Ammonium Sulfate Precipitation Method Research Articles

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein

Background and Aim: African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. Materials and Methods: SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). Results: The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). Conclusion: This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well. Keywords: African swine fever, diagnosis, indirect enzyme-linked immunosorbent assay, p30, protein expression, purification.

Production of Nattokinase from Bacillus Amyloliquifaciens MRS18: A Bacterial Strain Isolated from Fermented Beans.

Nattokinase (NK) is a naturally occurring fibrinolytic protease enzyme obtained from the traditional Japanese food called Natto and has several uses in the pharmaceutical and medical industries. Nowadays, the most often used thrombolytic agent in the clinical field is NK, in part because it is less expensive than other thrombolytic medicines. The objective of this study is to investigate the screening, isolation and characterization of the NK enzyme-producing Bacillus strain from fermented Soya beans. The sample of fermented soya beans were tested for the presence of fibrinolytic protease- producing bacteria, followed by the screening, extraction, characterization and clot lysis assays. A total of three isolates were screened for caseinolytic activities by casein hydrolysis assay. Out of those isolates, MRS18 was found to be potent in producing the enzyme proteinase. To determine the taxonomy and phylogeny of these isolates, biochemical and molecular characterization has been carried out. Bacillus amyloliquiefaciens MRS18 has been found with the highest caseinolytic activity. The clot lysing ability of the potent strain Bacillus amyloliquiefaciens was found to be 61.7% after 120 min, and on further purification, by ammonium sulphate precipitation method, the lysis percentage was found to be 65.6 % after 120 min. From the results of the present study, we concluded that Bacillus amyloliquefaciens isolated from the fermented soya beans produced an NK enzyme that exhibits immense potential to lyse blood clots.

Expression and Purification of SMGL-1.

Recombinant protein expression is a crucial technique in biology, with E. coli being the most widely used expression system. However, due to growth pressure, the expression of large molecular weight proteins in E. coli has remained a challenging task. SMGL-1, a newly discovered protein in C. elegans with Rabin 8 function, plays an important role in biology. To better understand the function of SMGL-1, we first predicted and analyzed its protein structure and properties using artificial intelligence. We then conducted studies on its expression and purification. Through optimization of IPTG concentration and expression strains, we successfully expressed SMGL-1 in E. coli, providing guidance for the expression of large proteins in E. coli. Furthermore, we explored the purification of SMGL-1 using GST affinity chromatography, Nickel affinity chromatography, and ammonium sulfate precipitation methods, laying the foundation for future purification work on SMGL-1.

Anti-Cryptosporidium oocysts polyclonal antibodies for cryptosporidiosis diagnosis and protection

Cryptosporidiosis is an intestinal infection that is triggered by the protozoan parasite Cryptosporidium spp. Cryptosporidium oocysts can spread from one host to another either through direct contact with infected hosts' faeces or through indirect means (consumption of contaminated water or food). Significant numbers of oocysts are produced as a result of the rapid growth of the parasite within the infected hosts. For proper care of cryptosporidiosis, a laboratory diagnosis is necessary. Therefore, this study aimed to produce anti-Cryptosporidium parvum (C. parvum) oocyst immunoglobulin (Ig)G polyclonal antibodies (pAbs). The produced pAbs were used in the detection of C. parvum oocysts antigens in stool and serum samples of infected calves. Moreover, pAbs were tested in protection of balb-c male mice from cryptosporidiosis infection. C. parvum oocysts were used in the preparation of antigens to be used in the immunization of New Zealand white rabbits. pAb was purified by ammonium sulphate precipitation method, caprylic acid purification method and diethylaminoethyl (DEAE) anion exchange chromatographic method. Sandwich enzyme-linked immunosorbent assay (ELISA) (using prepared pAb) scored higher sensitivity (85% and 95% for serum and stool samples) than that (80%) of microscopic examination of stool samples. Moreover, pAb significantly reduced the oocysts shedding, decreased inflammatory cytokines and enhanced the loss in the body weight of protected animals. The prepared pAb succeeded in the diagnosis of cryptosporidiosis in calves with high sensitivity either in the serum or stool samples. Our results indicated the usefulness of using the prepared pAb in protection against cryptosporidiosis.

