poland is characterised by a rather low concentration of steviosides, although proper nitrogen fertilisation can improve various characteristics of the grown plants. The addition of 100 kg or 150 kg of nitrogen per hectare of the field in the form of urea or ammonium nitrate increased the yield of the stevia plants. The stevioside content can be increased by applying fertilisation using 100 kg or 150 kg of nitrogen per hectare in the form of ammonium sulfate. The total yield of the stevia plants grown in Poland was lower than the yield typically recorded in warmer countries, and the low concentration of steviosides in the plant suggests that more research about growing Stevia rebaudiana in Poland would be needed to develop profitable methods of stevia cultivation.

An improved method for measuring catalase activity in biological samples.

Catalase (CAT) is an important enzyme that protects biomolecules against oxidative damage by breaking down hydrogen peroxide (H2O2) into water and oxygen. CAT is present in all aerobic microbes, animals, and plants. It is, however, absent from normal human urine but can be detected in pathological urine. CAT testing can thus help to detect such urine. This study presents a novel spectrophotometric method for determining CAT activity characterized by its simplicity, sensitivity, specificity, and rapidity. The method involves incubating enzyme-containing samples with a carefully chosen concentration of H2O2 for a specified incubation period. Subsequently, a solution containing ferrous ammonium sulfate (FAS) and sulfosalicylic acid (SSA) is added to terminate the enzyme activity. A distinctive maroon-colored ferrisulfosalicylate complex is formed. The formation of this complex is a direct result of the reaction between FAS and any residual peroxide present. This leads to the generation of ferric ions when coordinated with SSA. The complex has a maximum absorbance of 490 nm. This advanced method eliminates the need for concentrated acids to stop CAT activity, making it safer and easier to handle. A comparative analysis against the standard ferrithiocyanate method showed a correlation coefficient of 0.99, demonstrating the new method's comparable effectiveness and reliability. In conclusion, a simple and reliable protocol for assessing CAT activity, which utilizes a cuvette or microplate, has been demonstrated in this study. This interference-free protocol can easily be used in research and clinical analysis with considerable accuracy and precision.

Effect of ammonium sulfate combined with aqueous bio-chelator on Cd uptake by Cd-hyperaccumulator Solanum nigrum L.

The efficacy of using plants to phytoremediate heavy metal (HM) contaminated soils can be improved using soil amendments. These amendments may both increase plant biomasses and HMs uptake. We aimed to determine the composite effect of ammonium sulfate ((NH4)2SO4) combined with the application of an aqueous stem-extracted bio-chelator (Bidens tripartita L) on the plant biomasses and cadmium (Cd) phytoextraction by Solanum nigrum L. The constant (NH4)2SO4 application mode plus bio-chelator additives collectively enhanced the shoot Cd extraction ability owing to the increased plant biomass and shoot Cd concentration by S. nigrum. The shoot Cd extraction and the soil Cd decreased concentration confirmed the optimal Cd phytoextraction pattern in K8 and K9 treatments (co-application of (NH4)2SO4 and twofold/threefold bio-chelators). Accordingly, Cd contamination risk in the soil (2 mg kg-1) could be completely eradicated (<0.2 mg kg-1) after three rounds of phytoremediation by S.nigrum based on K8 and K9 treatments through calculating soil Cd depletion. The microorganism counts and enzyme activities in rhizosphere soils at treatments with the combined soil additives apparently advanced. In general, co-application mode of (NH4)2SO4 and aqueous bio-chelator was likely to be a perfect substitute for conventional scavenger agents on account of its environmental friendliness and cost saving for field Cd contamination phytoremediation by S. nigrum.

Influence of ammonium nitrate on the crystallisation of ammonium sulfate.

