This document describes the isolation and purification of endopolygalacturonase (EPG) enzymes produced by Alternaria cepulae fungus. The extracellular EPG was isolated from A. cepulae cultured in onion medium. Ammonium sulfate fractionation was used to precipitate proteins from the crude enzyme extract. Further purification was achieved using DEAE-cellulose ion exchange chromatography, which separated the EPG into two isoenzymes, EPG I and EPG II. Additional purification using gel filtration chromatography resulted in EPG I and EPG II being purified 245-fold and 270-fold respectively, as single protein bands on polyacrylamide gel electrophoresis. The purified isoenzymes had isoelectric points
17.Physicochemical characterization of Indole acetic acid oxidase from Altern...Annadurai B
This document describes a study that characterized the physicochemical properties of indole acetic acid oxidase (IAA oxidase) from Alternaria cepulae. The researchers found that the optimum pH for IAA oxidase activity was 5.5, and the optimum temperature was 40°C. Gel chromatography determined the molecular weight of IAA oxidase to be 30,000 daltons. The study provides information on the purification and characterization of IAA oxidase from A. cepulae which is involved in leaf blight disease of onions.
The document summarizes research analyzing two proteins, AvrGf1 and AvrGf2, that are crucial for the bacterial pathogen Xanthomonas citri to infect citrus plants. Genetic analysis identified the genes for these proteins. Sequence analysis found motifs indicating the proteins are injected into chloroplasts and interact with a citrus cyclophilin after a conformational change. The goal is to express and purify the proteins to study their interaction with the cyclophilin and understand how it activates them during infection.
Ultrasonication Assisted Extraction of Isolation Characterization of Berb...GuttiPavan
1. The project aims to isolate and characterize berberine from Anamirta cocculus stem through ultrasonication assisted extraction. The biological activities of the extracts will also be evaluated.
2. Berberine will be isolated through column chromatography and characterized using spectroscopy. Antioxidant, anti-inflammatory, and antimicrobial activities of the extracts and isolated berberine will be determined.
3. The project involves preparation of stem extracts, isolation of berberine, and evaluation of antioxidant, anti-inflammatory, and antimicrobial properties to understand the biological potential of Anamirta cocculus stem.
This study examined the functional state of epidermal growth factor (EGF) receptors during internalization in A-431 cells. The key findings were:
1) EGF-receptor complexes retained their phosphorylated state and connection to EGF during internalization, as shown using photoaffinity labeling and antibodies to phosphotyrosine.
2) Internalized EGF receptors, like those in the plasma membrane, were able to phosphorylate synthetic peptide substrates introduced into the cell, indicating they maintained kinase activity.
3) Approximately 15% of internalized EGF-receptor complexes were phosphorylated on tyrosine residues, suggesting functional heterogeneity of EGF receptors in A-431 cells.
1. The study examines how the substrate L-serine interacts with and stabilizes the active site of serine hydroxymethyltransferase (SHMT) enzymes from sheep liver and E. coli.
2. Results show L-serine enhances the thermal stability of both SHMT enzymes, increasing their apparent melting temperatures. Binding studies also indicate L-serine induces conformational changes in the enzymes without altering their overall structure.
3. The conformational changes upon L-serine binding, monitored by various spectroscopic methods, suggest L-serine compacts the enzyme structure, leading to increased thermal stability.
This document examines the process parameters for the biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) using the yeast Torulaspora delbrueckii. The maximum L-PAC yield of 331 mg/100 ml was obtained with 8 hours of reaction at 30°C using 600 mg of benzaldehyde. Growing the yeast in 3% glucose reduced the reaction time to 120 minutes. Addition of 0.6% acetaldehyde increased the L-PAC yield to 450 mg%. Semi-continuous feeding of benzaldehyde and acetaldehyde produced 683 mg L-PAC/100 ml. The cell mass was reusable for biotransformation up to nine times when substrate concentrations were
This document summarizes an investigation into the importance of amino acids 117 and 118 in regulating the kinetics and stability of E. coli alkaline phosphatase. Site-directed mutations were made to EcAP to mimic the amino acid sequences found in human placental alkaline phosphatase. Purification of wild-type EcAP and mutants N117F, G118Q, and G118R was successful, while purification of N117Y was inconclusive. Arrhenius analysis found similar activation energies for WT, G118Q, and N117F, but a higher energy for G118R, though results had high error. Incubation time inconsistencies affected reaction rates. While general trends were observed, conclusions could not be definitively drawn
This document summarizes research on the production of ferulic acid esterase by Streptomyces sp. Key findings include:
- Streptomyces sp. isolate S10 was identified that produced ferulic acid esterase when grown in de-starched wheat bran medium.
