Acetone Precipitation Research Articles

Size-controlled interfacial crystallization of A-type resistant starch microparticles via acetone precipitation

Short-chain glucan (SCG) recrystallization has emerged as an effective approach for producing resistant starch microparticle (RSP), which hold promise in both food science and clinical nutrition. This study introduces a novel method utilizing Tween 80 (TW80) to mediate the interfacial recrystallization of SCG into A-type crystalline RSP (ARSP) through acetone precipitation. TW80 plays a crucial role by stabilizing water-in-oil microemulsion droplets within acetone-oil mixtures, facilitating the formation of uniform and spherical ARSP ranging in size from 0.2 μm to 2 μm. The surface characteristics of ARSP, including porosity and smoothness, are notably influenced by SCG concentration. As SCG concentration increases, the melting temperature of ARSP rises from 117 °C to 145 °C, accompanied by a decrease in average surface area from 12.07 to 8.35 m2/g, alongside an increase in crystallinity. Moreover, the in vitro digestion test revealed that the digestion rate of ARSP was decreased from 0.73 h−1 to 0.39 h−1 with increasing SCG concentration, while increasing the resistant starch (RS) content in ARSP from 48.6% to 70.5%. This enhancement offers a strategic approach for modulating the digestion rate of ARSP, presenting opportunities for tailored release profiles and improved stability in food science applications. These findings underscore the potential of ARSP as a functional ingredient with significant implications for nutritional and clinical settings.

Mesenchymal stem cell-derived protein extract induces periodontal regeneration: MSC-protein induces periodontal regeneration

BackgroundPeriodontal disease is characterized by chronic inflammation and destruction of supporting periodontal tissues, ultimately leading to tooth loss. In recent years, “cell-free treatment” without stem cell transplantation has attracted considerable attention for tissue regeneration. This study investigated the effects of extracts of mesenchymal stem cells (MSC-extract) and their protein components (MSC-protein) on the proliferation and migration of periodontal ligament (PDL) cells and whether MSC-protein can induce periodontal regeneration. MethodsMSC-extract and MSC-protein were obtained by subjecting mesenchymal stem cells (MSCs) to freeze–thaw cycles and acetone precipitation. Cell proliferation was examined using a WST-8 assay and Ki67 immunostaining, and cell migration was examined using Boyden chambers. The MSC-protein content was analyzed using liquid chromatography-mass spectrometry, protein arrays, and enzyme-linked immunosorbent assays (ELISAs). Gene expression in MSC-protein-treated PDL cells was examined using RNA-sequencing and Gene Ontology analyses. The regenerative potential of MSC-protein was examined using micro-computer tomography (CT) and histological analyses after transplantation into a rat periodontal defect model. ResultsMSC-extract and MSC-protein promoted the proliferation and migration of PDL cells. Protein array and ELISA revealed that MSC-protein contained high concentrations of basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF). Exogenous bFGF promoted the proliferation and migration of PDL cells. Furthermore, the transplantation of MSC-protein enhanced periodontal tissue regeneration with the formation of new alveolar bone and PDLs. ConclusionsThese results indicate that the MSC-protein promotes the proliferation and migration of PDL cells and induces significant periodontal tissue regeneration, suggesting that the MSC-protein could be used as a new cell-free treatment for periodontal disease.

Mass spectrometry-based proteomics for source-level attribution after DNA extraction

Biological traces recovered from crime scenes serve as vital evidence in forensic investigations. While DNA evidence is frequently used to address the sub-source level of the hierarchy of propositions, the biological source of the DNA can be highly probative at the source level. Current body fluid detection methods pose certain limitations, such as reports of false positive results from some of the presumptive and/or confirmatory tests in current use. These tests are also individual tests for the detection of one body fluid, meaning that if the sample is suspected to be a mixture of multiple body fluids, then different tests would need to be conducted to confirm the body fluid(s) present, which may exhaust small amounts of available biological trace. Proteomics applications for the identification of body fluids have been previously explored, and potential biomarkers indicative of body fluids discovered from liquid-chromatography tandem mass spectrometry (LC-MS/MS) methods have been reported. This work focuses on developing a mass spectrometry-based proteomics approach for the identification of body fluids by targeting discriminating peptide biomarkers from the non-DNA component left over after DNA extraction of samples. The non-DNA component is typically a waste product but with unappreciated evidential value. Our methodology for the purification of proteins from the post-DNA extraction waste includes an acetone precipitation and single-pot solid-phase-enhanced sample preparation (SP3) technique, microwave-assisted trypsin digestion, and LC-MS/MS analysis of the resultant peptides. Preliminary results from this proof-of-concept study include a list of potentially discriminating proteins and peptides for blood, saliva, and semen developed from the analysis of post-DNA extraction waste. Our method allows for multiple analytes to be targeted simultaneously from a DNA profiling waste stream and we anticipate that it could eventually be incorporated into standard forensic laboratory workflows.

