Ammonium Sulfate Precipitation Research Articles

A novel cyclic dipeptide from Lactiplantibacillus plantarum MC39 inhibits proliferation of multidrug-resistant Klebsiella pneumoniae W8 and Enterobacter hormaechei U25

The present study pays special attention to lactic acid bacteria products, cyclic dipeptides (CDPs), as an effective prevention strategy that can be used to eradicate the threats posed by multidrug-resistant (MDR) Enterobacteriaceae. Herein, Lactiplantibacillus plantarum MC39, a strain isolated from an artisanal Egyptian cheese, was a producer of a novel CDP that could strongly inhibit, in vitro, MDR Klebsiella pneumoniae W8 and Enterobacter hormaechei U25, for the first time. Following partial purification with 80% ammonium sulphate precipitation and Sephadex G-100 chromatography, this biomolecule was identified as the cyclic dipeptide cyclo(Ala–Glu) using IR, HNMR, and LC–MS. Cyclo(Ala–Glu) was thermostable up to 121 °C, insensitive to pepsin and α-chymotrypsin, tolerant to organic solvents, active at acidic pH, and stable for 45 days of storage. Thus, it shows clear evidence to be usable as a bio-preservative in dairy products and as a potential therapeutic agent, controlling MDR K. pneumoniae and E. hormaechei.

Concurrent purification of phycobiliproteins from Leptolyngbya sp. and their selective enhancement in response to different wavelengths of LED light

The present study identifies freshwater cyanobacterium Leptolyngbya sp. CMSACR2013 as a potential producer of phycobiliproteins. Concurrent purification of phycobiliproteins using the three-stage ammonium sulfate precipitation technique was employed to recover phycocyanin and phycoerythrin simultaneously. This was followed by anion exchange chromatography, resulting in an analytical grade C-phycoerythrin (Purity = 4.6) and reactant grade C-phycocyanin (Purity = 3.5). Moreover, to understand the relative pigment production in Leptolyngbya sp., the cultures were exposed to different wavelengths of LED lights and cultivated for 28 days. C-phycoerythrin content was higher under green light (74.77 mg L−1). In contrast, C-phycocyanin and C-allophycocyanin were higher under red light (72.94 mg L−1 and 63.50 mg L−1). The results also revealed the highest biomass content under red light (2.20 g L−1). These results suggest exploiting the complementary chromatic adaptation exhibited by the strain to improve the selective accumulation of phycobiliproteins.

Cross-linked enzyme aggregates (combi-CLEAs) derived from levansucrase and variant inulosucrase are highly efficient catalysts for the synthesis of levan-type fructooligosaccharides

Efficient and convenient access to short to medium chain length levan-type fructooligosaccharides (LFOS) is needed in order realise the nutritional potential of this class of bioactive oligosaccharides. While LFOS are synthesised by fructansucrase enzymes, these reactions are routinely associated with high molecular weight fructan polymer formation. Recent studies have shown that FOS production can be enhanced by the combination of levansucrase and inulosucrase in a one-pot reaction. In the present study, the novel mixed enzyme cross-linked enzyme aggregates (combi-CLEAs) based on levansucrase (Lev) and N543A variant inulosucrase (Inu) were prepared by ammonium sulfate precipitation, followed by glutaraldehyde cross-linking. The effect of Lev and Inu ratio on the activity of combi-CLEAs was explored. The results showed that >70% of total sucrase activity was recovered after immobilization and that the combi-CLEAs produced high amounts of LFOS (degree of polymerisation 3 to 21), while high molecular weight polysaccharide production was much reduced. Biochemical characterisation indicated that the optimum pH and temperature of combi-CLEAs (pH 5.5 and 50ᵒC, respectively) were comparable to those of free enzyme; however, the stability of the enzyme was improved. In addition, these combi-CLEAs have operational stability for several reaction cycles, which makes them very attractive for biotechnology applications.