Production and characterization of egg yolk antibodies against the ZIKV NS2B expressed in Nicotiana benthamiana

The emergence of Zika virus (ZIKV) and its associated neonatal and congenital complications pose a threat to global health, particularly in tropical and subtropical regions with co-circulation of related flaviviruses and intense vector proliferation. Diagnosis of ZIKV by RT-PCR is limited to the viraemic phase and is not always accessible in low-income tropical settings, while serological tests often show cross-reactivity with other flaviviruses. Given the similarity of ZIKV symptoms to those of other arboviruses, but the different prognosis and risks, it is important to develop specific and accessible diagnostic tools. Egg yolk antibodies (IgY) were obtained from Leghorn laying hens immunized with recombinant ZIKV NS2B protein produced in agroinfiltrated Nicotiana benthamiana. After three immunizations, total IgY was recovered from the eggs by the 20% ammonium sulfate precipitation method. After characterisation by SDS-PAGE, dot blotting and ELISA, the IgY was adsorbed to dengue virus (DENV) from cell culture supernatants and tested for its ability to specifically detect ZIKV-positive sera samples. High yield and purity were observed on SDS-PAGE for polyclonal IgY, which reacted with NS2B at high titres in ELISA and detected both NS2B and ZIKV in dot blotting. However, a cross-reaction with DENV was observed and the anti-NS2B IgY was unable to discriminate ZIKV from DENV positive sera samples, even after adsorption with DENV. This is probably due to the phylogenetic relationship of the viruses and the shared identity of their proteins.

Bioefficacy of engineered Beauveria bassiana with scorpion neurotoxin, LqqIT1 against cotton mealybug, Phenacoccus solenopsis and cowpea aphid, Aphis craccivora.

Cotton mealybug, Phenacoccus solenopsis (Tinsley) and cowpea aphid Aphis craccivora (Koch) are notorious polyphagous, hemipteran sap sucking insect pests. A recombinant toxin gene 'LqqIT1' from the scorpion Leiurus quinquestriatus quinquestriatus (Ehrenberg) was cloned in the pAL1 fungal expression vector and then expressed in the entomopathogenic fungus Beauveria bassiana (Balasmo) using genetic modification techniques. The genetically transformed B. bassiana strain (BbLqqIT1-3) and its un-transformed parent strain (Bb-C) were screened to infect the third instar nymphs of P. solenopsis and first instar nymph of A. craccivora through leaf treatment and topical application (spray) method at 1 * 107 spores per ml concentration. The recombinant strain BbLqqIT1-3 was highly pathogenic against A. craccivora but non pathogenic to P. solenopsis. BbLqqIT1-3 induced 72 and 43.33% mortality in A. craccivora nymphs 96 h after leaf treatment and topical application, respectively. The nymphs of A. craccivora infected with BbLqqIT1-3 displayed classical neurotoxic symptoms such as sluggishness, solublize and liquification of the body. Crude soluble toxin protein, BbLqqIT1a-CSE and Bb-WT-CSE was extracted from the BbLqqIT1-3 and Bb-C, respectively using ammonium sulphate precipitation method, and their oral toxicity was analyzed at 5 µg/ml concentration. The survival of the studied insects was negatively affected by the crude soluble toxin extracts. The LT50 values of BbLqqIT1a-CSE against P. solenopsis and A. craccivora were 22.18 and 17.69 h, respectively. Exposure to crude soluble toxin extracts also accounted for the imbalance of ionic concentrations in the hemolymph of treated insects such as hyperpotassemia (3.53-8.18 meq/ml) in the P. solenopsis and hypopotassemia (7.52-0.47 meq/ml) in A. craccivora. The transformed fungus BbLqqIT1-3 strain exhibited promising results in invitro study.

