Ammonium Sulfate Precipitation Method Research Articles

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.

Extraction and Purification of R-Phycoerythrin Alpha Subunit from the Marine Red Algae Pyropia Yezoensis and Its Biological Activities.

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe or as a colorant in the food and cosmetic industries. In this study, phycoerythrin was extracted from the red algae Pyropia yezoensis and purified by ammonium sulfate precipitation and various chromatography methods. The purified phycoerythrin was analyzed by UV-visible and fluorescence spectroscopy. The isolated pigment had the typical spectrum of R-phycoerythrin, with a trimmer state with absorbance maxima at 497, 536, and 565 nm. It was further purified and identified by LC-MS/MS and Mascot search. It showed a 100% sequence similarity with the R-phycoerythrin alpha subunit of Pyropia yezoensis. The molecular mass was 17.97 kDa. The antioxidant activity of the purified R-phycoerythrin alpha subunit was analyzed. It showed significant antioxidant activity in ABTS and FRAP assays and had significant cytotoxicity against HepG2 cells.

Extraction, purification of tyrosinase and tyrosinase inhibitory activity of four medicinal plants from Nanded district (MS), India

The systematic study aims to explore the tyrosinase inhibitory activity of selected plants. The study included extraction, purification, and inhibition of the enzyme. Tyrosinase has an important role in melanin formation, is responsible for the production of colour pigments of skin, hair, and eye. In the presents study, tyrosinase was isolated from Mushrooms, isolation of enzyme was done by acetone precipitation procedure and precipitation of enzyme was done with ammonium sulphate precipitation method. Plants selected for extraction were Azadirachta indica (Neem), Manikara zapota (Chiku), Annona squamosa (Sitaphal), and Hibiscus Rosa-sinesis (China rose). For phytochemical screening Alkaloids-Mayer’s Test, Flavonoids (Shinoda Test, Alkaline Reagent Test), sugar (Benedict’s reagent Test), Glycosides (Borntrager's Test), Phenolic compounds Test (Ferric chloride Test, Gelatin Test, Lead Acetate Test). Mushroom tyrosinase inhibitory assay was determined by the spectroscopic method. The enzyme precipitates on 85% saturation of ammonium sulphate, dialyzed and loaded on preequilibrated DEAE cellulose column for further purification at 1ml/min flow rate. Purification shows the 26 frac-tions giving very good enzyme activity. The specific activity of the enzyme after all purification steps was found to be 2 U/µg proteins with 17.09- fold purify-cation.

Purification of Glutathione Reductase From Some Tissues of Capoeta umbla and the Inhibitory Effects of Some Metal Ions on Enzyme Activity

The aim of this study was to determine the in vitro inhibitory effects of some metal ions (silver ion (Ag+), cadmium ion (Cd2+), cobalt ion (Co2+), copper ion (Cu2+), nickel ion (Ni2+), lead ion (Pb2+) and zinc ion (Zn2+)) on glutathione reductase (GR) enzyme activities that purified from the gill, kidney and liver tissues of Capoeta umbla. For this purpose, the enzyme was purified from the gill, kidney and liver of C. umbla freshwater fish using ammonium sulfate precipitation and affinity column chromatography methods using 2′,5′-ADP Sepharose 4B. Within this study, the GR enzyme was purified for the first time from the tissues of C. umbla. Enzyme purity and molecular weight were determined using the sodium dodecyl sulfate polyacrylamide gel electrophoresis method. In addition, the inhibitory effects of different metal ions (Ag+, Cd2+, Co2+, Cu2+, Ni2+, Pb2+ and Zn2+) on GR enzyme activities of the gill, kidney and liver tissue of C. umbla were investigated under in vitro conditions. The metal ion concentrations inhibiting 50% of enzyme activity (IC50) were obtained by plotting activity percentage versus [I] figures. Finally, the dissociation constants of the enzyme inhibitor complex (Ki), and the inhibition types, were calculated from Lineweaver–Burk plots. In vitro inhibition rank order was determined as Ag+>Co2+>Pb2+>Zn2+>Cu2+ for C. umbla gill GR; Ag+>Pb2+>Co2+> Ni2+>Zn2+ for C. umbla liver GR; Ag+>Cu2+>Co2+>Pb2+>Ni2+ for C. umbla kidney GR. From these results, we showed that Ag+ metal ion is the most potent inhibitor of GR enzyme on gill, liver and kidney tissues. Our results also demonstrate that these metals might be dangerous at low micromolar concentrations for C. umbla GR enzyme.