Development of a microbial protease for composting swine carcasses, optimization of its production and elucidation of its catalytic hydrolysis mechanism

BackgroundDead swine carcass composting is an excellent method for harmless treatment and resource utilization of swine carcass. However, poor biodegradation ability of traditional composting results in poor harmless treatment effect. Researches report that the biodegradation ability of composting can be improved by inoculation with enzyme-producing microorganisms or by inoculation with enzyme preparations. At present, the researches on improving the efficiency of dead swine carcass composting by inoculating enzyme-producing microorganisms have been reported. However, no work has been reported on the development of enzyme preparations for dead swine carcass composting.MethodologyThe protease-producing strain was isolated by casein medium, and was identified by 16 S rRNA gene sequencing. The optimal fermentation conditions for maximum protease production were gradually optimized by single factor test. The extracellular protease was purified by ammonium sulfate precipitation and Sephadex G-75 gel exclusion chromatography. The potential for composting applications of the purified protease was evaluated by characterization of its biochemical properties. And based on amino acid sequence analysis, molecular docking and inhibition test, the catalytic hydrolysis mechanism of the purified protease was elucidated.ResultsIn this study, a microbial protease was developed for swine carcass composting. A protease-producing strain DB1 was isolated from swine carcass compositing and identified as Serratia marcescen. Optimum fermentation conditions for maximum protease production were 5 g/L glucose, 5 g/L urea, 1.5 mmol/L Mg2+, initial pH-value 8, inoculation amount 5%, incubation temperature 30 °C and 60 h of fermentation time. The specific activity of purified protease reached 1982.77 U/mg, and molecular weight of the purified protease was 110 kDa. Optimum pH and temperature of the purified protease were 8 and 50 °C, respectively, and it had good stability at high temperature and in alkaline environments. The purified protease was a Ser/Glu/Asp triad serine protease which catalyzed substrate hydrolysis by Glu, Arg, Ser, Asp and Tyr active residues.ConclusionsIn general, the microbial protease developed in this study was suitable for industrial production and has the potential to enhance composting at thermophilic stage. Moreover, the catalytic hydrolysis mechanism of the protease was further analyzed in this study.

Isolation, Characterization, and Effect on Biofilm Formation of Bacteriocin Produced by Lactococcus lactis F01 Isolated from Cyprinus carpio and Application for Biopreservation of Fish Sausage.

The aim of this work was the screening of bacteriocin-producing LABs isolated from fish, the selection of promising/prominent strain(s), the characterization of the bacteriocin produced, and the evaluation of its potential to be used as biopreservative(s). Amplification and sequencing of the 16S rRNA gene of the bacteriocin-producing strain was performed. Then a partial purification of the produced bacteriocin, using a combination of ammonium sulfate and chloroform-methanol precipitation, was done. Its molecular weight was determined by SDS-PAGE. In addition, the action spectrum, the hemolysis test, and its ability to inhibit biofilm formation were analyzed. A total of 88 isolates of lactic acid bacteria (LAB) including one bacteriocin producer, which was identified as Lactococcus lactis F01, were collected. The bacteriocin was partially purified with an estimated yield of 40%. Regarding the SDS-PAGE profile, the secreted bacteriocin has molecular weight of about 3.5 kDa and was identified as class I bacteriocin. The antimicrobial test showed that the bacteriocin inhibits pathogenic and/or spoilage bacteria, 10 Gram-positive and 16 Gram-negative bacterial species. Moreover, it can inhibit biofilm formation from 1.3% (Escherichia coli) to 63.92% (Pseudomonas aeruginosa ATCC15692) depending on the strain. The hemolytic activity of novel bacteriocin was observed at the concentration of 10 μg/ml of bacteriocin crude extract, which was 0.7 ± 0.0029%. In addition, it exhibited good thermal and pH stability with retained antibacterial activity of 85.25% after treatment at 121°C for 20 min, as well as at a pH range between 2.0 and 10.0. Moreover, this bacteriocin showed the ability to inhibit the growth of bacterial culture load in fish sausage stored at 8°C for 28 days. Considering the results obtained, bacteriocin could be potentially exploited as an alternative to chemical preservatives or as a substitute for antibiotics.

Purification of Lipase from Pseudomonas aeruginosa VSJK R-9 and Its Application in Combination with the Lipolytic Consortium for Bioremediation of Restaurant Wastewater.