Extraction and Purification of Bromelain Enzyme from Fruits and its Therapeutic Application Study

Food processing industries release a lot of fruit wastes mainly of seed, skin, rind, pomace, crown etc. containing potential bioactive compounds. Bromelain is a crude protein extracted from certain fruits. Bromelain is a sub-type of cysteine protease enzyme extensively investigated for its therapeutic properties such as anti-microbial and anti-oxidant activity with good ferric reducing power. The present study deals with isolating and purifying bromelain from kiwi and avocado fruits and assessing their therapeutic application. Fruit samples were taken from a juice shop in VIT, Vellore, for performing all the experimental analysis. Different parts such as stem, pulp, peel and leaves of the avocado and kiwi fruits were collected for the bromelain extraction process. The extraction of protein was done by the ammonium sulfate precipitation method. Estimation of protein content was performed and the concentration of crude enzyme was found to be 970μg/ml and 900μg/ml for avocado peel and kiwi pulp respectively. Avocado peel showed good activity in ferric reducing power and also a potent radical scavenging activity of 40% at 200μg concentration. Crude bromelain extracted from both fruits showed a broad spectrum of antimicrobial activity against Escherichia coli and Staphylococcus aureus. Hence it can be concluded that bromelain can be used as an effective health supplement.

A Chitosan-Based Flocculation Method for Efficient Recovery of High-Purity B-Phycoerythrin from a Low Concentration of Phycobilin in Wastewater.

Increasing the yield and purity of B-phycoerythrin (B-PE) can improve the economic state of microalgae industrial processing. One method of cost reduction involves the recovery of remaining B-PE from wastewater. In this study, we developed a chitosan (CS)-based flocculation technique for the efficient recovery of B-PE from a low concentration of phycobilin in wastewater. We investigated the effects of the molecular weight of chitosan, B-PE/CS mass ratio, and solution pH on the flocculation efficiency of CS and the effects of phosphate buffer concentration and pH on the recovery rate of B-PE. The maximum flocculation efficiency of CS, recovery rate, and purity index of B-PE were 97.19% ± 0.59%, 72.07% ± 1.37%, and 3.20 ± 0.025 (drug grade), respectively. The structural stability and activity of B-PE were maintained during the recovery process. Economic evaluation revealed that our CS-based flocculation method is more economical than the ammonium sulfate precipitation method is. Furthermore, the bridging effect and electrostatic interaction play important roles in B-PE/CS complex flocculation process. Hence, our study provides an efficient and economical method to recover high-purity B-PE from a low concentration of phycobilin in wastewater, which promoted the application of B-PE as a natural pigment protein in food and chemical applications.

BIOCHEMICAL CHARACTERIZATION AND PARTIAL PURIFICATION OF A BACTERIOCIN LIKE-INHIBITORY SUBSTANCE PRODUCED FROM Bacillus sp. T68 STRAIN

Bacteriocins are peptides produced by various types of bacteria. Members of the Bacillus genus are known to produce compounds with inhibitory activity in protein structure against pathogenic and non-pathogenic microorganisms. In this study, a bacteriocin-like inhibitory substance (BLIS) synthesized by Bacillus sp. T68 strain, which was previously isolated from soil, was characterized. T68 strain was grown on different media to produce bacteriocin. Crude BLIS obtained was tested by well diffusion method against indicator bacterium. It was investigated within the temperature range of 10-121 C and pH range from 3.0 to 10.0. It was examined in terms of different organic solvents, enzymes and chemicals. Extracellularly produced BLIS was partially purified by ammonium sulphate precipitation method and analyzed on SDS-PAGE. Activity of partially purified BLIS was investigated. It was observed that BLIS produced in Luria Bertani Broth medium was higher as compared to the other media against indicator bacterium at 30 °C for 24 h. BLIS activity maintained at low temperatures (10-40 °C) and lost completely at high temperatures (> 60 °C). It was detected that BLIS exhibited activity in the pH range between 5.0 and 10.0. The effect of EDTA on BLIS activity was slightly positive. Proteinase K and trypsin inhibited BLIS activity. Among the detergents, sodium dodecyl sulphate and Triton X-100 reduced BLIS activity, while Tween 20 and Tween 80 retained it. Additionally, it was determined that application of Tween 20 at 30 °C for 5 hours increased the BLIS activity by 40%. It was found that the solvents used other than butan 1-ol preserved the BLIS activity over 80%. Chloroform and isopropanol increased the BLIS activity slightly. An inhibitory zone formed by the BLIS corresponding to a molecular weight of about 15 kDa was detected. This BLIS exhibited stability over wide pH and temperature ranges and in organic solvent treatments.