Recombinant laccase biosynthesis for efficient polydopamine coating

Laccases are versatile biocatalysts with interest for various industrial applications. This study reports the expression of Trametes versicolor laccase in Komagataella phaffii X33. The cultivation parameters (methanol and CuSO4 concentration, and temperature) for recombinant laccase production were studied in an orbital shaker. Enhanced laccase production was achieved by adding 1 % (v/v) methanol daily, supplementing 0.1 mM CuSO4 and incubating at 25 °C. Under these conditions, laccase production was scaled-up in a 4 L stirred tank bioreactor. Subsequently, laccase was concentrated and purified using a combined protocol of ultrafiltration and acetone precipitation, achieving a purification factor of 3.02. The laccase produced exhibited robust stability within a pH range from 4.0 to 8.0 and thermal stability up to 30 °C. Michaelis Menten kinetic revealed Michaelis constant (KM) and maximum rate of reaction (Vmax) values of 44.5 µM and 110.9 µM/min, respectively. Finally, laccase was employed as a biocatalyst to assist the polymerization of dopamine to polydopamine, allowing the one-step coating of cellulose filter paper, as confirmed by diffuse reflectance spectroscopy (UV-Vis DRS) and scanning electron microscopy (SEM). This work represents an advance in the field of laccase production in both orbital shaker and bioreactor, while demonstrating, for the first time, the laccase-assisted polymerization of dopamine for the coating of filter paper with polydopamine.

ZASP: A Highly Compatible and Sensitive ZnCl2 Precipitation-Assisted Sample Preparation Method for Proteomic Analysis

Universal sample preparation for proteomic analysis that enables unbiased protein manipulation, flexible reagent use, and low protein loss is required to ensure the highest sensitivity of downstream liquid chromatography-mass spectrometry (LC-MS) analysis. To address these needs, we developed a ZnCl2 precipitation-assisted sample preparation method (ZASP) that depletes harsh detergents and impurities in protein solutions prior to trypsin digestion via 10 min of ZnCl2 and methanol-induced protein precipitation at room temperature (RT). ZASP can remove trypsin digestion and LC-MS incompatible detergents such as SDS, Triton X-100, and urea at high concentrations in solution and unbiasedly recover proteins independent of the amount of protein input. We demonstrated the sensitivity and reproducibility of ZASP in an analysis of samples with 1 μg to 1000 μg of proteins. Compared to commonly used sample preparation methods such as SDC-based in-solution digestion, acetone precipitation, FASP, and SP3, ZASP has proven to be an efficient approach. Here, we present ZASP, a practical, robust, and cost-effective proteomic sample preparation method that can be applied to profile different types of samples.

Biophysical Properties of Thermostable Amidase Produced by Aspergillus fumigatus in Submerged Fermentation

Amidases, also known as amidohydrolases (EC 3.5.1.4), are enzymes within the nitrilase superfamily or amidase signature family. They hydrolyze amides and nitriles into their corresponding acids while releasing ammonia. These enzymes are widely used in biotechnology and industry. However, their production is limited to a few organisms, which cannot meet industrial demand. Therefore, exploring new amidase sources is crucial. This study focuses on purifying and characterizing amidase from Aspergillus fumigatus using cold acetone precipitation and column chromatography with Sephadex G-100. The effects of temperature, pH and metal ions, on the enzyme activity were examined for both crude and purified amidase. The thermal and pH stability of the enzyme alongside the enzyme kinetics were also assessed. The purified amidase exhibited optimal activity of 13.1 U/mL at 70°C, with 65% residual activity after 5 hours at this temperature. The pH studies showed optimal activity at pH 5.0 for the purified enzyme (7.8 U/mL) and at pH 4.0 for the crude enzyme (5.5 U/mL). Metal ion effects indicated that Zn²⁺, Mg²⁺, and EDTA enhanced, while Cu²⁺ inhibited, the activities of both crude and purified amidase. Kinetic analysis showed that the enzyme followed Michaelis-Menten kinetics, with Km and Vmax values of 0.104 mM and 1.884 U/mL for crude amidase, and 0.017 mM and 1.872 U/mL for purified amidase. These findings suggest that amidase from Aspergillus fumigatus holds potential for industrial applications where amidases are essential.

