Ammonium Sulfate Fractionation Research Articles

Transglutaminase from Asian seabass liver: purification, characterization and cross-linking activity against natural actomyosin

The study investigated the purification and characterization of transglutaminase (TGase) from Asian seabass liver. In addition, protein cross-linking ability of partially purified TGase was also elucidated against the natural actomyosin extracted from threadfin bream surimi. During ammonium sulfate fractionation, the highest specific activity (11.31 ± 0.17 units/mg protein) was obtained from the 80–100% ammonium sulfate (AS) fraction (partially purified TGase; pTGase). Purity increased 1.46 times compared to crude samples with a 73% yield. Further purification using DEAE-Sepharose enhanced the specific activity (904.71 ± 4.65units/mg protein) by 117.19-fold as compared to the crude sample. The molecular weight (MW) of the purified TGase was 43.4 kDa, which was slightly different than the MW determined by SDS-PAGE (41.1 kDa). Optimal TGase activity was found at 45–50 °C and pH 8, with significant declines beyond these ranges. Furthermore, TGase maintained activity (80%) when heated till 55 °C, whereas it showed wide-range pH stability. NaCl and CaCl2 at 3% and 20 mM, respectively enhanced TGase activity. When natural actomyosin (NAM) was treated with pTGase at various concentrations (1–10 unit/mg protein), decreasing solubility and ɛ-amino groups confirmed its cross-linking ability. The result was also supported by the formation of aggregates of NAM proteins as determined by transmission electron microscopic images. Thus, Asian seabass liver could be a new source of purified and partially purified TGase, which can be employed for various food and pharmaceutical applications.

Characteristics of lectin receptors on erythrocyte membranes of patients with glioblastoma

Aim. This work is devoted to the search the abnormalities of the receptor-lectin reactions associated with glioblastoma for diagnostic purposes. Methods. Red blood cell (RBC) specimens were taken from 22 persons distributed into the two cohorts: donors without an oncological diagnosis; patients with glioblastoma (GB). Panel of 9 commercial lectins (LECTINOTEST, Lviv, Ukraine) was used: ConA, WGA, PHA-P; PHA-E; PHA-L, SNA, VAA, STA, and LABA. Also the original lectin from persimmon sepals (named PSL) was used. It was obtained by isoelectric focusing and ammonium sulfate fractionation. Intensity of lectin-receptor interactions was determined in reaction of hemagglutination. Results. Тwo lectins showed some directed divergence in patients from donors. For wheat germ agglutinin (WGA) сhanges in the intensity of lectin-receptor binding were downward. In the case of persimmon L (PSL) the alternative result was observed: the intensity of hemagglutination showed a trend to exceed the level of control. Conclusions. Some individual features in the reaction of erythrocytes with a set of lectins are observed in all examined persons. For two studied lectins, namely WGA and PSL, unidirectional deviations were observed in the reaction of erythrocytes of patients with GB compared to individuals without an oncological diagnosis.

Characterization of a Halostable Metalloprotease from the Halophilic Bacterium Bacillus clausii J1G-0%B

Protein plays a crucial role as a biocatalyst in various industries, particularly in breaking down proteins into amino acids. The demand for proteases capable of functioning under extreme conditions, such as high salinity, temperature, and pH, is increasing. To address this, the exploration of bacteria that produce stable proteases in such environments is essential. Bacillus clausii J1G-0%B, a halophilic bacterium isolated from Madura salt ponds, thrives in salinity levels of 0-20% NaCl. This study aims to obtain and characterize the protease produced by Bacillus clausii J1G-0%B, focusing on its activity and stability under extreme conditions. The research involved screening, production, and purification of the protease using ammonium sulfate fractionation and dialysis. Protease activity was measured using the Kunitz method, and protein content was determined using the Lowry method. Characterization included optimizing enzymatic conditions (pH, temperature, NaCl concentration), identifying metalloprotease types, and analyzing enzyme kinetics and thermodynamics. The study successfully produced protease using a halophilic medium with casein and 5% NaCl. After 96 hours of incubation, the protease exhibited a specific activity of 654.737 U/mg. Optimal activity was observed at pH 7, 50°C, and 10% NaCl, with stability between 2.5% and 15% NaCl concentration. Enzyme kinetics revealed a high affinity for casein, with a KM value of 0.164 mg/mL and Vmax of 13.182 µmol/mL·min. Thermodynamic analysis indicated high stability, as shown by a positive ΔGi value (+105.84 kJ/mol), a low inactivation constant (ki = 0.0031 min-1), and a long half-life (t½ = 223.548 minutes). EDTA chelation tests confirmed that the protease is a metalloprotease. The halostable protease from Bacillus clausii J1G-0%B shows significant potential for industrial applications and bioremediation in high-salinity environments.