Partial Purification and Characterization of Endoxylanase from a fungus, Leohumicola incrustata

Xylanases are glycoside hydrolases (GH) that degrade β-1,4-xylan, a linear polysaccharide found as hemicellulose in cell wall of plants. Endoxylanase (Endo-1,4-β-xylanase, EC 3.2.1.8) randomly catalyses xylan to produce varying short xylooligosaccharides (XOS). This study aimed to determine the characteristics of a partially purified endoxylanase from Leohumicola incrustata. Enzyme production was carried out using beechwood (BW) xylan, after which the cell-free crude filtrate was concentrated using the ammonium sulphate precipitation method. The hydrolysed products were analysed by thin-layer chromatography (TLC) and zymography. The result showed that the enzyme produced varying smaller-sized linear xylooligosaccharides with Rf values corresponding to those of xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose and other higher oligomers. The endoxylanase had a molecular mass of 72 kDa. The enzyme is stable in the presence of K+, Na+, Ca2+, Fe2+, Mg2+, Zn2+, Co2+, pH of 5.0 and temperature of 37oC. However, the activity gradually decreased after 60 min at 50oC and retained over 69% activity after 120 min, while at 60 and 70oC, the enzyme activity sharply decreased (pre-incubation periods). Endoxylanase from L. incrustata is comparable to those of other microorganisms and should be considered an attractive candidate for future industrial applications.

Isolation and characterization of a thermostable alkaline chitinase-producing Aeromonas strain and its potential in biodegradation of shrimp shell wastes

Chitinases are employed to the conversion of chitin and are produced by a wide range of bacteria. The objective of this study was to isolate chitinase-producing microorganisms with high chitinolytic activity. A thermostable alkaline chitinase producing isolate strain CQNU6-2 was obtained from soil samples and showed potential in biodegradation of shrimp shell wastes. The optimal culturing conditions of isolate CQNU6-2 is at 25°C and pH 7 for 24 h. The chitinase produced by strain CQNU6-2 exhibited maximum activity at pH 6.0 and 40°C and it could tolerate the treatment of high temperature (up to 80°C) and high pH (over 10). Taxonomic study, based on biochemical and morphological analysis and phylogenetic analysis of 16S rDNA, showed that strain CQNU6-2 was belongs to the genus Aeromonas sp. The isolate can effectively hydrolyze colloidal chitin with degradation rate of 100% and also can directly degrade the shrimp shells. Ammonium sulfate precipitation method can be used to preliminary purify the chitinase. In conclusion, strain CQNU6-2 had a promising potential for biodegradation of chitin under harsh pH or temperature conditions and could be employed to the comprehensive utilization of shrimp shell wastes.

Comparison of Two methods for precipitation Mycobacterium tuberculosis protein and Investigation of its protein profiles

Aims and objectives: Mycobacterium tuberculosis (Mtb) is a bacterium that causes tuberculosis (TB) in humans. tuberculoprotein are a widely used for detection of primary infection with Mtb. The production of tuberculoprotein is accompanied by some difficulties that require a series of modifications in the production and purification processes. In this study, we aimed to compare two methods for precipitation Mycobacterium tuberculosis tuberculoprotein and investigated its protein profiles. Methods: Mtb C strain grown in Löwenstein-Jensen and Dorset Henley liquid medium. Bacteria were inactivated by subjecting to 68°C for 90 min. After inactivation the culture was filtered and tuberculoprotein were precipitated by ammonium sulfate (AS) and Trichloroacetic acid (TCA). Protein concentrations were determined by using the Lowry protein assay. In finally, the approximate molecular weights in the purified fractions were determined by sodium dodecyl sulfate (SDS)-PAGE. Results: The results showed that concentration of extracted tuberculoprotein by AS precipitation method was lower than TCA method. SDS-PAGE pattern of proteins precipitation with TCA showed proteins with wide range of molecular weight but proteins precipitation with AS showed low molecular weight proteins. Conclusions: Finally the results of this study showed that optimum methods besides a novel approach of AS precipitation in production of tuberculoprotein are suitable and applicable.