Physicochemical Properties, Functionalities, and Antioxidant Activity of Protein Extracts from New Zealand Wild Sea Cucumbers (Australostichopus mollis).

This study investigated the physicochemical properties, functionalities, and antioxidant capacities of protein extracts from wild sea cucumber Australostichopus mollis collected from four distinct locations in New Zealand. Protein was extracted from sea cucumber body walls using trypsin enzymatic extraction, followed by cold acetone precipitation. The amino acid analysis revealed high glycine (189.08 mg/g), glutamic acid (119.45 mg/g), and aspartic acid (91.91 mg/g) concentrations in all samples. The essential amino acid indexes of the protein extracts (62.96, average) were higher than the WHO/FAO standard references, indicating the excellent protein quality of A. mollis. Furthermore, protein extracts from A. mollis demonstrated superior emulsifying activity (202.3-349.5 m2/g average) compared to commercial soy and whey protein isolates under all tested pH conditions, and enhanced foaming capacity (109.9-126.4%) and stability (52.7-72%) in neutral and acidic conditions. The extracts also exhibited good solubility, exceeding 70% across pH 3-11. Antioxidant capacities (ABTS and DPPH free radical scavenging activity and ferric reducing antioxidant power) were identified in A. mollis protein extracts for the first time, with clear variations observed among different locations. These findings elucidate the advantageous functional properties of protein extracts from wild New Zealand A. mollis and highlight their potential application as high-quality antioxidant food ingredients.

Production and partial purification of an innovative heat resistant α-keratinase with some remarkable medical and industrial applications

Background and objectives Keratinase has ultimate practical importance in industry, medicine, food industry and waste management fields. Their applications in wool and silk are as good cleaners and in leather industry as the best ever green dehairing agents providing high leather quality, as well they are used as crucial components of sophisticated detergents. Their medical prospective applications are in prion degradation and human callus removal. They convert keratinaceous wastes to valuable products saving the environment from hard keratin waste pollution. Bacteria, fungi and actinomycetes are effective keratinase producers and they are considered the most suitable sources. This study aims to formulate the production medium and to pinpoint the proper physiological conditions for the potent microorganism producing an efficient α-keratinase enzyme. The partial purification of the crude enzyme was successfully performed. The effect of the reaction temperature on both the crude and the partially purified enzyme (PPE) was duly studied with the thermostability of PPE. Some important applications have been implemented on PPE and these include leather dehairing, cloth stain removal, and topical treatment of human callus. Materials and Methods Thirteen recommended microbial strains were screened for effective and applicable α-keratinase productivity. Optimization of the cultural conditions for extracellular enzyme production and also the partial purification of the crude enzyme by ammonium sulphate salting out or by ethanol or acetone precipitation were carried out. The effect of reaction temperature on the enzyme and its thermostability were studied. Finally, the efficiency of the PPE on leather dehairing, stain removal, and human callus treatment was explored. Results and conclusion Among the 13 organisms screened, the fungal strain Trichoderma polysporum HZ-31 was the most potent producer of an influencial α-keratinase. The maximum α-keratinase activity of 58.2 UmL−1 was obtained by the previous-mentioned strain after 5-days fermentation medium containing (%, w/v): whole chicken feathers 0.5, glucose 0.2, peptone 0.5, yeast extract 0.5, K2HPO4 0.1, KH2PO4 0.3, CaCl2 0.1, MgSO4 0.1, and pH 7.0. Acetone fractionation of the crude keratinase was the most proper and offered the most promising keratinase fraction PPE at 80–90% acetone. This fraction had high thermostability and was kept at 55°C for more than 98% of its original activity after 60 min heating and this temperature (55°C) was also the optimum for 2 h enzymatic reaction. Conclusively, the present study succeeded in the achievement of a constitutive extracellular alkaline α-keratinase, which successfully proceeded to complete leather unhairing after 12–16 h at 37°C, afforded high performance to cloth blood stain removal with Arial detergent after 2 h at 50°C and complete degradation of the human callus after 4 h at 50°C.