LIPASE PRODUCTION FROM WASTE FRUIT PEELS FOR BIODIESEL PRODUCTION

Waste pineapple and passion fruit peels have potency as lipase enzyme sources. The combination of both peels with a ratio of 6:4 has the highest enzyme lipase activity after 3 days of fermentation and it has increased to 28.33 ± 0.0 U/ml after purification using the ammonium sulfate precipitation method. The response surface method design experiment was applied to determine the effect of the ratio of molar oil to methanol (1:3 – 1:9), reaction time (1 – 24 hours), and temperature (30 - 60C). The maximum biodiesel conversion of 82.7% could be achieved using the reaction condition of a ratio molar of 1:9, 13.65 hours reaction time, and a temperature of 46.5C for enzymatic transesterification of PO. Further, for bio-catalysis biodiesel production from WCO, the maximum conversion of 88% was achieved under reaction conditions of a ratio molar of 1:9, 14 hours reaction time, and a temperature of 48C.

Production of Anti-Camel IgY for diagnosis of infectious diseases affecting camels located in Kingdom of Saudi Arabia

Chicken can be used for the production of hyper-immune serum for many type of antigens specially, the antigens of mammals and other species. Therefore, the current study aimed to use the chickens for the productions of anti-camel IgY- antibodies using ammonium sulphate and ammonium sulphate - caprylic acid methods for detection of any infectious disease affecting camels.One ml of purified camel IgG of mixed with complete freund's adjuvant was injected intramuscularly and S/C to four groups of laying hens (Hisex strain, 140 days old). The first booster dose was administrated two week after first immunization with one ml of purified camel IgG mixed with incomplete freund's adjuvant and the hens were repeatedly boostered (three times) at two weeks intervals. During the immunization periods we collect the eggs every day and the anti-camel immunoglobulins Y was separated by using two extraction methods; the ammonium sulphate precipitation method and the ammonium sulphate caprylic acid method, the sensitivity and the specificity of the recovered antibody against the camel IgG were evaluated using SDS-PAGE and ELISA.Two weeks after the initial dose of immunizing the hens the anti-camels IgY-antibodies were detected then the mean antibody titer increased significantly (P < 0.05) till became the highest level at 10 weeks post immunization. The antibody titer remain at high level without significant increase (P > 0.05) up to 12 weeks after the first dose of immunization then started to decrease.Therefore, there are several benefits of using immunized chicken to produce IgY antibodies rather than using other species such as mammals in order to prepare anti-camels antibodies conjugates for diagnosis of infectious disease affecting camels.

Toxicity of crude toxin protein produced by Cordyceps fumosorosea IF-1106 against Myzus persicae (Sulze)

The entomopathogenic fungus Cordyceps fumosorosea IF-1106 is a potential biocontrol agent with high pathogenicity to the aphid Myzus persicae. We extracted the crude toxin protein from a liquid culture broth of an isolated C. fumosorosea strain using the ammonium sulfate precipitation method, and its toxicity to Myzus persicae was measured by injection, oral exposure, and topical exposure. The crude toxin protein of C. fumosorosea had insecticidal activity against M. persicae. Body cavity injection and oral exposure had significantly higher insecticidal activity against adults than contact sprays. The highest cumulative corrected mortality of adults after injection was 81.85 ± 13.45 %, and the highest cumulative corrected mortality of adults after ingestion was 85.45 ± 11.88 %. The proportion of plasmatocytes in adult blood lymphocytes reached the highest at 3 days after injection and feeding, and the proportion of granulocytes was the highest at 2 days after injection and feeding. These data confirmed the toxicity of the crude toxin protein of C. fumosorosea toxin to M. persicae and helped clarify the pathogenic mechanism of the strain. Population management of M. persicae may be possible by using a natural toxic compound produced by C. fumosorosea that is selective to this pest species.