- Maximum ferulic acid esterase production of 2.0 mU/ml occurred in MBS medium containing 1.5% de-starched wheat bran at 30°C and pH 6.5 with agitation after 96 hours of fermentation.
- Hydrolysis experiments confirmed the enzyme produced was ferulic acid esterase as it released ferulic acid from the de-starched wheat bran substrate.
17.Physicochemical characterization of Indole acetic acid oxidase from Altern...Annadurai B
This document describes a study that characterized the physicochemical properties of indole acetic acid oxidase (IAA oxidase) from Alternaria cepulae. The researchers found that the optimum pH for IAA oxidase activity was 5.5, and the optimum temperature was 40°C. Gel chromatography determined the molecular weight of IAA oxidase to be 30,000 daltons. The study provides information on the purification and characterization of IAA oxidase from A. cepulae which is involved in leaf blight disease of onions.
The document summarizes research analyzing two proteins, AvrGf1 and AvrGf2, that are crucial for the bacterial pathogen Xanthomonas citri to infect citrus plants. Genetic analysis identified the genes for these proteins. Sequence analysis found motifs indicating the proteins are injected into chloroplasts and interact with a citrus cyclophilin after a conformational change. The goal is to express and purify the proteins to study their interaction with the cyclophilin and understand how it activates them during infection.
Ultrasonication Assisted Extraction of Isolation Characterization of Berb...GuttiPavan
1. The project aims to isolate and characterize berberine from Anamirta cocculus stem through ultrasonication assisted extraction. The biological activities of the extracts will also be evaluated.
2. Berberine will be isolated through column chromatography and characterized using spectroscopy. Antioxidant, anti-inflammatory, and antimicrobial activities of the extracts and isolated berberine will be determined.
3. The project involves preparation of stem extracts, isolation of berberine, and evaluation of antioxidant, anti-inflammatory, and antimicrobial properties to understand the biological potential of Anamirta cocculus stem.
This study examined the functional state of epidermal growth factor (EGF) receptors during internalization in A-431 cells. The key findings were:
1) EGF-receptor complexes retained their phosphorylated state and connection to EGF during internalization, as shown using photoaffinity labeling and antibodies to phosphotyrosine.
2) Internalized EGF receptors, like those in the plasma membrane, were able to phosphorylate synthetic peptide substrates introduced into the cell, indicating they maintained kinase activity.
3) Approximately 15% of internalized EGF-receptor complexes were phosphorylated on tyrosine residues, suggesting functional heterogeneity of EGF receptors in A-431 cells.
1. The study examines how the substrate L-serine interacts with and stabilizes the active site of serine hydroxymethyltransferase (SHMT) enzymes from sheep liver and E. coli.
2. Results show L-serine enhances the thermal stability of both SHMT enzymes, increasing their apparent melting temperatures. Binding studies also indicate L-serine induces conformational changes in the enzymes without altering their overall structure.
3. The conformational changes upon L-serine binding, monitored by various spectroscopic methods, suggest L-serine compacts the enzyme structure, leading to increased thermal stability.
This document examines the process parameters for the biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) using the yeast Torulaspora delbrueckii. The maximum L-PAC yield of 331 mg/100 ml was obtained with 8 hours of reaction at 30°C using 600 mg of benzaldehyde. Growing the yeast in 3% glucose reduced the reaction time to 120 minutes. Addition of 0.6% acetaldehyde increased the L-PAC yield to 450 mg%. Semi-continuous feeding of benzaldehyde and acetaldehyde produced 683 mg L-PAC/100 ml. The cell mass was reusable for biotransformation up to nine times when substrate concentrations were
This document summarizes an investigation into the importance of amino acids 117 and 118 in regulating the kinetics and stability of E. coli alkaline phosphatase. Site-directed mutations were made to EcAP to mimic the amino acid sequences found in human placental alkaline phosphatase. Purification of wild-type EcAP and mutants N117F, G118Q, and G118R was successful, while purification of N117Y was inconclusive. Arrhenius analysis found similar activation energies for WT, G118Q, and N117F, but a higher energy for G118R, though results had high error. Incubation time inconsistencies affected reaction rates. While general trends were observed, conclusions could not be definitively drawn
This document summarizes research on the production of ferulic acid esterase by Streptomyces sp. Key findings include:
- Streptomyces sp. isolate S10 was identified that produced ferulic acid esterase when grown in de-starched wheat bran medium.
- Maximum ferulic acid esterase production of 2.0 mU/ml occurred in MBS medium containing 1.5% de-starched wheat bran at 30°C and pH 6.5 with agitation after 96 hours of fermentation.
- Hydrolysis experiments confirmed the enzyme produced was ferulic acid esterase as it released ferulic acid from the de-starched wheat bran substrate.