Hierarchical mesoporous TiO2/starch-based microparticles used as an efficient and reusable adsorbent for removal of water-soluble dye

The widespread use of organic dyes in various industrial applications, driven by rapid industrialization, has become a significant environmental concern. Thus, highly efficient and reusable adsorbent for removal of pollutant dyes have gained increasing attention in water treatment. In this study, we present TiO2 nanoparticle-embedded mesoporous starch-based microparticle (TiO2@MSMP) with hierarchical rose-like structure were synthesis by using acetone precipitation of short-chain glucan (SCG) obtained from waxy maize starch. The resulting TiO2@MSMP exhibits an A-type crystalline polymorph and mean particle size of approximately 2 μm, displaying a type IV adsorption isotherm with a mean pore diameter of 19 nm and an average surface area of 12.34 m2/g. The adsorption ability of TiO2@MSMP towards methyl orange (MO) and crystal violet (CV) were 85.8 mg/g and 103.8 mg/g, respectively. The reusability of TiO2@MSMP was achieved by UV irradiation, which resulted in photodegradation of the adsorbed dye over 80 % while maintaining good absorption ability and structural stability during the recycling process. Given its cost-effectiveness, high adsorption capacity, and excellent reusability, TiO2@MSMP holds promise as an effective and environmentally friendly adsorbent with significant potential for removing dyes from aqueous solutions and purifying water.

Extraction and Purification of Squalene from Virgin Olive Oil via Catalytic Transesterification and Molecular Distillation

Squalene, a highly valuable compound abundant in various natural sources, shows great potential in pharmaceutical, cosmetic, and nutraceutical applications. This research article outlines a comprehensive methodology for extracting and purifying squalene from virgin olive oil, a rich source of the compound. The extraction process begins with degumming, which involves heating the olive oil to 60-70°C to reduce viscosity, followed by the addition of 2-3% warm water to hydrate phospholipids. Food-grade phosphoric acid is then added to react with the phospholipids, forming precipitates. The mixture is stirred for 20-30 minutes and allowed to rest for an additional 20-30 minutes, enabling impurities to settle. The upper layer of degummed oil is separated via decantation or centrifugation and washed with warm water for pH adjustment. Next, transesterification is performed by mixing 100 ml of virgin olive oil with 25% methanol (w/w) and a catalyst (0.5% sodium methoxide or PTSA), and heating the mixture to 80-90°C under reflux for 1-2 hours. Following transesterification, the solvent and acetone are distilled out, and acetone precipitation is repeated 2-3 times to remove unsaponified matter, which is then filtered and evaporated. The concentrated oil undergoes molecular distillation at 180°C and 0.0033 bar pressure for 1 hour, yielding the distillate and residue for further analysis. Qualitative analysis using Thin Layer Chromatography (TLC) involves Merck TLC plates with silica gel 60 F254 and a hexane: chloroform (9:1) mobile phase. Spots are developed with a 10% HCl solution, confirming the presence of squalene with an RF value of 0.93. Quantitative analysis via High-Performance Thin-Layer Chromatography (HPTLC) employs Merck TLC plates with cyclohexane as the mobile phase and CAMAG at 254 nm and 366 nm wavelengths, revealing a squalene purity of 67% and a recovery rate of 69.8%. The initial purification through transesterification facilitated the conversion of ester groups, yielding squalene-rich fractions, while acetone precipitation effectively removed saponified matter. Molecular distillation further enhanced squalene purity. TLC analysis confirmed the qualitative presence of squalene, and HPTLC provided precise quantitative measurements. The obtained squalene purity of 67% significantly enriches the initial content in virgin olive oil, though further optimization could enhance purity and yield. Complementary techniques like GC-MS or HPLC could validate the purification process. This study presents an efficient, replicable procedure for extracting and purifying squalene from virgin olive oil, with significant implications for pharmaceutical, cosmetic, and nutraceutical industries. The findings support a sustainable and ethical shift towards vegetable-derived squalene, meeting market demands while ensuring high-quality production.