The crude oil biodegradation activity of Candida strains isolated from oil-reservoirs soils in Saudi Arabia

Crude oil (petroleum) is a naturally occurring complex composed of hydrocarbon deposits and other organic materials. Bioremediation of crude oil-polluted sites is restricted by the biodiversity of indigenous microflora. They possess complementary substrates required for degrading the different hydrocarbons. In the current study, four yeast strains were isolated from different oil reservoirs in Riyadh, Saudi Arabia. The oil-biodegradation ability of these isolates showed variable oxidation effects on multiple hydrocarbons. The scanning electron microscopy (SEM) images showed morphological changes in Candida isolates compared to the original structures. The drop-collapse and oil emulsification assays showed that yeast strains affected the physical properties of tested hydrocarbons. The content of biosurfactants produced by isolated strains was quantified in the presence of different hydrocarbons to confirm the oil displacement activity. The recovery assays included acid precipitation, solvent extraction, ammonium sulfate, and zinc sulfate precipitation methods. All these methods revealed that the amount of biosurfactants correlates to the type of tested hydrocarbons, where the highest amount was produced in crude oil contaminated samples. In conclusion, the study highlights the importance of Candida isolated from contaminated soils for bioremediation of petroleum oil pollution. That raises the need for further analyses on the microbes/hydrocarbon degradation dynamics.

Antidiabetic bio-peptides of soft and hard wheat glutens

The effects of various purification techniques on kiwifruit enzyme characteristics (protease activity, kinetic parameters, and protein patterns) and production of wheat gluten bio-active peptides were investigated. The enzyme extract purified by ammonium sulfate precipitation method exhibited the highest protease activity (26), Km (0.04 ± 0.002 mM), Kcat/Km (40), and yield (96%). Using actinidin, the hard and soft wheat gluten subunit proteins produced antidiabetic inhibitory (α-glucosidase and α-amylase) peptides. The smallest Mw fraction of soft wheat gliadin peptide (<1 kDa) showed the highest inhibitory capacity against α-glucosidase (18.4 ± 0.7%) and α-amylase (53.3 ± 1.9%). The presence of high levels of amino acids with hydroxyl groups and proline in P3 sub-fraction had a critical role on α-glucosidase (47.2%) and α-amylase (71.2%) inhibitory activities. In conclusion, wheat gluten subunit peptides showed significant metabolic effects relevant to glucose and insulin control in vitro.

ISOLATION, SCREENING, AND PARTIAL CHARACTERIZATION OF OXALATE OXIDASE ENZYME FROM PSEUDOMONAS STRAIN UNDER INDUCED OXALATE STRESS CONDITION

Oxalate oxidase (OxO), a manganese dependent enzyme, is involved in the catalysis of oxalate oxidation to carbon dioxide with the formation of hydrogen peroxide. OxO is present in the cell wall of plants. It increases the resistivity against diseases and external stress. Oxalate is found as waste metabolites in mammals, excess accumulation of oxalate results in hyperoxaluria and urolithiasis in humans. These disorders can be diagnosed by the help of OxO in many analytical methods. The present study is aimed on isolation of OxO enzyme from Pseudomonas strain under high salt stress. The OxO enzyme was produced in bulk under selected salt stress. It was partially purified by ammonium sulphate precipitation method, dialysis and ion-exchange chromatography. The OxO enzyme on enzymatic analysis showed potent activity concerning oxalic acid substrate. The microtiter plate analysis confirmed the ability of OxO enzyme purified from Pseudomonas strain in the diagnosis of oxalate-related disorders and in the future could be used to prepare the diagnostic biosensors.