This study aims to explore the influence mechanism of ammonium nitrate produced by ozone denitrification on the crystallisation of ammonium sulfate, a by-product of ammonia desulfurisation. The laser method was used to study the influence of ammonium nitrate on the solubility and metastable zone width of ammonium sulfate. An experiment on the influence of ammonium nitrate on the particle size of ammonium sulfate was designed, and the influence mechanism was explored through scanning electron microscopy and X-ray diffraction. The findings showed that the addition of ammonium nitrate increased the size and aspect ratio of ammonium sulfate crystals. The addition of ammonium nitrate inhibited the dissolution of ammonium sulfate and widened its metastable zone. The addition of ammonium nitrate covered the active sites of crystal nucleus growth, which inhibited the formation of crystal nuclei to a certain extent, and crystal growth dominated the crystallisation process. Moreover, the addition of ammonium nitrate induced the preferred orientation of the specific crystal plane of ammonium sulfate, and the addition of a small concentration of ammonium nitrate decreased the crystallinity of ammonium sulfate. The research results can provide a reference for crystallisation optimisation and quality improvement of ammonium sulfate in the ammonia desulfurisation process.

Optical manipulation and fusion of aqueous droplets containing inorganic and organic solutes in air using the dual-beam laser trapping technique.

The comparative evaluation of two aerosol droplets of different chemical compositions using the dual-beam laser trapping technique can be employed for highly sensitive and accurate measurements of the water activities of such droplets. However, it is technically difficult to load droplets of different chemical compositions into adjacent optical traps that are only a few tens of micrometers apart. To overcome this challenge, a chamber with an overhanging roof was created. This roof prevented the initially trapped droplets from being contaminated by aerosol droplets that were subsequently introduced into the chamber. Herein, we report the simultaneous laser trapping of an aqueous ammonium sulfate (AS) droplet and an aqueous succinic acid (SA) droplet in air using the dual-beam laser trapping technique. Two droplets were successfully fused through optical manipulation to form a mixed inorganic/organic droplet in air. This experimental approach is advantageous because it forms mixed inorganic/organic droplets under constant relative humidity (RH) conditions. However, in previous studies, it was necessary to compensate for changes in RH prior to and after droplet fusion. To assess the validity of theoretical predictions of the water activity of droplets containing AS and SA, the equilibrium radii of the droplet were compared with those calculated using certain theoretical models.

Investigating the migration hypothesis: Effects of trypsin-like protease on the quality of muscle proteins of red shrimp (Solenocera crassicornis) during cold storage.

This study aimed to investigate the effect of trypsin-like protease (TLP) on the quality of muscle proteins in red shrimp (Solenocera crassicornis) during cold storage. The results indicated that the activity of TLP decreased significantly in the head of shrimp but increased significantly in the muscle tissues during the cold storage. The myofibril fragmentation index (MFI) value of intact shrimp was significantly higher than that of beheaded shrimp, while the Ca2+-ATPase activity of intact shrimp was significantly lower than that of beheaded shrimp. SDS-PAGE analysis showed that the molecular weight of purified TLP from the shrimp head was about 24 kDa, and the TLP showed high activity at 50 °C and pH 8, indicating that the TLP belongs to the trypsin family. Results from in vitro simulation experiments indicated that the process of TLP incubation significantly reduced the particle size and enlarged the distribution of myofibrillar proteins (MPs) in shrimp muscle tissues. The comparisons were made with respect to the control samples. It can be inferred that TLP migrated from the shrimp head to the muscle tissues during storage and thus promoted the degradation of MPs in red shrimp. The beheading treatment could be an effective mean to maintain better quality and extend the commercialization of shrimp products.

Cricket Protein Isolate Extraction: Effect of Ammonium Sulfate on Physicochemical and Functional Properties of Proteins.

Crickets are known to be a promising alternative protein source. However, a negative consumer bias and an off-flavor have become obstacles to the use of these insects in the food industry. In this study, we extracted the protein from commercial cricket powder by employing alkaline extraction-acid precipitation and including ammonium sulfate. The physicochemical and functional properties of the proteins were determined. It was found that, upon including 60% ammonium sulfate, the cricket protein isolate (CPI) had the highest protein content (~94%, w/w). The circular dichroism results indicated that a higher amount of ammonium sulfate drastically changed the secondary structure of the CPI by decreasing its α-helix content and enhancing its surface hydrophobicity. The lowest solubility of CPI was observed at pH 5. The CPI also showed better foaming properties and oil-holding capacity (OHC) compared with the cricket powder. In conclusion, adding ammonium sulfate affected the physicochemical and functional properties of the CPI, allowing it to be used as an alternative protein in protein-enriched foods and beverages.