Isolation, purification, and characterization of lectins from medicinal plant Combretum glutinosum seeds endowed with analgesic and antiulcer properties

In the pursuit of safer and more effective treatments, there is a growing interest in plant-derived compounds, particularly lectins, because of their diverse pharmacological properties. This study focused on isolating, purifying, and characterizing lectin from Combretum glutinosum seeds (CGSLs) to assess its potential as an analgesic and antiulcer agent. CGSL extraction involved defatting and buffer extraction, followed by purification using ammonium sulfate fractionation and fetuin-agarose affinity column chromatography. The isolectins (iso-CGSLs), each consisting of 60 kDa and 57 kDa heterodimeric subunits, displayed glycoprotein properties with a 40 % neutral sugar content. They exhibited peak activity at 55 °C and remained stable for up to the fifth day at room temperature. The activity exhibited a pH dependence, peaking between 7.5 and 10.5, and all seemingly operated independently of metal ions. CGSL, at optimal doses ranging from 6 to 12 mg/kg, had significant analgesic effects on acetic acid-induced writhing and hot plate tests in mice. Evaluation using 0.7 % acetic acid resulted in notable pain reduction across all doses (P < 0.05). The analgesic effect of lectin was partially reversed by naloxone (a morphine antagonist), indicating partial involvement of the opioid receptor system. Furthermore, CGSL exhibited antiulcer effects in ethanol-induced gastric ulcer models in rats, highlighting its therapeutic potential as a natural alternative for analgesic and antiulcer treatments.

Determination of the molecular weight of native molecules of γ-hydroxybutyrate dehydrogenase isoforms obtained from maize (Zea mays L.) seedlings by gel chromatography

It is known that under oxygen deficiency the functioning of the tricarboxylic acid cycle is impaired, resulting in the activation of an alternative pathway, the GABA shunt. It maintains the functioning of the citric acid cycle by providing succinic acid. The key phase of this bypass is a reaction catalysed by succinate semialdehyde dehydrogenase (SSADH, EC 1.1.1.16). Under oxygen deficiency, SSADH ceases to function efficiently. This leads to the accumulation of succinic acid semialdehyde in the mitochondrial matrix and its high level adversely affects the plant cell metabolism. γ–hydroxybutyrate dehydrogenase (HBDH, EC 1.1.1.61) is an enzyme belonging to the group of oxidoreductases. It transforms γ–hydroxybutyrate into succinic acid semialdehyde, participating in the process of its detoxification, which is important in the maintenance of plant metabolism under oxygen deficiency. To date, unfortunately, there are no data on the biochemical and kinetic features of γ–hydroxybutyrate dehydrogenase. In this regard, our laboratory developed a method for purification of HBDH from green maize leaves, which makes it possible to study the physicochemical properties of this enzyme. During the study, we obtained γ-hydroxybutyrate dehydrogenase from 7-day-old seedlings of Zea mays L. by a five-stage purification. Homogenised plant material with extracted proteins was subjected to two-stage ammonium sulphate fractionation. The catalytic activity was determined spectrophotometrically at λ=340 nm by the amount of reduced NAD+. To remove ammonium salts, we used gel filtration through Sephadex G-25. Proteins were separated according to their charge by DEAE-Sephacel ion exchange chromatography. The enzyme was desorbed using a linear sodium chloride gradient (100-300 mM). The use of gel chromatography through Sephadex G-200 made it possible to determine the molecular mass of the purified enzyme isoforms. The homogeneity of enzyme preparations was confirmed by electrophoresis in polyacrylamide gel with universal AgNO3 staining. We used the tetrazolium method to confirm that the obtained protein preparations were γ-hydroxybutyrate dehydrogenase. As a result, homogeneous preparations of two isoforms of the enzyme (HBDH1 and HBDH2) were obtained. The first isoform of γ-hydroxybutyrate dehydrogenase was 185.7 times purified with a yield of 10% and had a specific activity of 343.6 U/mg of protein. The purification rate of the second isoform was 209 times with a yield of 7.74%. The specific activity of the obtained preparation was 386.7 U/mg of protein. Using gel chromatography through Sephadex G-200, we determined the molecular mass of native molecules of hydroxybutyrate dehydrogenase. We found that in 7-day-old maize seedlings the investigated enzyme was presented in low-molecular and high-molecular forms: for HBDH1 the Mr value was ⁓60.3 kDa, while for HBDH2 the molecular mass of the enzyme was 286 kDa.