QUORUM SENSING ANALYSIS AND EFFECT OF BACTERIOCIN IN CONTROLLING THE BIOFILM FORMATION OF PSEUDOMONAS AERUGINOSA

Objective: Pseudomonas aeruginosa is the commonest causative agent of Hospital Acquired Infection (HAI). Resistance of Pseudomonas aeruginosa towards disinfectants and antibiotics is high when compared to other organisms. There are various reviews on antibacterial effect of herbal extracts and nanoparticles against Pseudomonas aeruginosa. Thus, this study aims to prove the effectiveness of bacteriocin against Pseudomonas aeruginosa, which may help in benefiting the health care center by replacing normal disinfectants.
 Methods: 100 strains of Pseudomonas aeruginosa were collected from different patients, with a study period of 6 mo. Ammonium sulfate precipitation method was used to extract bacteriocin and its efficiency was checked by paper disc diffusion assay. Biofilm formation assay and quorum sensing analysis was performed by Microtitre plate methods and Thin Layer Chromatography (TLC), respectively.
 Results: In this study, 91%of P. aeruginosa strains were strong, 8% were intermediate, and 1% were weak biofilm producers. From TLC analysis, 67% of the strains produced Acyl Homoserine Lactone molecules. Out of which, 49% has shown unknown analytes of Retardation factor (Rf) value greater than 1. The Rf values identified were 3 unsubstitutedC4 (5%), 3 unsubstituted C6 (4%), 3 oxo C8 (3%), 3 oxo C4 (3%), 3 oxo C6 (2%), 3 oxo C1 (1%). Biofilm production, before and after bacteriocin exposure, was proved significant by paired t-test.
 Conclusion: Quorum sensing was confirmed to play a major role in biofilm formation. As bacteriocin was found to be effective in controlling the biofilm formation, it can be incorporated in any disinfectant, which helps in controlling the transmission of infection caused by Pseudomonas aeruginosa.

Production of biocatalyst α-amylase from agro-waste ‘rice bran’ by using Bacillus tequilensis TB5 and standardizing its production process

Abstract Processing (milling, oil extraction) of agricultural products result in accumulation of massive amount of agro-waste residues. A sustainable alternative technique is required to utilize those agro-waste residues through biotechnology to convert them into useful products. α-Amylase is an enzyme of glycoside hydrolases family which hydrolyzes the α-D-(1,4) glycosidic bond. In the present research, Bacillus tequilensis TB5 was hired for the α-amylase production through SmF by utilizing agro-waste substrate rice bran. Also, the role of varying Physico-chemical parameters on α-amylase production was evaluated to determine the optimal conditions required for its maximum production. The findings of this research revealed that the optimal conditions for maximum yield (39.736 ± 0.296 U/ml) were found at pH 6.0, temperature 37 °C and incubation period of 72 h. On analyzing influence of various nutritional supplement on enzyme production, it was found that some of the nutrients like; peptone, beef extract, ammonium chloride, ammonium sulphate can enhance enzyme yield at a particular concentration. Purification of α-amylase was also done through ammonium sulphate precipitation method and then molecular weight of 54 kDa was determined by SDS-PAGE. The present research carried strongly supports, that rice bran is an efficient agro-waste substrate can possibilities of the commercial production of α-amylase.

Partial purification and characterization of a broad-spectrum bacteriocin produced by a Lactobacillus plantarum zrx03 isolated from infant's feces.

