Ammonium Sulfate Fractionation Research Articles

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.

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.

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 > phosphatidylethanolamine > phosphatidylserine > sphingomyelin > phosphatidylinositol > 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.

Characteristics and application in cheese making of newly isolated milk-clotting enzyme from Bacillus megaterium LY114

Milk-clotting enzyme (MCE) is a crucial active agent in cheese making. It is necessary to find traditional MCE substitutes due to the limited production of traditional MCE (e.g., calf rennet) and increased cheese consumption. Bacillus megaterium strain LY114 with good milk-clotting activity (MCA) (448 SU/mL) and a high MCA/proteolytic activity (PA) ratio (6.0) was isolated and identified from agricultural soil in Laiyang (Shandong, China) through 16S rRNA sequencing of 45 strains. The Bacillus megaterium LY114 MCE had a remarkable specific activity (7532 SU/mg) and displayed a 4.83-fold purification yield with 34.17% recovery through ammonium sulfate fractionation and DEAE-Sepharose Fast Flow. The purified LY114 MCE was a metalloprotease with a molecular weight of 30 kDa. LY114 MCE was stable at pH 5.0–7.0 and temperature <40 °C. The highest MCA appeared at a substrate pH of 5.5 with 30 mM CaCl2. The Michaelis constant (Km) and maximal velocity (Vm) for casein were 0.31 g/L and 14.16 μmol/min, respectively. LY114 MCE preferred to hydrolyze α-casein (α-CN) rather than β-casein (β-CN) and had unique α-CN, β-CN and κ-casein (κ-CN) cleavage sites. LY114 MCE hydrolyzed casein to generate significantly different peptides compared with calf rennet and fungal MCE as determined by SDS-PAGE analysis. Chemical index analysis and sensory evaluation confirmed the usefulness of LY114 MCE in cheese making. LY114 MCE had the potential to be used in dairy processing and enriched traditional MCE substitutes.

Purification Scheme and Biochemical Profile of Metallo-alkaline Protease Produced by B.t. Dendrolimus IP 4A/4B

Objective: Purify and classify alkaline protease (AP), produced by Bacillus thuringiensis (B.t.) dendrolimus 4A/4B. Methods: Inoculum preparation, production media, enzymes assays, protein determination, ammonium sulfate precipitation, acetone fractionation, Sephadex Gel chromatography filtration. Results: The purification scheme, (a) ammonium sulfate fractionation, the most active fraction with 1.93 purification fold was at 60-75% saturation; (b) acetone precipitation, where the most active fraction was at 30-45% with purification fold of about 3.92 times; (c) Column chromatography on G-100 Sephadex with final purification folds 5.36 times of the crude enzyme with final yield recovery 1.9%. Purified AP showed stability against H2O2 1%, 5% and 10% (v/v), and retained 111%, 101% and 66% of its activity, respectively. AP retained 82%, 87% and 101% of its activity with 10, 5 and 1mM PMSF, respectively. Enzyme activity was completely inhibited by 10mM EDTA, while the enzyme retained about 125% of its activity with 10mM Mercapto-ethanol. CaCl2 and MgCl2 (10mM) showed activation effect, increasing the activity to 136% and 147% compared to the control, respectively. The optimum pH are 8.0 and 10.0. AP activity was increased through 30ºC to 50ºC. At 60˚C, the enzyme retained ~48% of its original activity. AP concentration 0.014mg protein/mL has maximum activity (6.750U/mL/min). The highest maximum velocity (Vmax) was 31.821μg tyrosine/mL/min. Km value 0.833%, w/v casein concentration. Conclusion: Thus, this inhibition profile suggested that the AP produced belongs to the class of metalo-proteases.

A novel protein extracted from Hemerocallis citrina Borani inhibits hepatocellular carcinoma cell proliferation by regulating mitochondria-dependent apoptosis and aerobic glycolysis.

Hemerocallis citrina Borani is a commonly consumed food in Asia and possesses many biologically active ingredients. In this study, a protein named Hemerocallis citrina Borani protein (HcBP) was purified using ammonium sulfate fractionation and anion exchange chromatography. Protease assays revealed that HcBP has peroxidase activity. Meanwhile, the UV absorption spectrum showed that HcBP contains heme. Notably, HcBP showed significant inhibitory effects on human hepatoma cancer cell proliferation. Mechanism investigations indicated that HcBP treatment resulted in overproduction of reactive oxygen species (ROS) and induced mitochondria-dependent apoptosis in human hepatoma cancer cells. Furthermore, we found HcBP not only downregulated pyruvate kinase M2 (PKM2) activity but also decreased the expression and nuclear levels of PKM2. The inhibition of PKM2 led to the downregulationof GLUT1, LDHA and PDK, and thus caused the suppression of glycolysis. In summary, our results suggested that HcBP has potential anti-hepatocellular carcinoma activity.