Production of a new chitinase from Nocardiopsis halophila TN-X8 utilizing bio-waste from the blue swimming crab: enzyme characterization and immobilization.

In accordance with the framework of the Circular Blue Bioeconomy in the Mediterranean region, the objective of this study was to evaluate the biotransformation of blue swimming crab (Portunus segnis) residues obtained from the port of Sfax by an extracellular chitinase produced by Nocardiopsis halophila strain TN-X8 isolated from Chott El Jerid (Tozeur, Tunisia). From the analysis of multiple extremophilic Actinomycetota, it was determined that strain TN-X8 exclusively utilized 60g/L of raw blue swimming crab as its carbon and energy source, achieving a chitinase activity of approximately 950 U/mL following a 6-day incubation period at 40°C. Pure chitinase, designated as ChiA-Nh30, was obtained after heat treatment, followed by ammonium sulfate fractionation and Sephacryl® S-200 column chromatography. The maximum ChiA-Nh30 activity was observed at pH 3 and 75°C. Interestingly, compared with cyclohexamidine, ChiA-Nh30 showed a good antifungal effect against four pathogenic fungi. Furthermore, when using colloidal chitin as substrate, ChiA-Nh30 demonstrated a higher degree of catalytic efficiency than the commercially available Chitodextrinase®. In addition, ChiA-Nh30 could be immobilized by applying encapsulation and encapsulation-adsorption techniques. The kaolin and charcoal used acted as excellent binders, resulting in improved ChiA-Nh30 stability. For the immobilized ChiA-Nh30, the yield of N-acetyl-D-glucosamine monomers released from 20% (w/v) blue swimming crab residues increased by 3.1 (kaolin) and 2.65 (charcoal) times, respectively.

Purification and biochemical characterization of a novel thermostable endo-polygalacturonase from Aspergillus niger strain HO32 and its suitability for clarification of orange juice

A novel endo-polygalacturonase (PGC-AN64) from Aspergillus niger HO32 was produced (88.22 U/mL) on orange peel by-product (OPP), purified, biochemically characterized, and its utility in clarification of orange juice was elucidated. The enzyme was purified by ammonium sulfate fractionation (80 %) and gel filtration chromatography on Sephacryl® S-200 HR column. Its pH and temperature optima were 6.5 and 70 °C, respectively, and was stable over a pH range of 5–7 and temperature 60–80 °C. The 26-residue NH2-terminal sequence of PGC-AN64 showed high homology with those of Aspergillus pectinases. Metal ions (Na+, Ca2+, Mg2+, Mn2+, and Zn2+) positively affect PGC-AN64 activity, while Ba2+, Cd2+, and Cr3+ negatively affect its original activity. Interestingly, PGC-AN64 exhibited a considerable substrate specificity and catalytic efficiency compared to the commercial enzymes PECLYVE V (P1) and PECLYVE CP (P2). More interestingly and compared to P1 and P2, PGC-AN64 showed that the clarification process had a significant effect on the transmittance percentages with 84.68 ± 2.08, 88.05 ± 1.79, and 93.13 ± 3.58 % for PGC-AN64, P1, and P2, respectively. Furthermore, color parameters after the clarification process increase in the L* and b* values, and led to a decrease in the a* value. The obtained results suggest that PGC-AN64 revealed its potential in orange juice clarification.