Microorganisms are very important in biodegradation of edible oil contaminated effluents, and they find an excellent scope in restaurant wastewater bioremediation. The edible oil in such wastewater harms the environment in a number of ways. The native bacteria in the wastewater are less effective in degrading edible oil. It leads to the formation of blockage along the sewer line. This narrows the diameter of line and forms partial to complete blockage causing overflows of wastewater exposing humans and animals to diverse pathogens. A consortium of lipolytic bacteria and lipase enzyme gives a new approach for effective and environment friendly degradation of waste oil in restaurant wastewater. In the present study, the lipase produced by Pseudomonas aeruginosa VSJK-R9 isolated from restaurant wastewater was purified by ammonium sulfate precipitation, dialysis and gel exclusion chromatography-Sephadex G-100, with 11.45-fold purification to obtain a yield of 35.08%. Its molecular mass was around 50kDa as determined by SDS-PAGE analysis. The bioremediation of restaurant wastewater supplemented with 0.5% NH4Cl and 0.8% K2HPO4 was studied with lipolytic consortium formed by the combination of lipolytic isolates Acinetobacterjunii VSJK-R6, Pseudomonas compostiVSJK-R8 and Pseudomonas aeruginosa VSJK-R9. Further, the impact of lipase supplementation was also evaluated, and it was found that the action of consortium was boosted by lipase. The oil and chemical oxygen demand value of the restaurant wastewater was considerably decreased. These findings have shown the application of lipase for bioremediation of restaurant wastewater and its positive impact on the performance of lipolytic consortium.

Application of Anoxybacillus gonensins UF7 lipase as a catalyst for biodiesel production from waste frying oils

This study was carried out to purify and characterize the lipase of locally isolated bacterium Anoxybacillus gonensis UF7 (GenBank accession: OM328064), and to investigate its applicability as a catalyst for biodiesel production. After ammonium sulphate precipitation and ion-exchange chromatography steps, the lipase of A. gonensis UF7 was purified with a specific activity of 1370 U/mg protein, a purification fold of 13.1 and a recovery yield of 40.65 %. The molecular weight of the enzyme was determined as 73 kDa by SDS-PAGE. The lipase showed maximum activity and stability at pH 8.0 and 60 °C. Its activity and stability were also high in the presence of methanol, ethanol, SDS, Tween-80, Triton X-100, Tween 80, Ca2+ and Mg2+. To prepare biodiesel from waste frying oils using purified lipase, the optimum reaction conditions were determined as a reaction time of 24 h, an enzyme concentration of 3 mL and an oil:methanol ratio of 3:1 (30 mL:10 mL). Transesterification yield was calculated as 87.7 %. The main fatty acid methyl esters of the biodiesel were methyl oleate (50.8 %), methyl palmitate (20.2 %) and methyl linoleate (16.6 %). The usability of lipases of Anoxybacillus strains including A. gonensins as a catalyst in biodiesel production was investigated for the first time in the present study.

Potential use of proteolytic bacteria Paenibacillus dendritiformis (BT7) isolated from Batu tannery effluents for the detergent industry

This research was aimed at identifying a bacterium that can produce alkaline proteases. As a result, bacteria that produce proteases were isolated from Batu tannery effluents, tested for protease synthesis on skim milk agar plates, and validated with a protease assay. Microscopic and molecular phylogenetic analyses identified Paenibacillus dendritiformis (BT7) as the bacterial isolate with the highest alkaline protease production. The isolate's maximum enzyme production was obtained by 2% inoculum size, 40°C temperature, 9.0 pH, and a 48-hour incubation time with production media components such as glucose, casein, MgCl2, and 2% NaCl. The maximal enzyme activity was 270 U/mL under all optimum culture conditions. Concentrated ammonium sulfate precipitation (75%) and dialysis were employed to obtain a cell-free, partially purified protease. The specific activity of the dialysate, which accounts for 3% of the enzyme yield, was discovered to be 134 U/mL. The partially purified protease was used for application in blood stain removal. It was studied and found that the alkaline protease resistance under stringent conditions is very stable with bleach detergent. Also, this enzyme could clean blood-stained fabrics. This study shows that the alkaline protease from Paenibacillus dendritiformis - BT7 could be used in various ways in the detergent industry that are good for the environment.

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.