Utilization of Partially Purified Papain Enzyme in Mallika Black Soybean Tempeh Hydrolysate as Umami Seasoning

&lt;p&gt;&lt;span lang="EN-US"&gt;Tempeh made from Mallika black soybean (&lt;em&gt;Glycine max &lt;/em&gt;(L.) Merr. &lt;em&gt;var. &lt;/em&gt;Mallika) can be fermented for up to 4 days and can be further optimized by adding partially purified papain enzyme obtained from California variety papaya leaves (&lt;em&gt;Carica papaya &lt;/em&gt;(L.) &lt;em&gt;var. &lt;/em&gt;California). Enzyme can be added to the hydrolysates to degrade protein into short-chain peptides and free amino acids, contributing to umami taste sensory attributes. The study aimed to determine the best ammonium sulfate fractionation of crude papain enzyme and the best physicochemical characteristics of black soybean tempeh protein hydrolysate. The addition of ammonium sulfate fractionation used was 0% to 80%; fermentation time was 2 to 4 days; and the concentration of enzyme added was 0%(w/v) to 1.5%(w/v). The results showed that the 40% fractioned papain enzyme gave the highest protease activity value (0.98±0.04 U ml&lt;sup&gt;-1&lt;/sup&gt;) and most of the papain enzyme was precipitated in this fraction leaving impurities. The black soybean tempeh hydrolysates with 4 × 1% showed the best physicochemical characteristic because it produced the highest umami substance. The best characteristics were moisture content (17.97±0.46%), glutamic acid content (171.58±5.72 mg g&lt;sup&gt;-1&lt;/sup&gt;) that was caused by a transamination reaction, dissolved protein content (470.66±19.50 mg g&lt;sup&gt;-1&lt;/sup&gt;), degree of hydrolysis (43.64±1.99%) and lightness (46.02±0.97). The umami substance’s amino acids are high in content, such as glutamic and aspartic acids (59.89±0.31 mg g&lt;sup&gt;-1 &lt;/sup&gt;and 26.47±0.09 mg g&lt;sup&gt;-1&lt;/sup&gt;). Sensory evaluation showed that treatment 4 × 1% demonstrated no significant difference in umami intensity with MSG (monosodium glutamate)&lt;/span&gt;&lt;span lang="EN-US"&gt;.&lt;/span&gt;&lt;/p&gt;

A novel thermostable TP-84 capsule depolymerase: a method for rapid polyethyleneimine processing of a bacteriophage-expressed proteins

BackgroundIn spite of the fact that recombinant enzymes are preferably biotechnologically obtained using recombinant clones, the purification of proteins from native microorganisms, including those encoded by bacteriophages, continues. The native bacteriophage protein isolation is often troubled by large volumes of the infected bacterial cell lysates needed to be processed, which is highly undesired in scaled-up industrial processing. A well-known ammonium sulphate fractionation is often a method of choice during purification of the native bacteriophage protein. However, this method is time-consuming and cumbersome, and requires large amounts of the relatively expensive reagent. Thus, other effective and inexpensive methods of reversible protein precipitation are highly desirable. We have previously characterized thermophilic TP-84 bacteriophage, defined a new genus TP84virus within Siphoviridae family, conducted the TP-84 genome annotation and proteomic analysis. The longest Open Reading Frame (ORF) identified in the genome is TP84_26. We have previously annotated this ORF as a hydrolytic enzyme depolymerizing the thick polysaccharides host’s capsule.ResultsThe TP84_26 ‘capsule depolymerase’ (depolymerase) is a large, 112 kDa protein, biosynthesized by the infected Geobacillus stearothermophilus 10 (G. stearothermophilus 10) cells. The TP84_26 protein biosynthesis was confirmed by three approaches: (i) purification of the protein of the expected size; (ii) mass spectrometry (LC–MS) analysis and (iii) detection of the enzymatic activity toward G. stearothermophilus polysaccharide capsules. Streptomycin-resistant mutant of the host was generated and microbiological aspects of both the TP-84 and G. stearothermophilus 10 were determined. A new variant of polyethyleneimine (PEI)-mediated purification method was developed, using the novel TP-84 depolymerase as a model. The enzyme was characterized. Three depolymerase forms were detected: soluble, unbound proteins in the bacteriophage/cells lysate and another integrated into the TP-84 virion.ConclusionsThe novel TP-84 depolymerase was purified and characterized. The enzyme exists in three forms. The soluble, unbound forms are probably responsible for the weakening of the capsules of the uninfected bacterial cells. The form integrated into virion particles may generate a local passage for the invading TP-84. The developed PEI purification method appears well suited for the scaled-up or industrial production of bacteriophage proteins.