Immobilization of alkaline protease produced by Streptomyces rochei strain NAM-19 in solid state fermentation based on medium optimization using central composite design.

This study evaluated Streptomyces rochei strain NAM-19 solid-state fermentation of agricultural wastes to produce alkaline protease. Alkaline protease production increased with flaxseed, rice bran, and cheese whey fermentation reaching 147U/mL at 48h. Statistical optimization of alkaline protease production was performed using the central composite design (CDD). Results of CDD and the optimization plot showed that 4.59g/L flaxseed, 4.31g/L rice bran, 4.17mL cheese whey, and a vegetative inoculum size of 7.0% increased alkaline protease production by 27.2% reaching 186U/mL. Using the 20-70% ammonium sulfate fractionation method, the optimally produced enzyme was partially purified to fivefold. The partially purified alkaline protease was then covalently immobilized on a biopolymer carrier, glutaraldehyde-polyethylene-imine-κ-carrageenan (GA-PEI-Carr), with 90% immobilization efficiency. Characterizations revealed that immobilization improved thermostability, reusability, optimum temperature, and sensitivity towards metal ions of the free enzyme. The optimal temperature for free and immobilized enzymes was 40 and 50°C, respectively. Both enzymes had the same optimum pH of 10. Immobilization increased Km from 19.73 to 26.52mM and Vmax from 56.7 to 62.5mmolmin-1L-1. The immobilized enzyme retained 35% of its initial activity at 70°C, while the free enzyme retained only 5%. The immobilized enzyme kept 80% of its initial activity at the 20th cycle. After 7weeks of storage, the free enzyme lost all its initial activity, whereas the immobilized enzyme retained 50%. The free and immobilized enzymes were able to hydrolyze gelatin, and azo-casein demonstrating different relative activity, 85, 80, 90 and 95%, respectively, compared to casein (100%).

Identification and enzymatic properties of arginine decarboxylase from Aspergillus oryzae.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao-ADC1 belongs includes three other Ao-ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively.

Investigation of catalytic properties of glutathione reductase obtained by chromatographic methods from the liver of rats with paracetamol-induced liver damage upon administration of 6-hydroxy-2,2,2,4-trimethyl-1,2-dihydroquinoline