03.Studies on the role of cellwall degrading enzymes of lleafblight diseases ...Annadurai B
This document reports on a study of the role of cell wall degrading enzymes in leaf blight disease of onion caused by Alternaria cepulae. The study found that polygalacturonase (PG) enzyme was responsible for the disease. Maximum PG production occurred on the 16th day of fungal culture. PG activity was observed at both pH 5.0 and 7.0, suggesting the existence of isozymes. Optimum temperature for PG activity was 35°C. The study concludes that PG plays a key role in degrading the plant cell wall and allowing fungal invasion during infection.
Extraction And Characterization Of Protease From The Viscera Of Skipjack Tuna...jeni_anggrek10
The document summarizes research on extracting and characterizing protease from the viscera of skipjack tuna fish. Protease was extracted from the intestines, stomach, pancreas, and liver of skipjack tuna using potassium phosphate solution and precipitated using either cold acetone or ammonium sulfate. The protease showed highest activity when precipitated with a 1:2 ratio of extract to acetone. The optimal temperature and pH for the acetone-precipitated protease were 50°C and 8, respectively. Tests on temperature and pH stability as well as effects of additives like NaCl, CaCl2, and EDTA were also conducted to characterize the protease.
This document summarizes the AP Biology lab on diffusion and osmosis. The lab involves using dialysis tubing filled with starch-glucose solution to determine the effects of osmosis on solutions of different concentrations. Students observe how a semi-permeable membrane allows for diffusion and how solution concentration and molecule size affect movement through the membrane. The concepts of diffusion, osmosis, hypotonic, hypertonic and isotonic solutions are explored. Water moves from areas of high water concentration to low water concentration.
This document summarizes an experiment on isolating and characterizing the enzyme alkaline phosphatase (AP) from E. coli bacteria. Key steps included purifying AP using dialysis, salting-in/salting-out, and DEAE cellulose chromatography. SDS-PAGE was used to analyze purity and molecular weight. Kinetic experiments at varying pH levels used the substrate PNPP and spectrophotometry to generate Michaelis-Menten and Lineweaver-Burk plots, allowing determination of kinetic parameters like Vmax and Km. The goal was to understand AP enzymatic activity and affinity for substrate under different conditions.
Expression Purification and Immunodetection of a fusion protein Glutathione S...iosrjce
Glutathione S Transferase(GST) is an enzyme of a multi gene family which is involved in reducing
oxidative damage to cells and detoxification of Xenobiotic compounds and plays critical role in life processes.
The entire work was completely qualitative and the objective of my work was to deal with the induction,
extraction and purification of the GST fusion protein from pGEX 3X vector.In order to achieve high degree of
transformed cells,the E.Coli BL21 host strain was made competent using 0.1M CaCl2 and adding of pGEX 3X
vector into host made it transformed.With the induction of GST protein by 0.1mM IPTG,the desired protein was
purified through glutathione Cl agarose column and was detected by immunoblotting method with the use of
anti GST HRP conjugate Ab which expressed the desired protein.
This document summarizes an experiment studying the kinetics of the tyrosinase enzyme using L-dopa as a substrate. Sodium benzoate was used as an inhibitor at 0.1 mM and 0.2 mM concentrations. The optimal tyrosinase concentration was determined to be 6.8 units/ml. For uninhibited tyrosinase, the Km was 490.35 μM and Vmax was 500 μM/s. With inhibition, Km and Vmax values changed, indicating mixed inhibition with an emphasis on uncompetitive inhibition. Experimental conditions influenced results.
The document summarizes the purification of alkaline phosphatase from E. coli over four days using various methods. These include lysing cells, dialysis, heat denaturation, ammonium sulfate precipitation, EDTA column chromatography, Bradford assay, and SDS-PAGE gel electrophoresis. The specific activity of alkaline phosphatase increased with each purification stage, though it was not fully pure after the final stage. Additional ion exchange chromatography would be needed for complete purification.
Electrophoretic Patterns of Esterases in Eri silkworm Samia Cynthia riciniIOSR Journals
The present study was carried out to investigate the patterns of esterase isozymes extracted from the silk gland, haemolymph and mid gut of Eri silkworm (Samia Cynthia ricini). The qualitative analysis of esterases was carried out by 7.5% of native Polyacrylamide Gel Electrophoresis (PAGE). The inhibitor sensitivity of the enzymes towards paraxon, eserine and pCMB was used to classify the individual zones of esterases. Three zones of esterases were observed in different tissues of Eri silkworm. Silk gland esterases were classified as CHsp (Cholinesterase like enzymes) esterases. The haemolymph and mid gut esterases were classified into Esdp (Enzyme inhibited by paraxon and pCMB).