Cytotoxic and apoptotic induction activity of protein isolated from blood clam (Anadara (tegillarca) granosa, Linnaeus 1758) Against breast cancer t47d cell line

Background: Cancer has become a major cause of death today. These risk factors are also balanced with the development of drugs, one of which comes from nature. Medical products derived from natural resources such as blood clam (Anadara (tegillarca) granosa) have been already developed. Crude protein extracted from blood calm contains a 20-kDa protein which had been known to be able to inhibit the growth of HT-29 cell line. Purpose: This study aimed to determine cytotoxic activity and apoptotic effect of crude protein which is isolated from blood calm against breast cancer T47d cell line. Materials and Methods: This study used the ammonium sulfate precipitation method for the isolation of a crude protein of blood clams. MTT assay method was performed to determine cytotoxicity and to know the value of IC50, apoptosis test qualitatively and quantitatively was done by double-staining using ethidium bromide and acridine orange as well as flow cytometric dye using annexin V dyes and propidium iodide. Findings: Crude protein clams of blood have IC50 value of 11.11μg/ml and can induce apoptosis of breast cancer cell line (T47D) at 15 μg/ml concentration by double-staining method, but the calculation with flow cytometry method still shows different results in this study. Research Limitations: This study observed the inhibitory concentration of crude extract consisting of proteins with various molecular weights against the t47d cell line. Originality: The results of this study indicate that crude protein clams are cytotoxic and can induce apoptosis in breast cancer cell lines (T47D).

Antimicrobial activity of bacteriocin produced by a new Latilactobacillus curvatus sp.LAB-3H isolated from traditional yogurt.

In recent years, the use of bacteriocin-producing Lactobacillus species has received much attention in different areas, including using as probiotics, food preservation, and as broad antimicrobial spectrum activity. In this study, a bacteriocin-producingLactobacillusstrain was isolated from traditional yogurt. The isolate was identified by morphological, biochemical, 16S rRNA analyses, and designated asLatilactobacillus curvatusLAB-3H. The primary antimicrobial activity of the isolate was evaluated onMicrococcus luteusPTCC 1408 by the agar gel diffusion method. The growth ofL. curvatusLAB-3H in MRS broth at 37°C and its antimicrobial activity againstLactobacillus caseiATCC 39,392 was evaluated. In batch culture analysis, bacteriocin production starts at the early exponential growth phase and continues to the middle of the stationary phase about (660 AU/ml) at 28h and pH 3.8. Bacteriocin produced byL. curvatusLAB-3H showed antibacterial activity against some selected foodborne pathogens, includingListeria monocytogenesPTCC1294,Bacillus cereusPTCC1857,Staphylococcus aureusPTCC1917, andEscherichia coliPTCC1276. For purification of bacteriocin, ammonium sulfate precipitation and cation exchange chromatography methods were used. The maximum antimicrobial activity observed was about 3985.15 AU/mg of protein, which was a 249.22-fold increase, and 5.21% yield compared with that in the cell-free supernatant. The molecular weight of bacteriocin was approximately 55kDa by SDS-PAGE. The obtained results in this study demonstrate that bacteriocin fromL. curvatusLAB-3H is a potential candidate for controlling microbial contaminations and can be used in different sectors, such as food industries.