Purification and Characterization of a Novel D-Galactose Binding Lectin from Seeds of Meizotropis buteiformis.

The main aim of the present study is to find out the novelty and characteristics of a lectin purified from the plant Meizotropis buteiformis. Lectins are proteins that possess the ability to specifically bind glycans of glycoconjugates. Plants are considered rich sources of lectins and the determination of sugar specificity of a purified plant lectin is an important aspect in order to evaluate its potential area of application. In the present study, a novel D-Galactose specific lectin is purified from Meizotropis buteiformis through affinity chromatography and examined for its various physical and biochemical characteristics. The objective of the present study is to purify a novel lectin up to its homogeneity from the seeds of Meizotropis buteiformis and characterization of its various physical and biochemical properties. The lectin was purified by simple successive steps of lectin extraction, ammonium sulphate fractionation, and affinity chromatography. Activity of the purified lectin was determined by hemagglutination assay. Some physicochemical parameters of the purified protein were also determined along with identification of protein by LC-MS/MS and the spectra analysis using Mascot sequence matching software (Matrix Science) with the NCBI database. From the current investigation, it was found that the purified lectin has a native molecular weight of 75 kDa. Among the various sugars and sugar derivatives tested, lactose and D-galactose were found to be potent inhibitors of its activity. Its optimum pH range was found to be from 6.5 to 7.5 and also it exhibited full activity at a temperature from 0ºC to 50ºC. The purified lectin does not show any effects on its activities for metal ions tested. The protein view report of the LC-MS/MS result analysis showed a 50% sequence similarity with that of the lectin beta-chain of the Butea monosperma. In the present study, a novel D-Galactose specific lectin is purified from Meizotropis buteiformis by affinity chromatography using Sepharose 4B. The purified lectin is found to be heterodimeric and metal ion independent. The LC-MS/MS results suggested that the purified lectin has not been reported earlier.

Identification, kinetics and thermodynamic analysis of novel β-galactosidase from Convolvulus arvensis seeds: An efficient agent for delactosed milk activity

The β-galactosidase was extracted and purified from 100 g of C. arvensis seeds using a variety of protein purification procedures such as ammonium sulphate fractionation, gel filtration, and finally chromatography on a cationic ion exchanger. The effects of metal ions, kinetics parameters, and glycoprotein nature were determined, as well as the optimal pH and temperature of the purified enzyme. With a high specific activity (72 units/mg), β-galactosidase was isolated to a 24-fold apparent electrophoretic homogeneity. The molecular mass of β-galactosidase was determined as monomeric, which was further confirmed by SDS-PAGE and MALDI-TOF/MS analysis, with a 45 kDa molecular weight. The enzyme has a Km of 0.33 mM and a Vmax of 42 μmol/min Lactose in milk was reduced by 38.5 and 70 % after 4 h of incubation with β-galactosidase from C. arvensis. The β-galactosidase thermal inactivation kinetic parameters ΔH°, ΔS°, and ΔG° were calculated, indicating that the enzyme undergoes significant unfolding events during denaturation. Using β-galactosidase from C. arvensis seeds, lactose hydrolysis in milk up to approx. 50 % was observed. The findings indicate the potential use of C. arvensis seeds for the production of low/delactosed milk for lactose-intolerant population.

Bacteriocin from Lacticaseibacillus rhamnosus sp. A5: Isolation, Purification, Characterization, and Antibacterial Evaluation for Sustainable Food Processing