This document summarizes the validation of a quantitative LC/MSMS method to measure the cleavage of a 17-residue SNAP-25 epitope by botulinum neurotoxin serotype A (BoNT/A). Peptides representing the intact 17-mer epitope and its cleavage products (11-mer and 6-mer) were characterized. The method was validated and used to evaluate the kinetics of 17-mer cleavage, finding near-completion at 40-45 minutes. The validated method will be applied to evaluate potential BoNT/A inhibitors and detect contamination in samples.
Dna fingerprinting, chemical composition, antitumor and antimicrobial activit...Alexander Decker
This study analyzed the chemical composition and biological activities of essential oils and extracts from four Annona species grown in Egypt. Gas chromatography-mass spectrometry identified the major constituents in each species' essential oils. Ethanol extracts showed strong cytotoxic effects against breast, colon, and liver cancer cell lines, with lower IC50 values than the essential oils. Both essential oils and ethanol extracts displayed antimicrobial activity against various bacteria and fungi. The essential oil and ethanol extract of Annona cherimola exhibited the strongest antitumor and antimicrobial effects, suggesting potential as a natural medicine.
The document analyzes the pigments and antioxidant properties of red dragon fruit (Hylocereus polyrhizus).
High Performance Liquid Chromatography analysis identified betanin as the main pigment contributing to the fruit's deep purple color. Antioxidant assays found high levels of polyphenols (86.1 mg/0.5 g) and flavonoids (2.3 mg/g), as well as strong reducing power and DPPH radical scavenging activity, indicating dragon fruit has significant antioxidant activity. The results confirm betanin is the primary pigment in dragon fruit and that the fruit contains high levels of antioxidants with potential health benefits.
This document summarizes an experiment examining how mutations in the b subunit of the stator stalk of E. coli ATP synthase affect its function and stability. The experiment involved growing E. coli strains with different mutations under limited glucose conditions to assess oxidative phosphorylation ability. Growth was measured over time, with mutants showing less growth than the wild type. Membrane proteins were also prepared from the strains to allow for further ATPase assays and western blots to quantify protein expression and better understand how mutations impact ATP synthase function.
Antioxidant and-anticancer-activities-of-moringa-leavesSilentdisco Berlin
Moringa is a plantfood of high nutritional value, ecologically and economically beneficial and readily available in the countries hardest hit by the food crisis. http://miracletrees.org/ http://moringatrees.org/
This document describes experiments performed to sequence the human Apolipoprotein B (ApoB) gene. A portion of the ApoB gene was amplified via PCR and subcloned into E. coli plasmid vectors. The plasmid vectors containing the inserted ApoB fragment were then purified and sequenced. Sequence analysis revealed that human ApoB is highly similar to Canis lupus familiaris (dog) ApoB, indicating evolutionary conservation. The experiments aimed to accurately insert, track, and sequence the ApoB gene to better understand its structure, function, and evolutionary relationships.
This document summarizes research on the purification, characterization, and antifungal activity of a chitinase enzyme from Streptomyces venezuelae P10. Key findings include:
1) The chitinase was purified using ammonium sulfate precipitation, chitin affinity chromatography, and DEAE-cellulose anion exchange chromatography, yielding a purified enzyme with a molecular weight of 66 kDa.
2) The purified chitinase demonstrated optimal activity at 35°C and pH 6-8 and showed antifungal activity against phytopathogens such as Aspergillus niger and Alternaria alternate.
3) Thin layer chromatography analysis identified the chitin
Distribution of enzymes (rhodanese, 3 mercaptopyruvate sulphurtransferase, ar...Alexander Decker
The document analyzes the activities of four enzymes (Rhodanese, 3-Mercaptopyruvate Sulphurtransferase, Arginase, and Thiaminase) in nine commonly consumed plant tubers in Nigeria. It finds that all four enzymes exhibited activity in the plant tubers. 3-MST activity varied significantly between tubers, with white yam showing the highest. Arginase activity also varied significantly, with Irish potato having the highest and cassava the lowest. Thiaminase activity varied as well, with cassava stem the highest and bitter yam peel the lowest. The study confirms the presence and nutritional value of these enzymes in commonly consumed plant tubers.
The document provides details about the contents page of the Rock Sound magazine, including typography, layout, images, color, and make-up (mise-en-scene). The masthead and sell line use bold, distorted text in red and silver to represent the loud rock genre. Down the left column are page numbers and descriptions in standard black font. Across the right is a red background with band images wearing punk makeup and rock costumes. The colors red, black, white, and brown symbolize both the softer and louder sides of rock.
The document provides instructions for a 7th grade creation lab assignment where students are asked to choose one or two popular icons to create a pop art piece expressing their point of view on consumerism using irony. Students are prompted to sketch their piece and explain the elements chosen, how it reflects consumerism, and how it uses irony.