Characterization of novel bacteriocin produced by bacteriocinogenic Carnobacterium maltaromaticum isolated from raw milk

This study was designed to assess newly isolated bacteriocin-producing strain as potential food preservative. A bacteriocin producing lactic acid bacterium, named Carnobacterium maltaromatium KCA018, was screened from raw milk using deferred and spot-on-the-lawn assays. The crude cell free supernatant (CFS) was purified to obtain proteinaceous bacteriocin by ammonium sulfate precipitation (assigned as bacteriocin KCA) and tested for bacteriocin production, physical stability, antimicrobial activity, and bacteriocin-encoding gene detection. The growth curves of C. maltaromatium KCA018 reached late exponential phase after 15 h of incubation at 25 °C and 30 °C (Fig. 2). The maximum production of bacteriocin KCA was reached after 12 h of incubation at 25 °C, showing the antimicrobial activity of more than 3000 AU/ml against Listeria monocytogenes. The purified bacteriocin KCA was stable up to 67 °C for 30 min of exposure and between pH 4 and 7, showing more than 6000 AU/ml. The antibacterial activity of bacteriocin KCA was lost in the presence of pronase, proteinase K, and trypsin. Purified bacteriocin KCA showed higher antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. The CFS and purified bacteriocin KCA effectively inhibited the growth of L. monocytogenes ATCC 1911, E. faecalis ATCC 19433, and E. feacium ATCC 11576. The molecular weight of purified bacteriocin KCA was estimated at approximately 5 kDa. The positive amplification was observed for pisA and cbnBM1 with approximately between 100 and 200 bp. The newly identified bacteriocin can be a promising preservative for application in food.

Purification and Identification of Lipid-Lowering Protein from Barley Extract after Lactiplantibacillus plantarum dy-1 fermentation

Previous studies have found that the protein in barley extract fermented by Lactiplantibacillus plantarum dy-1 has the ability to inhibit lipid accumulation. However, the isolation, purification, and structural identification of the protein with lipid-lowering activity were still needed. In the present study, barley protein fermented by L. plantarum dy-1 with the optimal lipid-lowering ability was isolated and purified in three steps: using ammonium sulfate precipitation, anion-exchange chromatography, and size-exclusion chromatography. Combined with the model of HepG2 cells induced by oleic acid, the results showed that the pure protein LFBEP-C1 had the best lipid-lowering potential. Furthermore, our research found that LFBEP-C1 enriched the content of hydrophobic amino acids in LFBEP-C1. Ultraviolet spectroscopy analysis indicated that the glycosidic bond in LFBEP-C1 was an O-type glycosidic bond. The FTIR and circular dichroism spectra indicated that α-helix and random coil were the main secondary structures of LFBEP-C1. Mass spectrometry determined the theoretical molecular weight of LFBEP-C1 as 48 kDa, and its amino acid coverage was 63%. These findings suggest that the protein LFBEP-C1 with the best lipid-lowering activity was isolated and purified, and its structural characteristics were identified.

Purification of an antiviral protein from the seeds of quinoa (Chenopodium quinoa Willd.) and characterization of its antiviral properties.

Plant viral pathogens cause damaging diseases in many agriculture systems, and emerging viral infections are a serious threat for providing adequate food to a continuously growing population. Recent studies of biogenic substances have provided new opportunities for producing novel antiviral agents. The present work has been conducted to evaluate the antiviral activity of quinoa (Chenopodium quinoa Willd.) seeds crude extract. The antiviral activity was retained in different buffer solutions of various pH ranges (5.2-8.5) and remained after the diafiltration process. The putative virus inhibitor was sensitive to treatment with sodium dodecyl sulfate and trichloroacetic acid. An antiviral protein with ~ 25kDa molecular weight was isolated from the seed quinoa extract using ammonium sulfate precipitation, anion and cation exchange chromatography. The purified protein (Quinoin-I) significantly inhibited TMV on tobacco leaves with an IC50 value at a 6.81μg/ml concentration. Enzyme activity assay revealed the RNase activity of Quinoin-I, and this feature was retained in the presence of β-mercaptoethanol and ethylene diamine tetraacetic acid. This antiviral protein has been shown as a promising leading molecule for further development as a novel antiviral agent.