Comparison of Extraction Assays and Quantification of Protein from Ulva anandii (Cholorophycota)

Seaweeds contain many macronutrients including protein, therefore they can be utilized to fulfil the protein requirements of human beings. This research focused on extracting total protein in green seaweed Ulva anandii (Amjad et Shameel 1993), from the crude extracts, by using the trichloroacetic acid (TCA) and acetone precipitation methods, and the estimation of crude extract (water-soluble proteins), and those obtained from the two above-mentioned methods. The results indicate that the water-soluble proteins had the highest quantity (949.75µg/mL) followed by the TCA precipitation method (831µg/mL), while the acetone precipitation method had the least concentration of total protein (100 µg/mL). The study concludes that treatment with organic solvents lowers the quantity of protein extracted from U. anandii.

Evaluation of the Emulsifying Property of Detarium Microcarpum Gum in Ibuprofen Emulsion Formulations

An emulsion is a mixture of two or more immiscible liquids that adopt a macroscopic homogeneous aspect and a microscopic heterogeneous one under specific transforming processes. In an emulsion, one liquid is dispersed in the other and an emulsifying agent is usually added to stabilize it i.e., to prevent the immiscible layers of the mixtures from separating. In this study, the emulsifying property of Detarium microcarpum gum (DMG) in ibuprofen emulsion formulations was evaluated. DMG was obtained from the seeds of the plant through acetone precipitation of the filtrate obtained from distilled water maceration of powdered Detarium microcarpum seeds. Six Ibuprofen emulsions were formed using tragacanth, DMG or a combination the two as the emulsifying agent. Some of the formulations contained methyl paraben, propyl paraben, vanilla and tartrazine. Physicochemical properties such as pH, viscosity and the type of the emulsions, were evaluated. The pH of the emulsions ranged from 4.5 to 4.8 on the first day and 4.0 to 5.6 on day 15. The viscosity at 28oC ranged from 1200 to 5400 mPas on day 0 and 1370 to 5400 mPas on day 15. The emulsions were of the oil-in-water type and they exhibit shear thinning behaviour. DMG was used to prepare ibuprofen emulsions with good physicochemical properties that were comparable to those produced using tragacanth as the emulsifying agent

Ocular surface immune transcriptome and tear cytokines in corneal infection patients.

Microbial keratitis is one of the leading causes of blindness globally. An overactive immune response during an infection can exacerbate damage, causing corneal opacities and vision loss. This study aimed to identify the differentially expressed genes between corneal infection patients and healthy volunteers within the cornea and conjunctiva and elucidate the contributing pathways to these conditions' pathogenesis. Moreover, it compared the corneal and conjunctival transcriptomes in corneal-infected patients to cytokine levels in tears. Corneal and conjunctival swabs were collected from seven corneal infection patients and three healthy controls under topical anesthesia. RNA from seven corneal infection patients and three healthy volunteers were analyzed by RNA sequencing (RNA-Seq). Tear proteins were extracted from Schirmer strips via acetone precipitation from 38 cases of corneal infection and 14 healthy controls. The cytokines and chemokines IL-1β, IL-6, CXCL8 (IL-8), CX3CL1, IL-10, IL-12 (p70), IL-17A, and IL-23 were measured using an antibody bead assay. A total of 512 genes were found to be differentially expressed in infected corneas compared to healthy corneas, with 508 being upregulated and four downregulated (fold-change (FC) <-2 or > 2 and adjusted p <0.01). For the conjunctiva, 477 were upregulated, and 3 were downregulated (FC <-3 or ≥ 3 and adjusted p <0.01). There was a significant overlap in cornea and conjunctiva gene expression in patients with corneal infections. The genes were predominantly associated with immune response, regulation of angiogenesis, and apoptotic signaling pathways. The most highly upregulated gene was CXCL8 (which codes for IL-8 protein). In patients with corneal infections, the concentration of IL-8 protein in tears was relatively higher in patients compared to healthy controls but did not show statistical significance. During corneal infection, many genes were upregulated, with most of them being associated with immune response, regulation of angiogenesis, and apoptotic signaling. The findings may facilitate the development of treatments for corneal infections that can dampen specific aspects of the immune response to reduce scarring and preserve sight.