A new Lacticaseibacillus rhamnosus strain A5 was isolated from pickle soup and characterized for its probiotic suitability. Strain A5 was Gram-positive, catalase-negative, acid-producing, and exhibited potential antibacterial activity against Escherichia coli (inhibition zone 17.3 mm), Bacillus subtilis (inhibition zone 14.5 mm), Salmonella enterica (zone of inhibition 16.1 mm) and Staphylococcus aureus (zone of inhibition 14.2 mm) by performing investigations on the disc diffusion. The cell-free supernatant of newly isolated strain A5 retained its inhibition ability of the growth of test bacteria at pH 2.0 to 5.0, temperature 121 °C for 30 min and UV irradiation for 8 h. However, the inhibitory effects of cell-free supernatant disappeared when subjected to papain, trypsin, and pepsin enzymatic treatments. By eliminating the interferences of organic acid and hydrogen peroxide, the cell-free supernatant possessed antibacterial activity against two indicator bacteria (E. coli and B. subtilis) and showed high thermal tolerance. These results indicated that the antibacterial substances produced by strain A5 were proteinaceous in nature, namely bacteriocin. The antibacterial bacteriocins in the supernatant of the strain A5 culture were further purified by ammonium sulfate fractionation and gel filtration chromatography. The purified bacteriocins also showed a pronounced inhibitory effect against E. coli and B. subtilis. The approximated molecular weight of bacteriocins was less than 14 kDa after determining by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In conclusion, the newly isolated strain A5 and its bacteriocins could be potentially applied in food preservation to prevent the risk of foodborne illness.

Isolation and Characterization of Uricase Produced from Chicken Liver

Uricase is an enzyme that degrades uric acid into allantoin. One of the uricase sources is obtained from chicken species (Gallus gallus domesticus) liver which are broiler and native chicken. This study aims to determine the maximum uricase activity in broiler and native chicken liver. The uricase activity was obtained by measuring the uric acid concentration as uricase substrate using spectrophotometric method and wavelength at 291 nm. Uricase isolation was carried out into extraction process, ammonium sulfate fractionation (0-60% saturation of ammonium sulfate), and dialysis. During isolation process, centrifugation speed was also optimized to obtain the maximum uricase crude extract and uricase activity. The molecular weight of uricase was also determined by SDS PAGE. The result showed that the highest uricase activity remained using centrifugation speed of 15,000 rpm. The optimum uricase fraction for broiler chicken liver was obtained at 20-40% saturation of ammonium sulfate with uricase activity was 1.854 x 10-2 U/mg, and the uricase fraction for native chicken liver was obtained at 40-60% saturation of ammonium sulfate with uricase activity was 2.496 x 10-2 U/mg. The optimum fraction for uricase production and isolation is carried out to the dialysis process. The optimum uricase activity of broiler chicken liver crude extract was 4.921 x 10-4 U/mg, the uricase fraction was 3.989 x 10-3 U/mg, and the dialysate was 5.120 x 10-3 U/mg. While the native chicken liver crude extract was 2.980 x 10-4 U/mg, the uricase fraction was1.415 x 10-2 U/mg, and the dialysate was 1.753 x 10-2 U/mg. The molecular weight of the uricase was around 35 kDA according to the SDS PAGE result.

Proteolytic Sensitivity, In Vitro Glycation Efficiency of Diabetic and Non-diabetic Human Serum Albumin

Background: Glycation of human serum albumin (HSA) leads to disturbances in its stability, activity, and other properties which, in turn, affect the functional properties of HSA. Modification of albumin by glycation shows considerable potential as a significant biochemical biomarker for diagnosing diabetes. The characteristics of the glycation process in proteins have not been fully examined yet and, therefore, there is insufficient knowledge about them in the field. Objectives: This study aimed to clarify the differences between diabetic and non-diabetic HSA as well as their structure-function relationship. Methods: The physiological and laboratory characteristics of glycated albumin as well as HSA were explored. A total of 30 subjects were enrolled in this study in which 15 normal healthy individuals were assigned into the control group, and 15 type-2 diabetic patients were included in the diabetic group. Patients with type-1 diabetes, pregnant women, and individuals with other diseases were excluded from the study. Protein estimation, polyacrylamide gel electrophoresis, ammonium sulphate fractionation, dialysis, glycation of HSA followed by gel electrophoresis of glycated samples, digestion of BSA, as well as HSA by α-chymotrypsin and their documentation and stoichiometry were all performed. Results: Various characteristic differences were observed between diabetic and non-diabetic HSA including proteolytic susceptibility and in vitro glycation efficiency. Hypoalbuminemia was, particularly, observed in diabetic patients, which was suggestive of a relationship between hyperglycemia and hypoalbuminemia. Conclusion: Peculiar contrariety between diabetic and non-diabetic HSA, specific differences in their glycation efficiencies, as well as proteolytic susceptibility and their innuendos were precisely traced out. It was concluded that albumin may have been regarded as a significant clinical biomarker for diagnosing diabetes.