Ammonium Sulfate Fractionation Research Articles

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

<p><span lang="EN-US">Tempeh made from Mallika black soybean (<em>Glycine max </em>(L.) Merr. <em>var. </em>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 (<em>Carica papaya </em>(L.) <em>var. </em>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<sup>-1</sup>) 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<sup>-1</sup>) that was caused by a transamination reaction, dissolved protein content (470.66±19.50 mg g<sup>-1</sup>), 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<sup>-1 </sup>and 26.47±0.09 mg g<sup>-1</sup>). Sensory evaluation showed that treatment 4 × 1% demonstrated no significant difference in umami intensity with MSG (monosodium glutamate)</span><span lang="EN-US">.</span></p>

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.

Intracellular Aminopeptidase Activity Determination from the Fungus Sporisorium reilianum: Purification and Biochemical Characterization of psrAPEi Enzyme.

The aims of this study were to, first, determine the intracellular aminopeptidase activity (APEi) and second, purify and biochemically characterize one intracellular aminopeptidase enzyme from the phytopathogen fungus Sporisorium reilianum (psrAPEi), the causal agent of head smut in corn. The fungus produced APEi activity in all media cultures evaluated. The psrAPEi was purified by a procedure that involved ammonium sulfate fractionation and four chromatographic steps using an FPLC system (Fast Protein Liquid Chromatography). Results showed an estimated molecular mass of 52.2kDa. Enzymatic activity was optimal at pH 7.0 and 35°C and was inhibited by EDTA-Na2, 1,10-phenanthroline, bestatin, and PMSF. This aminopeptidase showed a preference for leucine, arginine, and lysine at the N-position. The Km and Vmax values were 3.72μM and 188.0μmol/min, respectively, for L-lysyl-4-nitroanilide. This is the first study to report on intracellular aminopeptidase activity in S. reilianum and the purification and characterization of an intracellular metallo-serine-aminopeptidase (psrAPEi).

Partial Purification of Phytocystatin from Moringa oleifera lam

Background: Phytocystatins are plants cysteine protease inhibitors (CPIs) that are known for their numerous uses in medicine and biotechnology. Methods: Different extraction media which include, Sodium Hydroxide, Hydrochloric acid, Sodium Chloride, Sodium phosphate buffer and distilled Water were used to evaluate flower, leave, root, latex, and stem bark of Moringa oleifera for inhibitory activity against cysteine protease (Papain enzyme). The phosphate buffer extract with higher protease inhibitory activity was concentrated by cold acetone precipitation and further subjected to partial purification using ammonium sulphate fractionation and Hydrophobic chromatography on Phenyl Sepharose column. The molecular weight of partially purified protein was estimated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis. Results: The extracts of Sodium phosphate buffer from the seeds of Moringa oleifera showed higher CPI activity of 11.23 units/mg protein. Cold acetone precipitated extract gave high yield of 94.6% protein with 60% inhibition of protease. The partially purified protein showed a fifty percent inhibitory concentration (IC50) of 1.22±0.3 mg/ml protein against the enzyme with 63% inhibition. An estimated molecular weight of 13.5kDa was obtained from electrophoretic analysis of partially purified protein. Conclusion: Moringa oleifera seeds could be a good source of low molecular weight protein Inhibitor of Cysteine protease and might be useful in biotechnology of traditional medicinal plants, in transgenic crops to arrest the negative pathogenic over expressions of cysteine proteases

Purification and characterization of dextranase from Penicillium cyclopium CICC-4022 and its degradation of dextran

A dextranase was purified from Penicillium cyclopium CICC-4022 by ammonium sulfate fractionation and secondary tangential flow filtration, and the enzymatic properties were studied. The purified dextranase was used to regulated the molecular mass and homogeneity of dextran. Weight-average molecular mass (Mw) and polydispersity index (Mw/Mn) of dextran were measured by gel permeation chromatography (GPC) coupled with a triple-detector array (GPC-TDA), which is composed of a multiple-angle light scattering, a viscometer, and a refractive-index detector. The dextranase was purified by 2.24-fold, the recovery rate was 45.84%, the specific activity was 1442.05 U/mg, and the Mw was 77 KDa. Dextranase showed maximum activity at pH of 5.0 and 55 °C. Na+, K+ and NH4+ can effectively improve the dextranase activity, Cu2+ and Pb2+ can strongly inhibit the dextranase activity. Dextranase specifically degraded the α-1,6 glycosidic bonds of dextran. By controlling the dextranase activity, substrate concentration, and time, the specific Mw dextran with good homogeneity was obtained. The structure of dextran was not altered before or after dextranase hydrolysis, but its conformation changed from a spherical chain to a compliant chain. When the Mw of the dextran product was about 5 KDa, it was a compact spherical chain conformation in solution.