03.Studies on the role of cellwall degrading enzymes of lleafblight diseases ...Annadurai B
This document reports on a study of the role of cell wall degrading enzymes in leaf blight disease of onion caused by Alternaria cepulae. The study found that polygalacturonase (PG) enzyme was responsible for the disease. Maximum PG production occurred on the 16th day of fungal culture. PG activity was observed at both pH 5.0 and 7.0, suggesting the existence of isozymes. Optimum temperature for PG activity was 35°C. The study concludes that PG plays a key role in degrading the plant cell wall and allowing fungal invasion during infection.
Extraction And Characterization Of Protease From The Viscera Of Skipjack Tuna...jeni_anggrek10
The document summarizes research on extracting and characterizing protease from the viscera of skipjack tuna fish. Protease was extracted from the intestines, stomach, pancreas, and liver of skipjack tuna using potassium phosphate solution and precipitated using either cold acetone or ammonium sulfate. The protease showed highest activity when precipitated with a 1:2 ratio of extract to acetone. The optimal temperature and pH for the acetone-precipitated protease were 50°C and 8, respectively. Tests on temperature and pH stability as well as effects of additives like NaCl, CaCl2, and EDTA were also conducted to characterize the protease.
This document summarizes the AP Biology lab on diffusion and osmosis. The lab involves using dialysis tubing filled with starch-glucose solution to determine the effects of osmosis on solutions of different concentrations. Students observe how a semi-permeable membrane allows for diffusion and how solution concentration and molecule size affect movement through the membrane. The concepts of diffusion, osmosis, hypotonic, hypertonic and isotonic solutions are explored. Water moves from areas of high water concentration to low water concentration.
This document summarizes an experiment on isolating and characterizing the enzyme alkaline phosphatase (AP) from E. coli bacteria. Key steps included purifying AP using dialysis, salting-in/salting-out, and DEAE cellulose chromatography. SDS-PAGE was used to analyze purity and molecular weight. Kinetic experiments at varying pH levels used the substrate PNPP and spectrophotometry to generate Michaelis-Menten and Lineweaver-Burk plots, allowing determination of kinetic parameters like Vmax and Km. The goal was to understand AP enzymatic activity and affinity for substrate under different conditions.
Expression Purification and Immunodetection of a fusion protein Glutathione S...iosrjce
Glutathione S Transferase(GST) is an enzyme of a multi gene family which is involved in reducing
oxidative damage to cells and detoxification of Xenobiotic compounds and plays critical role in life processes.
The entire work was completely qualitative and the objective of my work was to deal with the induction,
extraction and purification of the GST fusion protein from pGEX 3X vector.In order to achieve high degree of
transformed cells,the E.Coli BL21 host strain was made competent using 0.1M CaCl2 and adding of pGEX 3X
vector into host made it transformed.With the induction of GST protein by 0.1mM IPTG,the desired protein was
purified through glutathione Cl agarose column and was detected by immunoblotting method with the use of
anti GST HRP conjugate Ab which expressed the desired protein.
This document summarizes an experiment studying the kinetics of the tyrosinase enzyme using L-dopa as a substrate. Sodium benzoate was used as an inhibitor at 0.1 mM and 0.2 mM concentrations. The optimal tyrosinase concentration was determined to be 6.8 units/ml. For uninhibited tyrosinase, the Km was 490.35 μM and Vmax was 500 μM/s. With inhibition, Km and Vmax values changed, indicating mixed inhibition with an emphasis on uncompetitive inhibition. Experimental conditions influenced results.
The document summarizes the purification of alkaline phosphatase from E. coli over four days using various methods. These include lysing cells, dialysis, heat denaturation, ammonium sulfate precipitation, EDTA column chromatography, Bradford assay, and SDS-PAGE gel electrophoresis. The specific activity of alkaline phosphatase increased with each purification stage, though it was not fully pure after the final stage. Additional ion exchange chromatography would be needed for complete purification.
Electrophoretic Patterns of Esterases in Eri silkworm Samia Cynthia riciniIOSR Journals
The present study was carried out to investigate the patterns of esterase isozymes extracted from the silk gland, haemolymph and mid gut of Eri silkworm (Samia Cynthia ricini). The qualitative analysis of esterases was carried out by 7.5% of native Polyacrylamide Gel Electrophoresis (PAGE). The inhibitor sensitivity of the enzymes towards paraxon, eserine and pCMB was used to classify the individual zones of esterases. Three zones of esterases were observed in different tissues of Eri silkworm. Silk gland esterases were classified as CHsp (Cholinesterase like enzymes) esterases. The haemolymph and mid gut esterases were classified into Esdp (Enzyme inhibited by paraxon and pCMB).