[Influence of Ammonium Sulfate and Ammonium Nitrate on the Properties of Ambient PM2.5 in Zhenjiang].

Recently, the contribution of inorganic salts (nitrates in particular) to the mass concentration of particulate matter with an aerodynamic diameter of less than 2.5 µm (PM2.5) has been increasing across China. However, it is urgent to understand how the increased inorganic salts affect the crucial properties of PM2.5. Here, we conducted continuous field observations at Zhenjiang Ecology and Environment Protection Bureau from January 1 to December 31, 2021. The mass concentrations of ammonium sulfate[(NH4)2SO4] and ammonium nitrate (NH4NO3) were calculated using different methods. The contributions of (NH4)2SO4 and NH4NO3 to the extinction coefficient, hygroscopic growth, and acidity of PM2.5 were discussed in detail. Our results demonstrated that the mean mass concentrations of (NH4)2SO4 and NH4NO3 during the study period were (6.5±4.5) and (15.0±13.3) µg·m-3, which contributed (20.5±18.2)% and (34.5±18.4)% to the mass concentration of PM2.5, respectively. The total extinction coefficient of PM2.5 was (224.5±194.2) Mm-1, in which NH4NO3 was the largest contributor[(40.1±20.9)%] followed by (NH4)2SO4[(19.1±10.8)%]. (NH4)2SO4 and NH4NO3 were also the dominant contributors to the hygroscopic growth of PM2.5. In particular, NH4NO3contributed from (53.8±13.4)% to (61.6±14.6)% to the aerosol water content of PM2.5 under pollution conditions. Thus, NH4NO3 was a key air pollutant to be targeted for further improving the visibility and air quality in Zhenjiang in the future. However, the reduction in the precursors of NH4NO3 would lead to an increase in aerosol acidity, particularly in the spring and winter seasons. Our results help us understand the evolution of air quality and the related impacts and also provide important information on air quality improvement in Zhenjiang in the future.

Feasibility of ammonium sulfate recovery from wastewater sludges: Hydrothermal liquefaction pathway vs. anaerobic digestion pathway.

This study evaluated two pathways to recover the nitrogen-content of wastewater sludges as ammonium sulfate (AmS) for use as fertilizer. The first pathway entails sludge stabilization by hydrothermal liquefaction (HTL) followed by recovery of AmS from the resulting aqueous product by gas permeable membrane (GPM) separation. The second one entails stabilization of the sludges by anaerobic digestion (AD) followed by recovery of AmS from the resulting centrate by GPM separation. A bench-scale GPM reactor is shown to be capable of recovering >90% of N in the feed. Recoveries of NH3-N in the HTL-pathway ranged 96-100% in 5.5-7.5 h at mass removal rates of 0.2-0.3 g N/day, yielding 3.3-6.0 g AmS/L of feed. Recoveries of 98% were noted in the AD-pathway in 4 h at mass removal rates of 0.06-0.97 g N/day and a yield of 1.7-2.1 g AmS/L of feed. Inductively coupled plasma optical emission spectrometer analysis confirmed that both pathways yielded AmS meeting the US EPA and European region guidelines for land application. The GPM reactor enabled higher nitrogen-recoveries in the HTL-pathway than those reported for current practice of AD followed by ammonia stripping, ion exchange, reverse osmosis, and/or struvite precipitation (96-100% vs. 50-90%). A process model for the GPM reactor is validated using performance data on three different feedstocks.

Protein Extraction and Purification by Differential Solubilization.