A novel L‐asparaginase from the symbiotic Enterobacter aerogenes isolated from Eucheuma sp.

This study was aimed to purify and characterize the enzyme L-asparaginase from endophytic microorganisms of Eucheuma sp. in Puntondo Island, South Sulawesi, Indonesia. Research stages were ammonium sulfate precipitation, dialysis, and Sephadex G-75 gel filtration chromatography and Sephadex CMC-50 ion-exchange chromatography matrices. The result of test activities of ammonium sulfate fraction showed the highest range of 40%–60% the activity value of 6.71 IU/ml. The highest protein content was obtained on a 0%–20% fraction of 2.76 mg/ml. Temperature and pH assessments indicated that L-asparaginase had optimum activity at 37°C and pH 8, respectively. At the stage of dialysis, the highest protein content was 5.17 IU/ml in a 0%–20% fraction, and the highest enzyme activity was 1.23 IU/ml in a 20%–40% fraction. The results of gel filtration chromatography fractions G-75 were F5–F10, with a specific activity of 0.60 U/mg, 2.05 times higher purity level than the crude extract enzyme. The results of CMC-50 ion-exchange chromatography showed that F54 and F55 fractions had a specific activity of 106.97 and 295.38 U/mg, respectively, with a purity level of 366.34 and 1,011.57 times higher than the crude extract enzyme. Novelty impact statement Enterobacter aerogenes strain P5, an endophytic bacterium from Eucheuma sp., can produce pure L-asparaginase enzyme with a high specific activity applied in pharmaceuticals nutraceutical applications.

Biochemical characterization and thermodynamic principles of purified l-Asparaginase from novel Brevibacillus borstelensis ML12

l-Asparaginase from Brevibacillus borstelensis ML12 was purified and sequentially studied for biochemical characterization. A 1.94 fold purification using 80% ammonium sulphate fractionation was obtained. The molecular weight was found out to be 39.5 kDa following SDS-PAGE method. The purified l-Asparaginase showed enzyme stability in the alkaline range7.6–8.8 and maximum activity at pH 8.6. Co2+ proved to be a good activator in all the three respective concentrations (5mM10 mM, 20 mM) that were selected in the study. Effect of various surfactants, bile salt, organic reagents, chelators were also incorporated and Tween 80 and bile salt were found to enhance enzyme activity. Vmax and Km values were calculated by using Lineweaver-Burk plot and the values came out to be 204.081 μmol ml-1 min-1 and 0.253 mM respectively. Thermo kinetics study revealed that deactivation energy as obtained from Arrhenius equation was 10.882 kJmol-1. The z-value was calculated as 185.16K in case of thermal inactivation of asparaginase.

The Kunitz chymotrypsin inhibitor from Erythrina velutina seeds displays activity against HeLa cells through arrest in cell cycle.

Erythrina velutina is a species of arboreal leguminous that occurs spontaneously in the northeastern states of Brazil. Leguminous seeds represent an abundant source of peptidase inhibitors, which play an important role in controlling peptidases involved in essential biological processes. The aim of this study was to purify and characterize a novel Kunitz-type peptidase inhibitor from Erythrina velutina seeds and evaluate its anti-proliferative effects against cancer cell lines. The Kunitz-type chymotrypsin inhibitor was purified from Erythrina velutina seeds (EvCI) by ammonium sulphate fractionation, trypsin- and chymotrypsin-sepharose affinity chromatographies and Resource Q anion-exchange column. The purified EvCI has a molecular mass of 18kDa with homology to a Kunitz-type inhibitor. Inhibition assays revealed that EvCI is a competitive inhibitor of chymotrypsin (with K i of 4 × 10-8M), with weak inhibitory activity against human elastase and without inhibition against trypsin, elastase, bromelain or papain. In addition, the inhibitory activity of EvCI was stable over a wide range of pH and temperature. Disulfide bridges are involved in stabilization of the reactive site in EvCI, since the reduction of disulfide bridges with DTT 100mM abolished ~ 50% of its inhibitory activity. The inhibitor exhibited selective anti-proliferative properties against HeLa cells. The incubation of EvCI with HeLa cells triggered arrest in the cell cycle, suggesting that apoptosis is the mechanism of death induced by the inhibitor. EvCI constitutes an interesting anti-carcinogenic candidate for conventional cervical cancer treatments employed currently. The EvCI cytostatic effect on Hela cells indicates a promised compound to be used as anti-carcinogenic complement for conventional cervical treatments employed currently.

Characterization of \u03b2-galactosidase and \u03b1-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri

PurposeGracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them.MethodsG. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis.ResultFormation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745).ConclusionsG. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.