This document summarizes the validation of a quantitative LC/MSMS method to measure the cleavage of a 17-residue SNAP-25 epitope by botulinum neurotoxin serotype A (BoNT/A). Peptides representing the intact 17-mer epitope and its cleavage products (11-mer and 6-mer) were characterized. The method was validated and used to evaluate the kinetics of 17-mer cleavage, finding near-completion at 40-45 minutes. The validated method will be applied to evaluate potential BoNT/A inhibitors and detect contamination in samples.
Dna fingerprinting, chemical composition, antitumor and antimicrobial activit...Alexander Decker
This study analyzed the chemical composition and biological activities of essential oils and extracts from four Annona species grown in Egypt. Gas chromatography-mass spectrometry identified the major constituents in each species' essential oils. Ethanol extracts showed strong cytotoxic effects against breast, colon, and liver cancer cell lines, with lower IC50 values than the essential oils. Both essential oils and ethanol extracts displayed antimicrobial activity against various bacteria and fungi. The essential oil and ethanol extract of Annona cherimola exhibited the strongest antitumor and antimicrobial effects, suggesting potential as a natural medicine.
The document analyzes the pigments and antioxidant properties of red dragon fruit (Hylocereus polyrhizus).
High Performance Liquid Chromatography analysis identified betanin as the main pigment contributing to the fruit's deep purple color. Antioxidant assays found high levels of polyphenols (86.1 mg/0.5 g) and flavonoids (2.3 mg/g), as well as strong reducing power and DPPH radical scavenging activity, indicating dragon fruit has significant antioxidant activity. The results confirm betanin is the primary pigment in dragon fruit and that the fruit contains high levels of antioxidants with potential health benefits.
This document summarizes an experiment examining how mutations in the b subunit of the stator stalk of E. coli ATP synthase affect its function and stability. The experiment involved growing E. coli strains with different mutations under limited glucose conditions to assess oxidative phosphorylation ability. Growth was measured over time, with mutants showing less growth than the wild type. Membrane proteins were also prepared from the strains to allow for further ATPase assays and western blots to quantify protein expression and better understand how mutations impact ATP synthase function.
Antioxidant and-anticancer-activities-of-moringa-leavesSilentdisco Berlin
Moringa is a plantfood of high nutritional value, ecologically and economically beneficial and readily available in the countries hardest hit by the food crisis. http://miracletrees.org/ http://moringatrees.org/
This document describes experiments performed to sequence the human Apolipoprotein B (ApoB) gene. A portion of the ApoB gene was amplified via PCR and subcloned into E. coli plasmid vectors. The plasmid vectors containing the inserted ApoB fragment were then purified and sequenced. Sequence analysis revealed that human ApoB is highly similar to Canis lupus familiaris (dog) ApoB, indicating evolutionary conservation. The experiments aimed to accurately insert, track, and sequence the ApoB gene to better understand its structure, function, and evolutionary relationships.
This document summarizes research on the purification, characterization, and antifungal activity of a chitinase enzyme from Streptomyces venezuelae P10. Key findings include:
1) The chitinase was purified using ammonium sulfate precipitation, chitin affinity chromatography, and DEAE-cellulose anion exchange chromatography, yielding a purified enzyme with a molecular weight of 66 kDa.
2) The purified chitinase demonstrated optimal activity at 35°C and pH 6-8 and showed antifungal activity against phytopathogens such as Aspergillus niger and Alternaria alternate.
3) Thin layer chromatography analysis identified the chitin
Distribution of enzymes (rhodanese, 3 mercaptopyruvate sulphurtransferase, ar...Alexander Decker
The document analyzes the activities of four enzymes (Rhodanese, 3-Mercaptopyruvate Sulphurtransferase, Arginase, and Thiaminase) in nine commonly consumed plant tubers in Nigeria. It finds that all four enzymes exhibited activity in the plant tubers. 3-MST activity varied significantly between tubers, with white yam showing the highest. Arginase activity also varied significantly, with Irish potato having the highest and cassava the lowest. Thiaminase activity varied as well, with cassava stem the highest and bitter yam peel the lowest. The study confirms the presence and nutritional value of these enzymes in commonly consumed plant tubers.
The document provides details about the contents page of the Rock Sound magazine, including typography, layout, images, color, and make-up (mise-en-scene). The masthead and sell line use bold, distorted text in red and silver to represent the loud rock genre. Down the left column are page numbers and descriptions in standard black font. Across the right is a red background with band images wearing punk makeup and rock costumes. The colors red, black, white, and brown symbolize both the softer and louder sides of rock.
The document provides instructions for a 7th grade creation lab assignment where students are asked to choose one or two popular icons to create a pop art piece expressing their point of view on consumerism using irony. Students are prompted to sketch their piece and explain the elements chosen, how it reflects consumerism, and how it uses irony.