The aim of the present study was to analyze some catalytic and regulatory properties of the enzyme preparation of glutathione reductase (GR, EC 1.6.4.2.) obtained from rat liver using gel filtration and ion exchange chromatography under conditions of paracetamol-induced liver injury (PILI) and administration of 6-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline (DHQ). Laboratory animals (Rattus norvegicus rats of the Wistar line) were divided into 3 groups. The animals of the control group were orally administered vaseline oil. The second group consisted of rats with PILI induced by a single oral administration of paracetamol at a dose of 1000 mg/kg body weight dissolved in 1 cm3 of vaseline oil. Animals of the third group were modeled by PILI and orally administered with DHQ at a dose of 50 mg per 1 kg body weight dissolved in 1 cm3 of 1% starch, 1 hour and 12 hours after paracetamol administration. Liver and serum were collected 24 hours after paracetamol administration. GR activity was measured on a spectrophotometer at 340 nm wavelength. The enzyme was purified from rat liver by ammonium sulfate fractionation, gel filtration through Sephadex G-25, and ion exchange chromatography on DEAE-cellulose. The total amount of protein in the samples was measured using the BCA protein assay kit. GR was obtained from the liver of rats treated with DHQ on the background of PILI with a 32-fold purification degree. Using the method of double inverse Lineweaver-Burk coordinates it was shown that administration of DHQ to rats was accompanied by a decrease in the affinity of the enzyme for NADPH, as compared to the indicators at pathology. The introduction of isocitrate into the spectrophotometric medium resulted in a less pronounced increase in the activity of GR from the liver of animals treated with DHQ on the background of PILI, compared to the second group of rats. The enzyme from the liver of animals of the third group was characterized by a more significant decrease in activity when glucose-6-phosphate was introduced into the reaction medium. Citrate had a less pronounced regulatory effect on GR from the liver of rats treated with DHQ on the background of pathology, relative to the indicators of animals with PILI. The changes in enzyme properties demonstrated may be due to conformational changes in the molecule resulting from the inhibition effect of DHQ on the intensity of free radical-induced oxidation, which develops against the background of the toxic effect of paracetamol metabolites.

Thrombolytic protease characterization from leaves and fruit flesh of the jernang rattan plant (Daemonorops draco)

Thrombolytic agents are used for thrombolytic therapy to dissolve blood clots that form in a blood vessel. All currently used thrombolytic agents have unfavorable shortcomings, such as gastrointestinal bleeding, allergic reactions, and thrombolytic agent resistance, treatment for some of which can be quite expensive. As a result, the search for thrombolytic agents derived from plants is currently taking place. Some plants have been discovered to contain protease enzymes with thrombolytic activity; pharmaceuticals derived from plants are believed to be safer. Jernang rattan (Daemonorops draco) is a plant of the Arecaceae family and is known to produce resin. Jernang rattan resin is also known to have antioxidant, antiseptic, antitumor, antimicrobial, and cytotoxic activity, but very limited information on proteolytic activity of the protease from this plant. This research aims to isolate proteases from the leaves and fruit flesh of the rattan jernang plant (D. draco) and to investigate the proteolytic activity of the isolated proteases. The protease was isolated from the leaves and the fruit flesh, and then partially purified by ammonium sulfate precipitation. The radial caseinolytic assay showed that protease in a 60% ammonium sulfate fraction gave a clear zone, with diameters of 1.4 cm and 1.8 cm for the protease isolated from leaves and fruit flesh, respectively. A Folin‐Ciocalteau assay showed that the enzymes isolated were able to hydrolyze casein and release L‐tyrosine, with activity of 0.158 U/mL and 0.174 U/mL for the protease from the leaves and fruit flesh, respectively. A fibrinogenolytic assay showed that the protease from the fruit flesh hydrolyzed the A‐α, B‐β and the γ chain of human fibrinogen, while the protease from the leaves hydrolyzed the A‐α and γ chain. Both proteases were inhibited by 56% by phenylmethylsulfonyl fluoride (PMSF), indicating that the enzymes are serine proteases. Based on the assay results obtained, it can be concluded that proteases isolated from the leaves and fruit flesh have potential as thrombolytic proteases.

Hemagglutinin from the Root Tuber of Dioscorea preussii Pax.