Lactobacillus plantarum zrx03 was a bacteriocin‐producing strain isolated from infant's feces. The fermentation supernatant produced by this strain could strongly inhibit Escherichia coli JM109 ATCC 67387, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes CICC 21633, in which the diameter of inhibition zone was 12.83 ± 0.62 mm, 15.08 ± 0.31 mm, 6.75 ± 0.20 mm, respectively, compared with lactic acid bacteria N1, N2, M13, M21, M31, and M37. According to amplification of 16S rRNA gene and identification of phylogenetic tree, this strain had a 1,450 bp sequence and 100% identity to the L. plantarum strain. Based on the influence of different protease treatments, such as pepsin, trypsin, papain, and proteinase K on the antimicrobial activity, this antimicrobial substance was considered to be a natural protein. Using bacteriocin produced by this strain as study object of this experiment, it had been extracted from ammonium sulfate precipitation and different organic solvents. The results showed that ethyl acetate was selected as the optimal solution to crude extraction of bacteriocin after comparing ammonium sulfate precipitation method and organic solvent extraction method, such as n‐butanol, n‐hexane, dichloromethane, trichloromethane, in which the diameter of the inhibition zones was above 28 mm. Results also showed the inhibition spectrum of the obtained bacteriocin had a broad spectrum of inhibition which could inhibit Gram‐positive, Gram‐negative, yeast. Especially, it could effectively inhibit S. aureus ATCC 25923, Bacillus subtilis CICC 10002, Bacillus anthracis CICC 20443, E. coli JM109 ATCC 67387, and Salmonella CMCC 541, and the zone diameter of inhibition has reached more than 28 mm. Moreover, it had a good thermal stability which antibacterial activity was retained 70.58% after treatment at 121°C for 30 min, and pH‐stability was between pH 2.0–9.0. These results suggested bacteriocin produced by L. plantarum zrx03 had potential application prospects in food preservation.

Extraction of PLA2 and antibacterial activity test of lionfish (Pterois volitans) spine venom

The population of Pterois volitans has caused significant damage to other fish populations and coral reef ecosystems. Population control of P. volitans consumes a considerable cost so that the utilization of these fish needs to be sought to be useful along with controlling the population. This fish is known to contain the enzyme Phospholipase A2 (PLA2), which used as an antibiotic against some bacteria. This study will examine the antibacterial activity of the phospholipase A2 enzyme extracted from P. volitans venom to Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. The method used to isolate the enzyme PLA2 is by using precipitation of ammonium sulfate (AS) and precipitation with ethanol. The results of the precipitation tested with the Lowry protein concentration test, the Marinetti PLA2 activity test, and the identification of the SDS-PAGE protein. The agar diffusion disc method is used to test the antibacterial activity. The results obtained from this research are that 80% ammonium sulfate precipitation method has the highest protein and enzyme activity with a ratio of 1.32 times compared to toxic extract. For antibacterial activity test results, an 80% ammonium sulfate sample may inhibit the activity of S. aureus bacteria but does not affect B. subtilis and E. coli.

Evaluation of dissolution of purified bromelain from pineapple core extract (Ananas comosus [L.] Merr) in the form of chitosan-coated microspheres

The aim of this study is to coat the bromelain from pineapple’s core into chitosan microspheres crosslinked by glutaraldehyde to maintain its activity until it reaches the intestines. The crude enzyme was first purified by ammonium sulfate precipitation method, followed by the dialysis. The bromelain fraction of the dialysis has a specific activity of 294.44 U/mg with a purity level of 15.68 fold compared to the crude enzyme. Coating bromelain using glutaraldehyde crosslinked microsphere showed an efficiency of 87.14 %. The dissolution test results showed the release rate of bromelain in artificial gastric fluid is 9.35 % and in artificial intestinal environment is 79.92 %. The result of this study indicates that chitosan-coated microspheres are good enough as bromelain coatings for slow-release matrices.