Antagonistic activity of glucanolytic bacteria Bacillus subtilis W3.15 against Fusarium oxysporum and its enzyme characterization

Abstract. Putri RE, Mubarik NR, Ambarsari L, Wahyudi AT. 2021. Antagonistic activity of glucanolytic bacteria Bacillus subtilis W3.15 against Fusarium oxysporum and its enzyme characterization. Biodiversitas 22: 4067-4077. Biocontrol of Fusarium oxysporum, a phytopathogenic fungus that causes plant wilt can be approached with cell-wall degrading enzymes such as ?-glucanase. The aim of this study was to evaluate the prospective ability in glucanase production from several soil bacterial isolates and to characterize its ?-glucanase activity of ammonium sulfate precipitation, and to determine its antifungal activity against F. oxysporum in vitro. Twenty bacterial isolates were screened qualitatively and quantitatively as ?-glucanase producers. The results showed that the prospective isolate W3.15 can produce ?-glucanase on glucan agar as the selection medium. From 16S rRNA sequences identification, the isolate belongs to the genus Bacillus, closely related to Bacillus subtilis. The enzyme activity of the ammonium sulfate fraction of isolate W3.15 is optimum at a pH of 7 and temperature range of 60-80oC. B. subtilis W3.15 exhibits high inhibition against the mycelial growth of F. oxysporum and significantly reduced fungal biomass.

A New Lectin from Auricularia auricula Inhibited the Proliferation of Lung Cancer Cells and Improved Pulmonary Flora.

Lectins are widely distributed in the natural world and are usually involved in antitumor activities. Auricularia auricula (A. auricula) is a medicinal and edible homologous fungus. A. auricula contains many active ingredients, such as polysaccharides, melanin, flavonoids, adenosine, sterols, alkaloids, and terpenes. In this study, we expected to isolate and purify lectin from A. auricula, determine the glycoside bond type and sugar-specific protein of A. auricula lectin (AAL), and finally, determine its antitumor activities. We used ammonium sulfate fractionation, ion exchange chromatography, and affinity chromatography to separate and purify lectin from A. auricula. The result was a 25 kDa AAL with a relative molecular mass of 18913.22. Protein identification results suggested that this lectin contained four peptide chains by comparing with the UniProt database. The FT-IR and β-elimination reaction demonstrated that the connection between the oligosaccharide and polypeptide of AAL was an N-glucoside bond. Analyses of its physical and chemical properties showed that AAL was a temperature-sensitive and acidic/alkaline-dependent glycoprotein. Additionally, the anticancer experiment manifested that AAL inhibited the proliferation of A549, and the IC50 value was 28.19 ± 1.92 μg/mL. RNA sequencing dataset analyses detected that AAL may regulate the expression of JUN, TLR4, and MYD88 to suppress tumor proliferation. Through the pulmonary flora analysis, the bacterial structure of each phylum in the lectin treatment group was more reasonable, and the colonization ability of the normal microflora was improved, indicating that lectin treatment could significantly improve the bacterial diversity characteristics.

CBP22, a Novel Bacteriocin Isolated from Clostridium butyricum ZJU-F1, Protects against LPS-Induced Intestinal Injury through Maintaining the Tight Junction Complex.

A novel bacteriocin secreted by Clostridium butyricum ZJU-F1 was isolated using ammonium sulfate fractionation, cation exchange chromatography, affinity chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The bacteriocin, named CBP22, contained 22 amino acids with the sequence PSAWQITKCAGSIAWALGSGIF. Analysis of its structure and physicochemical properties indicated that CBP22 had a molecular weight of 2264.63 Da and a +1 net charge. CBP22 showed activity against E. col K88, E. coli ATCC25922, and S. aureus ATCC26923. The effects and potential mechanisms of bacteriocin CBP22 on the innate immune response were investigated with a lipopolysaccharide- (LPS-) induced mouse model. The results showed that pretreatment with CBP22 prevented LPS-induced impairment in epithelial tissues and significantly reduced serum levels of IgG, IgA, IgM, TNF-α, and sIgA. Moreover, CBP22 treatment increased the expression of the zonula occludens and reduced permeability as well as apoptosis in the jejunum in LPS-treated mice. In summary, CBP22 inhibits the intestinal injury and prevents the gut barrier dysfunction induced by LPS, suggesting the potential use of CBP22 for treating intestinal damage.