In this study, Dioscorea preussii root tuber hemagglutinin was purified and its physicochemical properties were determined. The antioxidative and anti-hemolytic activities of the hemagglutinin were also investigated. The hemagglutinating assay was used to detect the presence of lectin in the phosphate-buffered saline extract of the D. preussii root tuber. The lectin was purified using ammonium sulfate fractionation and molecular sieve chromatography. The optimum pH and temperature were determined. In addition, antioxidant activity was assessed using 2,2 diphenylpicrylhydrazyl (DPPH) radical scavenging, metal chelating, ferric reducing antioxidant power (FRAP), and lipid peroxidation inhibition assays. Red blood cells subjected to oxidative damage caused by H2O2 were employed to evaluate their antihemolytic ability. Starch inhibited hemagglutinin activity. Dioscorea preussii hemagglutinin (DPH) maintained full hemagglutinating activity from 30 °C to 60 °C and pH 5-13. Ethylene diamine tetraacetic acid did not affect the hemagglutinating activity of hemagglutinin. All denaturing agents (Guanidine-HCl, urea, and β-mercaptoethanol) reduced the hemagglutinating activity of the hemagglutinin to different degrees. The hemagglutinin scavenged the DPPH radical and chelated iron metal with half maximum inhibitory concentration (IC50) of 0.727 ± 0.035 mg/mL and 0.583 ± 0.078 mg/mL, respectively, while the FRAP assay showed that it contained 76 mg of ascorbic acid equivalent per gram of the purified hemagglutinin. In the absence of hemolytic agents and at lower concentration tested, hemagglutinin showed positive membrane integrity protection. This study provides information on the antioxidant properties of D. preussii root tuber hemagglutinin as well as its cell membrane protective ability. The lectin is a starch-binding, which makes it a novel lectin.

Novel Phospholipase C with High Catalytic Activity from a Bacillus stearothermophilus Strain: An Ideal Choice for the Oil Degumming Process

A novel thermoactive phosphatidylcholine-specific phospholipase C (PC-PLCBs) was identified from Bacillus stearothermophilus isolated from a soil sample from an olive oil mill. Enhanced PLCBs production was observed after 10 h of incubation at 55 °C in a culture medium containing 1 mM of Zn2+ with an 8% inoculum size and 6 g/L glucose and 4/L yeast extract as the preferred carbon energy and nitrogen sources, respectively. PLCBs was purified to homogeneity by heat treatment, ammonium sulfate fractionation, and anion exchange chromatography, resulting in a purification factor of 17.6 with 39% recovery. Interestingly, this enzyme showed a high specific activity of 8450 U/mg at pH 8–9 and 60 °C, using phosphatidylcholine PC as the substrate, in the presence of 9 mM sodium deoxycholate and 0.4 mM Zn2+. Remarkable stability at acidic and alkali pH and up to 65 °C was also observed. PLCBs displayed a substrate specificity order of phosphatidylcholine &gt; phosphatidylethanolamine &gt; phosphatidylserine &gt; sphingomyelin &gt; phosphatidylinositol &gt; cardiolipin and was classified as a PC-PLC. In contrast to phospholipases C previously isolated from Bacillus strains, this PLCBs substrate specificity was correlated to its hemolytic and anti-bacterial potential against erythrocytes and Gram-positive bacterial membranes, which are rich in glycerophospholipids and cardiolipin. An evaluation of PLCBs soybean degumming process efficiency showed that the purified enzyme reduced the phosphorus content to 35 mg/kg and increased the amount of diacylglycerols released, indicating its ability to hydrolyze phospholipids in the crude soybean oil. Collectively, PLCBs could be considered as a potential catalyst for efficient industrial oil degumming, advancing the edible oil industry by reducing the oil gum volume through transforming non-hydratable phospholipids into their hydratable forms, as well as through generating diacylglycerols, which are miscible with triacylglycerols, thereby reducing losses.

Non-enzymatic glycation and aggregation of camel immunoglobulins induce breast cancer cell proliferation.