ACETONE PRECIPITATION OF PROTEINS FOR DETERMINATION OF THE OF ENZYMES ACTIVITY IN MEDICINAL PLANTS

Plant phenolic compounds are widely used in medicine and agriculture. The study of the mechanisms of their biosynthesis and accumulation is a complex task. It is also true for the determination of the activity of the key plant enzymes such as shikimate dehydrogenase. At the same time, the isolation of proteins and enzymes from plant is complicated by the presence of phenolic compounds and pigments. The aim of the research was to evaluate the feasibility of the acetone precipitation of proteins of leaf extracts of plants to determine the activity of shikimate dehydrogenase. The object of the study was the leaves of sea buckthorn (H. rhamnoides) and silky dogwood (C. sericea). The proteins were precipitated from the crude extract with cold (-20 °C) acetone in varying ratio followed by washing three times with acetone and final dissolution in buffer. The activity of shikimate dehydrogenase was determined at pH=10.0 and expressed in ncat per mg of protein. Protein fractions with different activity of shikimate dehydrogenase were isolated from the leaves of sea buckthorn (H. rhamnoides) and silky dogwood (C. sericea). It was shown that the protein fraction with a ratio of 1 : 1.5 of extract : acetone (60% saturation) is most convenient to work with, has a high enzyme activity and does not contain colored impurities. This method of protein isolation and pre-purification is fast, requires a small amount of sample, and suitable for screening studies of the activity of various enzymes in medicinal plants.

Extraction of Apoplastic Peptides for the Structural Elucidation of Mature Peptide Hormones in Arabidopsis.

Various secreted peptides, including peptide hormones, are present in the apoplast, but their biochemical characterization remains a challenge due to their low abundance, difficulty in extraction, and interference from numerous secondary metabolites. Here, we describe a simple and straightforward protocol for the extraction of apoplastic peptides with a high purity. This protocol takes advantage of the fact that apoplastic peptides diffuse and accumulate in the culture medium when Arabidopsis seedlings are subjected to whole-plant submerged culture. The peptides in the culture medium are efficiently recovered by o-chlorophenol extraction followed by acetone precipitation. The recovered peptides can be subjected to nano-liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) without any additional clean-up. This procedure enables the structural elucidation of mature peptide hormones in the apoplast with the use of Arabidopsis plants that overexpress peptide hormone genes.

Rhizopus oligosporus alkaline protease in cassava fermentation: Characterization and detergent potential

The study addresses the investigation of a novel alkaline protease produced by a fungus involved in the fermentation of cassava and its potential application as a detergent additive. This study aims to investigate the potential of a newly discovered alkaline protease, Rhizopus oligosporus alkaline protease (ROAP), produced during cassava fermentation, as a detergent additive. The objective is to understand its characteristics, stability, and stain-removing capabilities. ROAP was partially purified using acetone precipitation. The study extensively assessed its optimal conditions, including temperature and pH, and explored the impact of inorganic salts, chemical agents, and surfactants on enzyme activity. Kinetic parameters (Vmax, Km, Kcat) were determined, along with thermal stability indicators. Cloth washing experiments using stained white cotton fabrics were conducted to evaluate stain removal efficiency. After purification, ROAP exhibited an impressive increase in specific activity. Optimal conditions for its activity were identified at 40 °C and pH 10.0. The enzyme showed varying responses to different salts, some of which did not affect its activity while others influenced it. Most notably, ROAP proved highly effective in removing stains, achieving a 94% removal rate for grease stains when used alone and an 86% efficiency when combined with Rin. This study underscores the potential of ROAP as a valuable detergent additive, especially for tackling challenging protein-based stains like blood and grease. The enzyme's stability, efficiency, and versatility in various conditions hold promise for its application in industries requiring effective stain removal, contributing to improved cleaning processes.

Antitumor activity of urease-free l-asparaginase with low glutaminase coactivity produced by marine-derived Aspergillus flavus

l-asparaginase, which has a high therapeutic effect in various cancer diseases, hydrolyzes l-asparagine to aspartate and ammonia and is mainly used in treating Acute Lymphoblastic Leukaemia. This study aims to investigate the l-asparaginase production potential of marine filamentous fungi and the partial purification and characterization of the l-asparaginase. The rapid plate screening method was used to measure the l-asparaginase activity of 94 marine fungi. l-asparaginase producers were then screened for glutaminase and urease activity. The selected extracellular l-asparaginase producer, Aspergillus flavus MF10, showed 2.82 U/mL l-asparaginase activity in small-scale fermentation. l-asparaginase was concentrated by ultrafiltration followed by acetone precipitation. The specific activity of the enzyme, purified 4,8 fold by partial purification, was determined as 259 U/mg. There were no urease and glutaminase activities in the fermentation broth, but low glutaminase activity (1.27 U/mL) was detected in the concentrated fermentation broth. The optimum pH and temperature of the l-asparaginase were 8.0 and 40 °C, respectively. l-asparaginase was stable at pH 7.0–8.0 and can maintain its activity at 70% at 40 °C after 1 h. While the enzyme decreased the viability of SH-SY5Y (neuroblastoma) to 43% and LNCap (prostate cancer) to 61%, it did not show cytotoxic effects in healthy cell line L929 (fibroblast). It was also determined that l-asparaginase showed low cytotoxicity in HaCaT (keratinocyte) cells. In conclusion, it was shown that urease-free l-asparaginase with low glutaminase coactivity, produced from marine-derived A. flavus MF10, has the potential for medical use.