This document describes an experiment comparing a control wine to a variable wine in which oxidation was introduced. Various analytical tests were performed on the wines during production, including using a hydrometer, refractometer, and multiparameter meter. The results showed little difference between the wines, likely because potassium metabisulfite was added to prevent oxidation in the variable wine. The control wine had a final alcohol concentration of 13.7% while the variable wine was 13.3%, a difference of 0.4%. Oxidation did not affect specific gravity but slight differences were found in pH and other multiparameter results between the wines.
The document discusses spiritual practices of African and Afro-Colombian tribes. It asks questions about differences and similarities between their spiritual practices, as well as about body alterations in African cultures and personal opinions on such alterations. Videos are provided to examine the spiritual connection of African body alterations, and which persist today. The reader is asked to share their personal opinion on body alterations and which, if any, they would consider and why.
Este documento presenta una sesión introductoria sobre orientación como deporte. La sesión introducirá conceptos teóricos sobre orientación mediante una presentación de PowerPoint y manipulación de mapas para identificar colores y símbolos. Los estudiantes hablarán sobre si conocen este deporte y sus utilidades cotidianas. Luego, trabajarán en grupos para crear lemas que promuevan la práctica de la orientación basándose en su utilidad diaria.
The study investigated the abundance and distribution of Red Waratah Anemones on the rocky shore of Hastings Point. Quadrat surveys were conducted in the low, mid, and high intertidal zones. Results showed the anemones were most abundant in the mid-intertidal zone, followed by the low zone, and least abundant in the high zone. Analysis of the distribution found most anemones were located in crevices or under rock cover, which provide shade. The findings supported previous research that Red Waratah Anemones prefer the lower mid-intertidal zone, though a few individuals in the high zone were possibly offspring that survived from a past colonization event with different weather conditions.
El documento explica la potenciación, que es una operación matemática entre una base y un exponente. La potenciación se escribe como An y se lee como "A elevado a n". Cuando el exponente es un número natural n, indica las veces que aparece la base A multiplicándose por sí misma. El documento también proporciona enlaces adicionales sobre potenciación y ejercicios relacionados.
Land resource inventory of India for development of sustainable agricultural ...GCARD Conferences
This document discusses investment opportunities in agricultural research and land use planning in India using geospatial techniques. It summarizes the following key points:
1. The National Bureau of Soil Survey and Land Use Planning of India has conducted land resource inventories of agricultural land on 1:10,000 scale to provide site-specific soil and land use information for planning.
2. This information is being used to develop customized soil-land use models and management practices to optimize agricultural productivity for different land holdings and improve livelihoods.
3. Case studies showcase how these plans have led to reduced soil degradation, increased employment, and improved incomes for farmers when implemented in different agro-climatic regions of India.
This document presents a chemistry investigation comparing water quality between a saltwater lake and freshwater pond in Emerald Lakes. It tests various water quality parameters including temperature, turbidity, salinity, pH, and dissolved oxygen. A multiparameter meter was used to measure temperature, pH, and salinity directly in water samples from each site. Turbidity was measured using a turbidity tube on site and a colorimeter back in the lab. Dissolved oxygen was measured using test kits on site and a dissolved oxygen colorimeter. The results showed the saltwater lake had higher dissolved oxygen and lower turbidity than the freshwater pond, contradicting some of the initial hypotheses.
Comment assurer une stratégie PRM efficace ? Quels canaux digitaux sont les plus performants ? Comment rythmer les temps de contact ? Quels dispositifs mettre en place pour collecter des données ciblées ?
Découvrez les points clés d’une bonne stratégie PRM abordés lors de notre atelier petit-déjeuner du 20 septembre 2016.
This chapter discusses organizational innovation, change, and learning. It provides an overview of key concepts like complex adaptive systems and organizational learning. It then describes the innovation model, a multi-stage process for managing innovation including agenda setting, matching innovations to needs, restructuring, clarifying, and routinization. Finally, it addresses implementing large-scale organizational change and Kotter's 8 step transformation model.
46.Purification of indole acetic acid oxidase produced by Alternaria cepulae ...Annadurai B
This document describes the purification of indole acetic acid oxidase (IAAO), an auxin-degrading enzyme, produced by the fungus Alternaria cepulae during leaf blight disease of onions. The enzyme was purified from culture filtrate of A. cepulae using ammonium sulfate precipitation and two column chromatography steps - CM-Trisacryl ion exchange column and Ultrogel gel filtration column. This resulted in an 85.5-fold purification of IAAO with a 58.6% yield. Tests including PAGE, SDS-PAGE, isoelectric focusing, immunodiffusion and immuno-electrophoresis confirmed the homogeneity of the purified enzyme.