A novel antimicrobial protein of the endophytic Bacillus amyloliquefaciens and its control effect against Fusarium chlamydosporum

Fusarium chlamydosporum is the causal agent of stem rot in Jacaranda acutifolia. Bacillus amyloliquefaciens is an endophytic bacterium that shows promising biological control activity against F. chlamydosporum. In this study, B. amyloliquefaciens was cultured in a fermentation broth, and the main antimicrobial substance in the fermentation broth was isolated using the ammonium sulfate precipitation method and purified by DEAE sepharose fast flow weak anion exchange chromatography and sephadex G-50 molecular sieve chromatography. Polyacrylamide gel electrophoresis revealed a single antimicrobial protein with a molecular weight of approximately 29.0 kDa. The antimicrobial protein is a strong antagonist against F. chlamydosporum, leading to mycelial deformity, unclear mycelial septa, and isolated and granulated intracellular protoplasm. The N-terminal eight-amino acid sequence, NH2-Gly-Arg-Pro-Leu-Pro-Leu-Ala-Ala, was determined using the automatic Edman degradation method. BLAST analysis indicated that the amino acid residue sequence at positions 154–161 shows 100% homology to the hypothetical protein of Chromobacterium sp. LK1. However, no known homologous antimicrobial protein was identified, indicating that we have isolated a novel antimicrobial protein.

Biochemical characterization of lysozyme extracted from Caspian kutum, Rutilus kutum, Kamensky 1901

This paper presents the findings of a basic study on biochemical characteristics and activity of lysozyme extracted from a commercially important fish, Caspian kutum, Rutilus kutum, Kamensky 1901. The enzyme was purified by the ammonium sulphate precipitation method and cationic exchange chromatography. Molecular weight of lysozyme was estimated as 15.8kDa by SDS-PAGE. The optimum pH and temperature were 6.0 and 45.0°C, respectively,while the Km and Vmax values were 0.00007g mL-1 and 200units min-1, respectively. Thermo-stability of the enzyme was low at the temperatures higher than 50°C. The enzyme activity increased in the presence of NaCl (1-50mM), KCl (1-50mM) and CrCl2 (1-5mM), then decreased at the higher concentrations. Furthermore, the enzyme activity significantly decreased in the presence of FeCl2 and CuCl2 (1-100mM), but not stable in the presence of MgCl2. SDS inhibited the activity, but the enzyme exhibited a good degree of resistance to urea. Fluorescence quenching was observed in the presence of FeCl2 and CuCl2. Emission intensities in the presence of NaCl, KCl, CrCl2 and SDS were elevated, while was negligible for the urea. Our results exhibitedthat NaCl and KCl are well-established salts for elevating the enzyme activity.However, the purified lysozyme did not display a moderate stability against others salts. Besides, the purified lysozyme from R. kutum was not heat stable. Therefore, some phenomena such as water hardness and warming can exert negative effects on the innate immunity of the fish. So, the enzyme structure can be made more stable by medium engineering through changing the salt composition of aqueous solution.

Production, purification, and evaluation of quail immunoglobulin Y against Salmonella typhimurium and Salmonella enteritidis

Salmonella species have been the major foodborne problems in food production systems, with Salmonella enterica serovars typhimurium (S. typhimurium) and enteritidis (S. enteritidis) being among the more common isolates. The oral administration of chicken egg yolk specific antibodies (IgYs) has been established as an efficient alternative for treatment and prevention of gastrointestinal pathogens including Salmonella. The present study was aimed to investigate the possible production of specific IgYs against Salmonella typhimurium and Salmonella enteritidis in quail egg yolks. Salmonella spp.-free female Japanese quails (Coturnix coturnix japonica) were intramuscularly immunized with formalin or heat-inactivated Salmonella immunogens (1.0 × 109 CFU/mL) emulsified with Freund adjuvants. Egg yolk IgYs were purified using ammonium sulfate precipitation method. Anti-Salmonella IgYs titer and specificity were determined using enzyme-linked immunosorbent assay (ELISA) and western blot analysis. Salmonella specific IgYs detected in the immunized quails were significantly higher than those of the control group, which confirmed the immunization procedure. Specific IgYs against S. typhimurium and S. enteritidis were identified in both groups immunized with heat or formalin-inactivated immunogens. However, formalin-inactivated immunogens induced relatively higher immune responses over the heat-inactivated ones. Quail anti-Salmonella IgYs showed a high specificity to their corresponding immunogens, with moderate cross-reactivity to other members of Enterobacteriaceae family. Quail can be regarded as a valuable and inexpensive source for producing large-scale of specific antibodies that can be used for immunodiagnostic and immunotherapeutic purposes.