Glycation of biomolecules results in the formation of advanced glycation end products (AGEs). Immunoglobulin G (IgG) has been implicated in the progression of various diseases, including diabetes and cancer. This study purified three IgG subclasses (IgG1, IgG2, and IgG3) from Camelus dromedarius colostrum using ammonium sulfate fractionation and chromatographic procedures. SDS-PAGE was performed to confirm the purity and molecular weight of the IgG subclasses. Several biochemical and biophysical techniques were employed to study the effect of glycation on camel IgG using methylglyoxal (MGO), a dicarbonyl sugar. Early glycation measurement showed an increase in the fructosamine content by ~four-fold in IgG2, ~two-fold in IgG3, and a slight rise in IgG1. AGEs were observed in all classes of IgGs with maximum hyperchromicity (96.6%) in IgG2. Furthermore, glycation-induced oxidation of IgGs led to an increase in carbonyl content and loss of -SH groups. Among subclass, IgG2 showed the highest (39.7%) increase in carbonyl content accompanied by 82.5% decrease in -SH groups. Far UV-CD analysis illustrated perturbation of β-sheet structure during glycation reaction with MGO. Moreover, glycation of IgG proceeds to various conformational states like aggregation and increased hydrophobicity. In addition, the cytotoxicity assay (MTT) illustrated the proliferation of breast cancer cells (MCF-7) with IgG2 treatment.

Purification and characterization of leucyl aminopeptidase from the Todarodes pacificus hepatopancreas

Here, leucyl aminopeptidase (LAP) was purified from the hepatopancreas extract (HPE) of Todarodes pacificus, with 63.7-fold purification and a yield of 6.5%. Ammonium sulfate fractionation and sequential chromatographic steps, involving Sephacryl S-300, Toyopearl DEAE-650 M, DEAE-Sepharose CL-6B, and Sephadex G-50, were used for purification. The bitter-taste improvement of trypsin casein hydrolysate (TCH) by the purified LAP was also investigated. The LAP was characterized as a monomer; its molecular mass was 29.0 kDa under denatured conditions and 27.1 kDa under native conditions. With an optimum pH of 7.5 and optimum temperature of 45 °C, it remained stable up to ∼50 °C, within a pH range of 6–8. Its activity was inhibited by Cu2+ and Hg2+, bestatin, and the metal-chelating agents EDTA and 1,10-phenanthroline, and slightly enhanced by Fe2+, Zn2+, Mn2+, and Mg2+. Moreover, the purified enzyme was classified as a leucyl aminopeptidase, as it preferentially hydrolyzed Leu, followed by Met, Phe, Ala, and Lys p-nitroanilide. The apparent Km and Vmax values of the enzyme were 0.385 mM and 796.2 U/mg, respectively, for LeuPNA. Additionally, LAP-treated TCH showed lower bitterness than untreated TCH. The bitterness improvement effect of LAP could be attributed to the hydrolysis of hydrophobic amino acids at the N-terminus of the peptide; this confirms the substrate specificity of LAP. It has been confirmed that enzymes such as LAP that effectively reduce the bitter taste of bitter hydrolysates are distributed in the hepatopancreas of Todarodes pacificus, and this protein hydrolysate will be used as a bitter taste improver and food industry.

Purification and characterization of an asialofetuin specific lectin from the rhizome of Xanthosoma violaceum Schott

Lectins are proteins or glycoproteins that bind specifically and reversibly to the carbohydrate or glycoconjugates. A new lectin is purified from the rhizome of Xanthosoma violaceum Schott. by successive steps of ammonium sulfate fractionation and affinity chromatography with asialofetuin as ligand. The purified lectin was found to be a homotetramer of approximately 49 kDa with a subunit molecular weight of 12 kDa linked by non-covalent bonds. Characterization of the lectin shows that the hemagglutination activity is inhibited by asialofetuin and d-galacturonic acid. Hemagglutination activity is shown only in rabbit RBC but not in the human RBC of all blood groups. It is a metal ion-independent glycoprotein of 1.87% carbohydrate content, stable upto 40 °C and pH from 5.5 to 9. The lectin shows its optimum hemagglutination activity at 0 °C–40 °C and pH 6 to 8.5. From LC-MS/MS analysis it is confirmed that the purified lectin was not purified and characterized earlier.