The preparation of purified soluble proteins for biochemical studies is essential and the solubility of a protein of interest in various media is central to this process. Selectively altering the solubility of a protein is a rapid and economical step in protein purification and is based on exploiting the inherent physicochemical properties of a polypeptide. Precipitation of proteins, released from cells upon lysis, is often used to concentrate a protein of interest before further purification steps (e.g., ion exchange chromatography, size exclusion chromatography etc).Recombinant proteins may be expressed in host cells as insoluble inclusion bodies due to various influences during overexpression. Such inclusion bodies can often be solubilized to be reconstituted as functional, correctly folded proteins.In this chapter, we examine strategies for extraction/precipitation/solubilization of proteins for protein purification. We also present bioinformatic tools to aid in understanding a protein's propensity to aggregate/solubilize that will be a useful starting point for the development of protein extraction, precipitation, and selective re-solubilization procedures.

Ammonium sulfate supplementation enhances erythromycin biosynthesis by augmenting intracellular metabolism and precursor supply in Saccharopolyspora erythraea.

In this study, the cellular metabolic mechanisms regarding ammonium sulfate supplementation on erythromycin production were investigated by employing targeted metabolomics and metabolic flux analysis. The results suggested that the addition of ammonium sulfate stimulates erythromycin biosynthesis. Targeted metabolomics analysis uncovered that the addition of ammonium sulfate during the late stage of fermentation resulted in an augmented intracellular amino acid metabolism pool, guaranteeing an ample supply of precursors for organic acids and coenzyme A-related compounds. Therefore, adequate precursors facilitated cellular maintenance and erythromycin biosynthesis. Subsequently, an optimal supplementation rate of 0.02 g/L/h was determined. The results exhibited that erythromycin titer (1311.1 µg/mL) and specific production rate (0.008 mmol/gDCW/h) were 101.3% and 41.0% higher than those of the process without ammonium sulfate supplementation, respectively. Moreover, the erythromycin A component proportion increased from 83.2% to 99.5%. Metabolic flux analysis revealed increased metabolic fluxes with the supplementation of three ammonium sulfate rates.

Insights into the interaction mechanism of acid phosphatase from Lateolabrax japonicus livers and rosmarinic acid using multispectroscopy and molecular docking.

Acid phosphatase (ACP) is a key enzyme that hydrolyzes inosinic acid. The mechanisms underlying the interaction between rosmarinic acid (RA) and ACP and the inhibition of the enzyme were investigated using inhibition kinetics, UV-visible and fluorescence spectroscopy, circular dichroism, and molecular docking. The results showed that RA was a reversible inhibitor of ACP and that the inhibition mechanism was uncompetitive. The ACP fluorescence was quenched by RA, and the quenching mode was static. The interaction of ACP with RA was driven by H bonds and van der Waals forces. The addition of RA increased the α-helix content and decreased the ß-sheet, ß-turn, and random coil contents in ACP, thereby altering the secondary structure of the enzyme. This study enriched our understanding of inhibitory and interaction mechanisms involving ACP and RA.

Effect of sulfur on pollen germination of Clemenules mandarin and Nova tangelo.

This study aims to elucidate whether sulfur can inhibit citrus pollination by affecting pollen grains. For this, four sulfur-based products (inorganic sulfur, water dispersible granular sulfur, ammonium sulfate, copper sulfate) were tested to evaluate their effect on pollen germination and pollen tube growth of two citrus varieties: Clemenules mandarin (Citrus clementina) and Nova tangelo (Citrus clementina x [Citrus paradisi x Citrus reticulata]). Pollen grains were extracted from the flowers of these two varieties and subsequently placed in Petri dishes with modified BK (boron and potassium) germination medium with six concentrations of the sulfur-based products (0.2, 2, 20, 200, 2,000, 20,000 mg l-1). All the dishes were incubated and the pollen germination rate was calculated. All the sulfur products showed progressive pollen germination inhibition with a rising sulfur concentration. CTC50 (50% cytotoxicity inhibition) was around 20 mg l-1, with significant differences among treatments. Total pollen germination inhibition took place at 20,000 mg l-1. These results demonstrate that sulfur application can affect citrus pollination.