Secretory Laccase from Pestalotiopsis Species CDBT-F-G1 Fungal Strain Isolated from High Altitude: Optimization of Its Production and Characterization

Microorganisms producing laccases may be used for the pretreatment of lignocellulosic biomass to recover fermentable sugar. Very few fungi and other microbes growing in high altitudes have been tested for this purpose. As part of this study, we have collected soil samples from different parts of the Kathmandu Valley and the Rautah at district of Nepal (1600 to 2303 m above sea level) and successfully cultured 53 different isolates of microorganisms. Among the 53 isolates obtained 30 were Actinomycetes, 20 were Streptomycetes, and three were fungi). These isolates were tested for laccase expression using guaiacol, tannic acid, and 1-naphthol as substrates. Twelve of the 53 isolates tested positive for the expression of laccase. Among the laccase- positive isolates, a fungal species designated as CDBT-F-G1was found to produce high levels of laccase. This isolate was identified as Pestalotiopsis species based on 18S rRNA sequencing. Pestalotiopsis spp. CDBT-F-G1 isolate grows efficiently in PDB media containing 1% Kraft lignin at pH 5 and 30 °C and secretes 20 ± 2 U/mL laccase in culture medium. Further optimization of growth conditions reveled that addition of (i) metal salts, e.g., 1 mM magnesium sulfate (51 ± 25 U/mL); (ii) agitation of cultures at 200 rpm (51 ± 9U/mL); (iii) surfactants, e.g., 0.75 mM Tween 80 (54 ± 14 U/mL); (iv) 40% dissolved O2 (57 ± 2 U/mL) and inducers, e.g., 1 mM gallic acid (69 ± 11 U/mL), further promote laccase production by Pestalotiopsis spp. CDBT-F-G1 isolate. On the other hand, 0.1 mM cysteine inhibited laccase production. The secretory laccase obtained from fermentation broth of CDBT-F-G1 was partially purified by ammonium sulfate (13-fold purification with specific activity 26,200 U/mg) and acetone (14-fold purification with specific activity 31,700 U/mg) precipitation methods. The enzyme has an approximate molecular mass of 43 kDa, pH and temperature optima werepH6 and 60 °C, respectively. Vmax and Km were 100 μmol/min and 0.10 mM, respectively, with ABTS as the substrate. Given the above characteristics, we believe Pestalotiopsis spp. CDBT-F-G1 strain native to high altitudes of Nepal could be used to pretreat lignocellulosic biomass to efficiently recover fermentable sugars.

Isolation of Polygalacturonase-Producing Bacterial Strain from Tomatoes (Lycopersicon esculentum Mill.).

Background Polygalacturonase (EC 3.2.1.15) enzyme aids in microbial spoilage of fruits and vegetables. It is very important to find economical ways to producing the enzyme so as to achieve maximum yield in industries due to its use at different areas of production process. Methods Isolation of polygalacturonase-producing bacterial strain from tomatoes (Lycopersicon esculentum Mill.) was studied. Polygalacturonase-producing bacterial strains were isolated and screened from tomatoes stored at normal laboratory temperature (25 ± 2°C). They were identified based on their morphological, biochemical, and molecular characteristics. The enzyme produced was partially purified by the ammonium sulphate precipitation method. Molecular weights and optimum conditions for best enzyme activity were obtained by SDS PAGE technique. Results Five bacterial isolates resulted after screening. Bacterial strain code B5 showed highest polygalacturonase activity. Optimum conditions for polygalacturonase PEC B5 were maintained at pH 4.5; temperature 35°C; substrate concentration 0.3 mg/ml, and best activity at less than 5 min of heating. The enzyme PEC B5 was found to weigh 65 kDa and 50 kDa for crude and partially purified aliquots, respectively. The result of 16S rRNA gene sequencing revealed bacterial strain code B5 as Enterobacter tabaci NR146667 having 79% similarity with the NCBI GenBank. Conclusion Microorganisms should be developed for large-scale production of enzymes in developing countries.