Purification, Characterization and Bioefficacy of Legume Lectins against Mustard Aphid

Background: Lectins are carbohydrate binding proteins which perform diverse roles in plants. One important role is in plant defense. These proteins hold great potential as entomotoxic proteins as a part of integrated pest management. Methods: Lectins were purified and characterized from seeds of two legumes, Glycine max-Soybean and Lens culinaris-Lentil, employing ammonium sulfate fractionation and ion exchange chromatography. Bioefficacy of the purified lectins was evaluated against mustard aphid.Result: Lectins isolated from seeds of soybean (Glycine max agglutinin GMA-I, II) and lentil (Lens culinaris agglutinin LCA-I) were purified upto 9.30 (GMA-I), 4.60 (GMA-II) and 8.70 (LCA-I) fold, respectively. Lectin characterization revealed that soybean agglutinin and lentil agglutinin were specific towards D-Galactose and D-mannose, respectively. Insect bioassay was carried out with five different concentrations (10, 20, 30, 40, 50 µg/ml) of purified lectins of soybean and lentil against mustard aphid. The lethal concentration LC50 value for GMA-I was obtained as 32.1 µg/ml with a 95% confidential interval of 18.2 to 40.5 µg/ml and that of LCA-I was 19.1 µg/ml with a 95% confidential interval of 9.3 to 26.8 µg/ml. Lentil lectin (LCA-I) with lower LC50 value, was found to be the potential candidate for integrated pest management.

Protease Inhibitors Purified from the Canola Meal Extracts of Two Genetically Diverse Genotypes Exhibit Antidiabetic and Antihypertension Properties

Valorization of vegetable oil waste residues is gaining importance due to their high protein and polyphenol contents. Protease inhibitors (PIs), proteins from these abundantly available waste residues, have recently gained importance in treating chronic diseases. This research aimed to use canola meal of genetically diverse Brassica napus genotypes, BLN-3347 and Rivette, to identify PIs with diverse functionalities in therapeutic and pharmacological applications. The canola meal PI purification steps involved: native PAGE and trypsin inhibition activity, followed by ammonium sulfate fractionation, anion exchange, gel filtration, and reverse-phase chromatography. The purified PI preparations were characterized using SDS-PAGE, isoelectric focusing (IEF), and N terminal sequencing. SDS-PAGE analysis of PI preparations under native reducing and nonreducing conditions revealed three polymorphic PIs in each genotype. The corresponding IEF of the genotype BLN-3347, exhibited three acidic isoforms with isoelectric points (pI) of 4.6, 4.0, and 3.9, while Rivette possessed three isoforms, exhibiting two basic forms of pI 8.65 and 9.9, and one acidic of pI 6.55. Purified PI preparations from both the genotypes displayed dipeptidyl peptidase-IV (DPP-IV) and angiotensin-converting enzyme (ACE) inhibition activities; the BLN-3347 PI preparation exhibited a strong inhibitory effect with lower IC50 values (DPP-IV 37.42 µg/mL; ACE 129 µg/mL) than that from Rivette (DPP-IV 67.97 µg/mL; ACE 376.2 µg/mL). In addition to potential human therapy, these highly polymorphic PIs, which can inhibit damaging serine proteases secreted by canola plant pathogens, have the potential to be used by canola plant breeders to seek qualitative trait locus (QTLs) linked to genes conferring resistance to canola diseases.

Novel non-specific lipid-transfer protein (TdLTP4) isolated from durum wheat: Antimicrobial activities and anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages

Lipid transfer proteins (LTP) are members of the family of pathogenesis-related proteins (PR-14) that play a key role in plant defense mechanisms. In this study, a novel gene TdLTP4 encoding an antifungal protein from wheat (cv. Om Rabiaa) was cloned, overexpressed in Escherichia coli BL-21 (DE3) and enriched using ammonium sulfate fractionation. The TdLTP4 fusion protein was then tested against a panel of pathogens, food-borne and spoilage bacteria and fungi in order to evaluate the antimicrobial properties. TdLTP4 was applied to 0.5 μg/mL LPS-induced RAW 264.7 macrophages in vitro at different concentrations (5, 10, 20, 50 and 100 μg/mL). Levels of nitric oxide (NO), pro-inflammatory cytokines interleukin (IL)-1β (IL-1 β), interleukin (IL)-6 (IL-6), tumor necrosis factor (TNF-α) and anti-inflammatory cytokine IL-10 in the supernatant fraction were measured using enzyme-linked immunosorbent assay (ELISA). Expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) were detected via Western blot. The inhibition zones and minimal inhibitory concentration (MIC) values of bacterial strains were in the range of 14–26 mm and 62.5–250 μg/mL, respectively. Moreover, a remarkable activity against several fungal strains was revealed. TdLTP4 (5–100 μg/mL) decreased the production of NO (IC50 = 4.32 μg/mL), IL-6 (IC50 = 11.52 μg/mL), IL-1β (IC50 = 7.87 μg/mL) and TNF-α (IC50 = 8.66 μg/mL) by lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. TdLTP4 could modulate the macrophages inflammatory mode by causing reduction in iNOS and COX-2. According to these findings, TdLTP4 fusion protein could be used as natural anti-inflammatory and antimicrobial agent in food preservation and human health.