ISOLATION, SCREENING, AND PARTIAL CHARACTERIZATION OF OXALATE OXIDASE ENZYME FROM PSEUDOMONAS STRAIN UNDER INDUCED OXALATE STRESS CONDITION

Oxalate oxidase (OxO), a manganese dependent enzyme, is involved in the catalysis of oxalate oxidation to carbon dioxide with the formation of hydrogen peroxide. OxO is present in the cell wall of plants. It increases the resistivity against diseases and external stress. Oxalate is found as waste metabolites in mammals, excess accumulation of oxalate results in hyperoxaluria and urolithiasis in humans. These disorders can be diagnosed by the help of OxO in many analytical methods. The present study is aimed on isolation of OxO enzyme from Pseudomonas strain under high salt stress. The OxO enzyme was produced in bulk under selected salt stress. It was partially purified by ammonium sulphate precipitation method, dialysis and ion-exchange chromatography. The OxO enzyme on enzymatic analysis showed potent activity concerning oxalic acid substrate. The microtiter plate analysis confirmed the ability of OxO enzyme purified from Pseudomonas strain in the diagnosis of oxalate-related disorders and in the future could be used to prepare the diagnostic biosensors.

Develop and characterize thermally reversible transparent gels from pea protein isolate and study the gel formation mechanisms

Gels with thermo-reversibility and transparency like gelatin have been applied in various products in food and pharmaceutical industry. The replacement of gelatin by ingredients of plant origin has attracted attentions owing to the emerging markets of vegan, Halal and Kosher products. This work reports a thermo-reversible protein gel prepared with pea protein isolate extracted using ammonium sulfate precipitation method. The protein concentration and pH were discovered to be critical, and within a window of pH 2.4–4.2 and protein concentration of 10–15%, thermo-reversible gels were obtained, many of which possessing good mechanical properties with compressive stress up to 6.32 ± 1.40 kPa. Dynamic rheological measurement disclosed the thermo-reversibility of the gel being stable upon repeated heating and cooling process from 80 °C to 4 °C. Lower pH favored the formation of transparent gels with fine-stranded network as observed by scanning electron microscopy (SEM), whereas with the increase of pH up to 4.2, the gel gradually became opaque when the gel network changed to particulate aggregates while gel thermo-reversibility retained. Hydrogen bonds were shown to be dominant in forming the gel network structure during the cooling process while hydrophobic interactions and disulfide bonds were not significantly involved. The pea protein gel has the characteristics of being transparent and thermo-reversible with good mechanical property, which has provided opportunities of replacing gelatin in food applications such as fruits, beverage and fermented food products with mild acidic pH environment.

Defluoridation of water with a coagulant, Strychnos potatorum L. seed –agglutinin

The aim of the present study is to assess the suitability of Strychnos potatorum L. seed protein for defluoridation of potable water. Protein was isolated from Strychnos potatorum L. seed by using Ammonium sulphate precipitation method and coagulation activity was confirmed. Later the extracted protein was used for defluoridation studies of aqueous fluoride solution and real potable water. The effect of three parameters including pH, time and coagulant dosage of protein on the fluoride removal were studied in fluoride aqueous solution. About 52 potable water samples in and around Kodaikanal were collected from tribal and non-tribal areas and water quality was assessed. The exceeding amount of fluoride among 5 water samples was found when compared with BIS standard for drinking water and subjected to defluoridation process with coagulant protein. Strychnos potatorum L. seed coagulant protein removed about 75% of fluoride from aqueous fluoride solution (2 ppm) by 2 h treatment time, at 6.6 pH with 0.1 g of coagulant dose, treatment. Potable water defluoridation also showed 75% of fluoride reduction with 0.1 g of coagulant dose treatment. Seed characterization was performed using GC–MS and FT-IR, also proteomic studies carried out using MALDI-TOF. The seed coagulant protein showed, horcolin, a lectin type agglutinin protein (15.1 kDa) with 146 amino acid residues. This study suggests, the horcolin agglutinin protein in seeds is capable to remove fluoride effectively from drinking water samples due to the presence of its metal binding sites. • Strychnos potatorum L. seed coagulant protein was introduced for defluoridation. • Effect of pH, time and dosage on fluoride removal of aqueous solution was studied. • Seed coagulant defluorinated maximum 75% of fluoride from water samples. • FT-IR and GC–MS confirmed amide group for defluoridation and antioxidant property. • Coagulant horcolin protein was identified at 15.1 kDa with 146 amino acids.