Extracellular Protease Production, Optimization, and Partial Purification from Bacillus nakamurai PL4 and its Applications

The major goal of the study was to isolate bacteria synthesizing protease enzyme from a soil sample taken in Dandeli, Karnataka, India. Furthermore, screening, production, and optimization of medium components for maximum protease activity, partial purification of crude enzymes, and application of protease produced by Bacillus nakamurai were carried out. At 72 hrs and pH 6, the optimum incubation time, pH, and temperature were evaluated (acidic and thermophilic). As a substrate, casein was employed. Plackett-Burman screening was performed, and KH2PO4, xylose, MnCl2, and peptone were discovered to be essential components in protease production media. Following the production and optimization processes, partial purification was performed using ammonium sulfate precipitation, with the maximum protease activity at 60% ammonium sulfate, and further dialysis was performed using precipitated enzyme, yielding enzyme activity of 0.747 U/mL. The protease enzyme proved effective at removing egg yolk stains as well as degrading (dehairing) chicken feathers and hairs from goat skin. Bacillus nakamurai PL4 can be utilized for the industrial-scale production of proteases to fulfill current demands. Thus, such optimized parameters can optimize protease production and their application across various industries.

Preparation, Purification and Performance Evaluation of Polyclonal Antibody Against SARS-CoV-2 Produced in Rat.

Among all known human coronaviruses, some viruses (e.g., SARS-CoV-2) cause severe pneumonia or even death. With the regard to its spread and the importance of its rapid identification/treatment, and because pAbs are relatively cheap, able to bind to more sites on antigens and even neutralize them, this study was done for the production and purification of anti-SARS-CoV-2 polyclonal antibodies (pAb) in rats. Viral antigen purification was performed by PEG/NaCl precipitation. The efficiency of the sucrose cushion method was also investigated to produce a purer antigen. Immunization was done and antibody purification was performed by ammonium sulfate precipitation (33%), dialysis, and ion-exchange chromatography. Western blotting and enzyme-linked immunosorbent assay (ELISA) were performed to verify the antibody specificity. All data were analyzed by SPSS software. The results showed that the amount of concentrated virus increased with the increase of PEG concentration. Moreover, the sucrose cushion method increased its purity. Besides, induction of immune response in rats for pAb production with high titers was reached via these antigens and ELISA/western blot results indicated a suitable antibody-antigen interaction. Additionally, it was shown that ion-exchange chromatography could be a suitable technique for IgG purification. Herein, we presented a simple and cheap method for the purification of whole viral particles with relatively high quality. The results verified that these antigens could elicit a good immune response in the rat. The obtained pAbs showed a good specificity toward SARS-CoV-2 antigens. Accordingly, this study proposes a promising method for viral vaccine development.

Citrus limon peroxidase-assisted biocatalytic approach for biodegradation of reactive 1847 colfax blue P3R and 621 colfax blue R dyes

One of the big environmental problems in today’s world is dye-contaminated toxic waste. Peroxidase is known as highly efficient for the degradation of various pollutants, including dyes. Environmental contamination caused by the discharge of dyes into water bodies is an onerous challenge that poses both human and ecological hazards. In the current studies, biocatalysts used for enzyme decolorization (1847 Colafx Blue P3R and 621 Colafx Blue) are regarded as an eco-friendly method utilizing commonly available low-cost material lemon peels (Citruslimon peroxidase). Peroxidase was extracted in a phosphate buffer of pH 7.0 and partially purified by 20–80% ammonium sulfate precipitation technique from Citruslimon peels. The soluble enzyme was characterized in terms of kinetic and thermodynamic parameters. The values of Km and Vmax (23.16 and 204.08 μmol/ml/min) were determined, respectively. The enzyme showed maximum activity at pH 5.0 and a temperature of 55 °C. Citruslimon efficiently degraded 1847 Colafx Blue P3R and 621 Colafx Blue R dyes with maximum degradation of 83 and 99%, respectively, with an initial dye concentration of 200 ppm at pH 4 and 35 °C temperature within 5–10 min of incubation time. The effect of the redox mediator on the degradation process was examined. Results showed that the peroxidase HOBT system efficiently enhanced the degradation of dyes from water. Hence, Citruslimon peroxidase is an efficient biocatalyst for the treatment of effluents.