Effect of Glycolipoprotein Biosurfactant from Enterococcus faecium on the viability of Breast cancer cell lines.

Objective : This study , was research for cytotoxicity of bio-surfactant created by Enterococcus faecium isolated from feces of Iraqi healthy breast-fed infants with age &lt; 6 months.&#x0D; Methods : Cold acetone precipitation was used to the extraction of extracellular Glycolipoprotein biosurfactant and partially purify it. Biosurfactant was then evaluated against two the cell lines, a Breast cancer MCF-7 cell line and a human normal fibroblast cell line NHF), specifically for cell survival and proliferation. &#x0D; Results : At all concentrations with varying percentage, The viability of the MCF-7 cancer cell line was shown to be reduced with the addition of biosurfactant.; maximum inhibition percentage was 74.2% at a 100 µg/ml concentration, which is lesser than 45.5% cytotoxicity Of NHF healthy fibroblasts cell line.&#x0D; Conclusions : The findings of this study are highly encouraging in terms of the potential of Glycolipoprotein biosurfactants to treat cancer and encourage additional research with different cell lines.

Purification of fibrinolytic enzyme from Bacillus amyloliquefaciens GUTU06 and properties of the enzyme

A producing-fibrinolytic enzyme strain was isolated with high yield. The strain was identified as Bacillus amyloliquefaciens. B. amyloliquefaciens GUTU06 fibrinolytic enzyme was purified by acetone precipitation and reverse micelle. Acetone precipitation condition and reverse micelle condition were examined. Results showed that the total reverse micelle extraction efficiency was 64.49 % ± 1.6 %. The purification fold of the entire process reached 13.38. The optimum pH of purified enzyme is 5, and the optimum temperature is 45 °C. Fe3+ and K+ can enhance the fibrinolytic activity of the enzyme. Compared to commercial fibrinolytic enzymes such as urokinase and lumbrukinase, GUTU06 fibrinolytic enzymes have a lower pH optimal range and higher temperature stability. The molecular weight of the enzyme was approximately 28 kDa. Reverse micelle extraction with cetyl trimethylammonium bromide as a surfactant combined with acetone precipitation is suitable for separating and purifying fibrinolytic enzymes and a promising technique for obtaining active proteins.

Biological activities of purified prolidase from pathogenic E.coli

Prolidase is a ubiquitous enzyme that plays a chief role in the metabolism of proline-rich proteins. The goal of this study extraction and purify prolidase from E. coli and evaluate some applications such as anti-biofilm and anticancer. Hundred stool Samples were collected from infants with breastfeeding, non-vomiting, and non-diarrhea to isolate E.coli bacteria. A 16S rRNA gene (585 bp) was found in all isolates of E. coli via PCR identification. Depending on the qualitative method on prolidase agar, only 40 (80%) isolates could produce prolidase from 50 isolates that were considered non-pathogens, then only 32 isolates revealed different levels in prolidase production with specific activity equal to (2.1U/mg) of E.coli. MS12. Sucrose, casein, and 40Co were the chosen isolate's best conditions for producing prolidase. Cold acetone precipitation and dialysis were used to extract the enzyme, and DEAE-cellulose and the Sephadex G-150 column were used in purification with specific activity (2 U/ml) and (6.6 U/mg) protein. Prolidase showed the highest effect on biofilm at 500 μg/ml concentration against P. aeruginosa, then E. coli, 65% and 60.3% respectively. Brain Tumor Cell Line (A127), Colorectal Adenocarcinoma cells (CaCo-2), and Normal embryonic liver cell line (WRL-68) were used to test the prolidase effect on these cell lines. An assay of MTT was used to detect the inhibiter concentration (IC50) values and cytotoxic effect of purified prolidase. Keywords: E.coli, Prolidase, purification, antibiofilm, anticancer activity