Development of Dot-ELISA Technique for Estimation of Milk Progesterone and Pregnancy Diagnosis using PVDF Membrane

An analytical hormone separation method and dot enzyme linked immunosorbant assay (dot-ELISA) for quantitation of hormone are described. For extraction of progesterone from milk chloroform methanol mixture was used. For Dot ELISA, conjugate was prepared by immunoglobulin isolated from whole serum by ammonium sulphate precipitation method and conjugated with Horse radish Peroxidase by modified gluteraldehyde procedure. For immobilization of progesterone to PVDF membrane a number of solvents were tried viz., Methanol, Methanol + Carbonate-bicarbonate buffer, Methanol + PBS, Ethanol and 70 per cent isopropanol. On observation following Dot ELISA procedure, 70 per cent isopropanol was selected as best. Dot ELISA of serially diluted progesterone was performed to get the best titre of conjugate selected for further studies. For standard colour of dot with different progesterone concentration, Dot ELISA with varying concentration of progesterone was performed. The new technique was thus developed for estimation of progesterone using PVDF membrane and 70 per cent isopropanol as solvent. The developed Dot ELISA was also used for estimation of progesterone and pregnancy diagnosis from cattle milk sample and technique was validated. This techniques may be useful for estimation of other steroid hormones.

Screening and Isolation of Fibrinolytic Enzymes from Bacteria using Agrowaste for Thrombolytic Treatment

Bacterial fibrinolytic enzymes find great applications to treat and prevent cardiovascular diseases (CVD). The novel food-grade microorganisms are useful for thrombolytic therapy. Agro-residues were used for the production of fibrinolytic enzyme in solid-state fermentation. In this study, four different food samples were used and two Bacillus sp were isolated. The bacterial isolates were cultured and screened for its fibrinolytic activity. Under SSF, the production of fibrinolytic enzyme was enhanced by using cow dung substrate. The enzyme was then further purified by ammonium sulphate precipitation and dialysis method. After the successive purification steps, the molecular weight was estimated. The efficiency of the fibrinolytic enzyme produced was determined by its clot lytic activity on fibrin clot with the known concentration of the standard ‘Streptokinase’.

Statin Therapy and Serum Testosterone in Men with Type 2 Diabetes

There is a high prevalence of hypogonadism in men with type 2 diabetes. Statins could potentially decrease testosterone levels by reducing the availability of cholesterol for androgen synthesis. In this study we compared testosterone levels and hypogonadal symptoms with statin use in men with type 2 diabetes. PATIENTS AND METHODS Total testosterone, sex hormone–binding globulin (SHBG), and estradiol were measured by an enzyme-linked immunosorbent assay. Bioavailable testosterone was measured by the modified ammonium sulfate precipitation method. Free testosterone was calculated using Vermeulen's formula. Symptoms of hypogonadism were assessed using the Androgen Deficiency in the Aging Male questionnaire. RESULTS Statins were associated with lower total testosterone and a trend toward lower SHBG compared with untreated patients. Bioavailable testosterone, free testosterone, estradiol, and hypogonadal symptoms were not affected. Atorvastatin was associated with reduced total testosterone and a trend toward reduced SHBG compared with no treatment, and there was a dose-response effect with the lowest levels of total testosterone seen in men treated with ≥20 mg atorvastatin .Simvastatin use was not associated with significant reductions in testosterone or SHBG levels. CONCLUSION Assessing androgen status using total testosterone in men with type 2 diabetes treated with statins, particularly atorvastatin, may potentially lead to diagnostic error. Levels of bioavailable testosterone or free testosterone are recommended for the assessment of hypogonadism in this group if total testosterone levels are borderline.