Purification of Xanthine Oxidase Enzyme and Investigation of Its Immobilization with Glutaraldehyde

In this study, the xanthine oxidase (XO) enzyme was purified by affinity chromatography technique using Sepharose-4B-L-tyrosine-4-aminobenzamidine gel and its immobilization with glutaraldehyde was investigated. Using ammonium sulfate precipitation and affinity gel, xanthine oxidase was purified 643.04-fold in an 11.5% yield. The purity of the enzyme was checked by SDS polyacrylamide gel electrophoresis and a single band around 150 kDa was observed. KM (the Michaelis constant) and VMax (the asymptotic reaction velocity at infinite substrate concentration) of the enzyme were determined at 1.67x10-4 M and 0.56 U/mL.min respectively by using a xanthine compound as a substrate. The in vitro effects of NH4F, NH4Cl, CaCl2, ZnCl2, HgCl2, Hg(NO3)2.H2O compounds and commercially named colchicum dispert, commonly used in the treatment of gout disease in the clinic, were investigated. The IC50 values of compounds showing inhibition effects were determined. Afterward, XO was immobilized with glutaraldehyde. The highest XO activity was observed in the sample of the immobilized enzyme at a rate of 6% glutaraldehyde. The kinetic constants (KM and VMax) of the immobilized enzyme were determined as 5.18x10-4 M and 0.73 U mL-1 min-1 respectively. These values revealed that the catalytic activity of the free enzyme was higher than the immobilized enzyme.

Screening and Characterization of L-asparaginase Producing Bacterial Strains from the Soil in Bangladesh

Although the enzyme L-asparaginase (L-ASNase) from Escherichia coli, Erwinia and Serratia has been applied to treat certain lymphomas and leukemias, several medical complications such as severe immunological responses leading to hypersensitivity, anaphylaxis, etc. have limited its application. The researchers have documented that such impediments are due to the different biochemical and kinetic properties of L-ASNase, which are directly dependent on genetic variations in microbial strains. Thus, there is a compelling need to explore novel L-ASNase producing microorganisms that would exhibit different serological properties while retaining similar and/or better therapeutic effects against cancer cells. Heretofore, L-ASNase producing bacterial strains from Bangladesh have never been isolated and characterized. Therefore, the main objective of this research was to isolate and characterize these strains from unexplored and ecologically different habitats that could lead to developing a potential therapeutic drug with fewer immunological responses and side effects over the existing drugs in order to treat cancer patients in the near future. Two L-ASNase producing bacterial strains were successfully isolated from the soil of Hatirjheel lake in Dhaka for the first time. Molecular characterization revealed that both strains belonged to Pseudomonas aeruginosa and their DNA sequences were submitted to NCBI GenBank. The accession number OK446669 was obtained for the strain of P. aeruginosa EWUKR-1 and OL307081 for P. aeruginosa EWUKR-2. The specific activity of L-ASNase from EWUKR-2 (212.1 ± 14.8 U/mg protein) was significantly higher than that of EWUKR-1 (16.3 ± 0.8 U/mg protein) when they were grown in modified M9 media containing 0.5 g/l glucose at 370C for 24 hours. The experimental results revealed that both of these bacterial strains were extracellular L-ASNase producers. The enzyme from P. aeruginosa EWUKR-2 was partially purified using saturated ammonium sulfate followed by dialysis and concentrated using Vivaspin-20 centrifugal concentrator having MWCO of 30 kDa. The optimum temperature and pH of the partially purified enzyme were 370C and 7.5, respectively. The purification-fold after ammonium sulfate precipitation and yield of the concentrated enzyme were 2.8 and 101%, respectively. SDS-PAGE analysis revealed that the molecular weight of L-ASNase from P. aeruginosa EWUKR-2 was around 43 kDa.