Purification and characteristics of a novel milk-clotting metalloprotease from Bacillus velezensis DB219

Milk-clotting enzyme (MCE) is the essential active agents in dairy processing. The traditional MCE is mainly obtained from animal sources, in which calf rennet is the most widely used in cheese industry. Traditional MCE substitute is becoming necessary due to its limited production and increased cheese consumption. A novel traditional MCE substitute was produced from Bacillus velezensis DB219 in this study. The DB219 MCE exhibited a notable specific activity of 6,110 Soxhlet units/mg and 3.16-fold purification yield with 28.87% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified DB219 MCE was a metalloprotease with a molecular weight of 36 kDa. The DB219 MCE was weak acid resistance and stable at pH 6.0 to 10.0 and temperature <45°C. The highest milk-clotting activity was observed in substrate at pH 5.5 added with 20 to 30 mM CaCl2. The Michaelis constant and maximal velocity for casein were 0.31 g/L and 14.22 μmol/min. The DB219 MCE preferred to hydrolyze β-casein instead of α-casein. The DB219 MCE hydrolyzed α-casein, β-casein, and κ-casein to generate significantly different peptides in comparison with calf rennet and ES6023 MCE (fungal MCE) through SDS-PAGE and reversed-phase HPLC analysis. The DB219 MCE mainly cleaved Thr124-Ile125 and Ser104-Phe105 bonds in κ-casein and had unique casein cleavage sites and peptide composition through LC-MS/MS analysis. The DB219 MCE was potential to be a new milk coagulant and enriched kinds of traditional MCE substitute.

Partial Purification and Characterization of the Acetylcholinesterase from Poecilia reticulata (Peters, 1859)

Poecilia reticulata (P. reticulata) is a species of tropical fish in the family Poeciliidae that is found all over the world and is a favorite in freshwater aquariums. The Malay name for this fish is "Ikan Gapi," although the English term "Guppy" has become more common. The Million Fish, or Rainbow Fish, as it's known in other parts of the world. The goals of this research were to char-acterize the acetylcholinesterase that will be partially purified from P. reticulata brain extract using ammonium sulphate fractionation followed by an affinity chromatography method. The result shows that ammonium sulphate fractionation gave better purification fold over the procainamide affinity chromatographic method with fold of purification of 3.38 compared to 0.64 for the latter. Polyacrylamide gel electrophoresis (SDS-PAGE) shows a dramatic reduction of protein bands compared to crude extract. A pH of 9 in a Tris-HCl buffer system and a temperature of 15 °C were found to be ideal for the activity of the enzyme. Based on its high Vmax and low Km with acetylthiocholine iodide (ATC) as a substrate compared to butyryl thiocholine iodide (BTC) and propionyl thiocholine iodide (PTC), the substrate specificity profile identified acetylcholinesterase (AChE) as the major enzyme present in the purified enzyme. The molecular weights of the en-zyme following affinity chromatographic purification are about 92 kDa.

Inhibitive Assay of Insecticides using the Acetylcholinesterase from Poe-cilia reticulata (Peters, 1859)

Poecilia reticulata (P. reticulata) is a species of tropical fish in the family Poeciliidae that is found all over the world and is a favorite in freshwater aquariums. The Malay name for this fish is "Ikan Gapi," although the English term "Guppy" has become more common. The Million Fish, or Rainbow Fish, as it's known in other parts of the world. The goals of this research were to char-acterize the acetylcholinesterase that will be partially purified from P. reticulata brain extract using ammonium sulphate fractionation The screening results show that at 1 mg/L insecticide, acephate, dimethoate, trichlorfon and bendiocarb leads to 100% inhibition, whilst parathion, methomyl and malathion cause &gt;90% inhibition. The AChE from P. reticulata is less sensitive to other species as far as the insecticides carbofuran, carbaryl, methomyl, bendiocarb, parathion, diazinon, chlorpyrifos with the exception of malathion, where the sensitivity of malathion to the AChE from was within the range of the AChE from other species such a E. electricus , Pangasius sp., Chan-na micropeltes, Clarias batrachus, H. nemurus, Tor tambroides, and Osteochilus hasselti.

Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes).

Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125kDa) and X2 variants (88 and 79kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2β, were estimated to have two domains, and X2β is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.