Partial Purification of Extracellular Amylase From Halotolerant Actinomycetes Streptomyces brasiliensis MML2028

Amylase is considered as an industrially important enzyme as it occupies the most important function in the food, paper, and pharmaceutical industries. The present study is concerned with the optimization, production and partial purification of halotolerant amylase from newly isolated Streptomyces brasiliensis MML2028, from Kelambakkam salt pan, Tamil Nadu, India. The primary screening was carried out by well diffusion assay to find the zone of lysis. The assay was observed for each media optimization by measuring the release of reducing sugar (RS) by the 3,5 dinitro salicylic acid (DNS) method and expressed in the international unit (UI). Ammonium sulphate precipitation was used to partially purify the enzyme and then lyophilized. SDS-PAGE was performed to identify the molecular weight. The production medium was optimized with 1% of the starch substrate, 3% of NaCl at 24˚C and pH 9, and incubation of 9 days. The total activity of the partially purified α-amylase was observed to be 1806.9U/mL. The partially purified enzyme was more active with 3% NaCl, pH 8, and 24˚C which is known to be a halotolerant alkaline α-amylase. The enzyme showed tolerance towards magnesium, manganese ions, Triton x-100, and urea. De-inking of α-amylase showed good results proving that the enzyme activity is more efficient. Hence, the alkaliphilic amylase from Halotolerant actinomycetes S. Brasiliensis MML2028 could be a better microbial source that can be used in many industries, especially in paper and textiles.

Cytotoxic activity of arginine deiminase purified from Lactobacillus sp

The goal of this research was to characterize ADI isolated from Lactobacillus sp. and test its anticancer activity in vitro. Because the arginine-degrading enzyme is a strong anticancer agent, it was isolated in two phases from a Lactobacillus sp. clinical isolate: 70% saturated ammonium sulphate precipitation followed by gel filtration chromatography (2x35cm). The most active fractions in enzyme activity (7.3 U/ ml), Purified enzyme had a special action activity of (48.6 U/mg) with nine folds of purgation and 61.4% enzyme recovery, The cytotoxic activity of purified arginine deiminase on PC3,A375 and WRL68 cells for 24 was examined. Purified arginine deiminase inhibited the proliferation of cancer cell lines PC3 and A375, with IC50 values of 68.64 g/ml and 136.3 g/ml, respectively. At a concentration of 6.25 ϻg/ml, 94.83% and 95.68% cell viability were observed of PC3 and A375 after treatment with purified arginine deiminase, respectively. However, cell viability reached to 42.52% and 48.77% using 400 ϻg/ml concentration purified arginine deiminase, respectively while ADI didn't show a significant effect on the viability of normal cell line.

Functional antibodies against G-protein coupled receptors in Beagle dogs infected with two different strains of Trypanosoma cruzi.

The interaction of the anti-beta1-adrenergic receptor autoantibodies (β1ARAb) and the anti-muscarinic M2 receptor autoantibodies (M2RAb) with cardiac neurotransmitter receptors were identified in human chronic Chagas cardiomyopathy (CCC) related to the ECG and dysautonomia disturbances. Dogs are considered gold model to the study of Trypanosoma cruzi infection due the clinical similarities with CCC. This study aims to evaluate whether anti-β1ARAb, anti-β2ARAb, and anti-muscarinic M2RAb are generated in Beagle dogs infected by T. cruzi using Y and Berenice-78 strains of T. cruzi. Animals were infected with 4.0 x 103 bloodstream trypomastigotes/kg of body weight and, after 25 months of infection, blood sample was collected, and serum stored at -80°C. Dog serum was treated by ammonium sulphate precipitation and the IgG antibodies isolated and added to the beating neonatal rats' cardiomyocytes. All T. cruzi-infected dogs developed agonistic β1ARAb, β2ARAb, and M2RAb. Animals infected by Berenice strain presented less β2ARAb and M2RAb activities than dogs infected by Y strain of the parasite. In cardiomyocytes culture, the antibodies recognized an epitope on the second extracellular loop of the receptors which were similar to findings in human Chagas disease. There was no detection of antibody against G protein-coupled receptor in serum from uninfected dogs. In conclusion, both Y and Berenice-78 strains of T. cruzi induced dog antibodies, whose targets located in the second extracellular loop of the adrenergic and muscarinic receptors were similar to those observed in individuals with CCC. Therefore, our findings highlight dogs as a promisor model to investigate pathogenic roles of functional Ab against G-protein coupled receptors.