33.Expression, Production and Purification of Proteinases from Aspergillus spp.Annadurai B
This document summarizes research on the expression, production, and purification of proteinases (proteases) from 18 strains of Aspergillus. The researchers found that Aspergillus flavus and Aspergillus sojae had the highest mycelial dry weight, indicating maximum growth. Aspergillus tamarii and Aspergillus awamori showed the highest protein content. Aspergillus nidulance had the highest proteinase content. The proteinases were purified using ammonium sulfate precipitation and column chromatography. SDS-PAGE analysis revealed a protease band with a molecular weight of 60,000 Da. The study demonstrates that certain Aspergillus strains are good producers of extracellular proteinases, which
54.Isolation and purification of cellulase from Aspergillus terreusAnnadurai B
This document describes the isolation and purification of cellulase enzymes from the fungus Aspergillus terreus. The intracellular cellulase was purified using ammonium sulfate precipitation, DEAE-cellulose chromatography, and gel filtration chromatography. This purification scheme achieved a 270-fold purification with a 22.11% yield. Tests including PAGE, SDS-PAGE, immunodiffusion, and isoelectric focusing confirmed the homogeneity of the purified enzyme. The purified cellulase showed optimal activity between pH 4-7 and temperatures of 40-50°C.
05.Pectic enzymes of A.cepulae in leaf blight disease of onionAnnadurai B
This document summarizes a study on pectic enzymes produced by Alternaria cepulae, a fungus that causes leaf blight disease in onions. Key findings:
- A. cepulae was isolated from infected onion leaves and found to grow well in several culture media, with maximum growth in Czapeck and Lindberg medium.
- Production of endo-polygalacturonase (EPG) enzyme was highest in natural onion medium and pectin medium. EPG activity peaked at day 16 of incubation.
- EPG activity was inhibited by calcium chloride, indicating its role in disease resistance. Two forms of EPG may be produced with optimal activity at pH 5 and 7.
This document summarizes an AP Biology lab review covering several topics:
- Lab 1 discusses diffusion and osmosis, describing experiments with dialysis tubing and potato cores in sucrose solutions. It concludes that water moves based on concentration gradients and molecule size.
- Lab 2 examines enzyme catalysis, measuring factors like pH and temperature that affect the rate of a reaction catalyzed by the enzyme catalase.
- Lab 3 covers mitosis and meiosis, describing experiments with onion root tips and fungi to observe cell stages and genetic recombination.
This document summarizes research on the production of ferulic acid esterase by Streptomyces sp. Key findings include:
- Streptomyces sp. isolate S10 was identified that produced ferulic acid esterase when grown in de-starched wheat bran medium.
- Maximum ferulic acid esterase production of 2.0 mU/ml occurred in MBS medium containing 1.5% de-starched wheat bran at 30°C and pH 6.5 with agitation after 96 hours of fermentation.
- Hydrolysis experiments confirmed the enzyme produced was ferulic acid esterase as it released ferulic acid from the de-starched wheat bran substrate.
Electrochemical, in-vitro in-vivo study of Co (II)-ofloxacin complexIOSR Journals
Ofloxacin complex has been synthesized and screened for its physicochemical, microbial as well as pharmacological activity have been done in solid and aqueous phase. On the basis of elemental analysis, polarographic studies, amperometric titration and IR spectral studies the probable formula for the complex has been determined at 30±1OC and ionic strength of μ= 1.0[KCl]. Raper’s paper disc method was used for microbial study against various pathogenic bacteria and fungi.Invivo syudy of Swiss mice [25-30gm] were used for antibacterial activity against ofloxacin and its complex on xyline-Alcoholic activity test Kidney, liver and serum of these rats were also studied. On the basis of observed result it could be concluded that Co(II)-Ofloxacin complex were found to be non-toxic and more potent than pure Ofloxacin.(1)
The document describes AP Biology Lab 1 on diffusion and osmosis. It would test the concentration of unknown sucrose solutions by placing potato cores in the solutions and observing their behavior based on principles of osmosis and diffusion across semi-permeable membranes. The experiment aims to determine which flask contains each concentration by comparing the results to expectations.
1) The study validated the use of five E.coli bioluminescent biosensor strains to rapidly detect and enumerate bacteria as an alternative to traditional plate counting methods.
2) The gene expression patterns of the biosensors, measured through bioluminescence, closely matched the growth curves and correlated strongly with colony forming unit counts.
3) The biosensors were then used to test the efficacy of sorbic acid as a preservative according to European Pharmacopeia standards, demonstrating over 99% light reduction representing a 3 log unit reduction of bacteria.
Analytical Monitoring Of Alcoholic Fermentation Using NIR SpectroscopyRichard Hogue
This document discusses using near infrared (NIR) spectroscopy to develop calibration models for monitoring various parameters during alcoholic fermentation by Saccharomyces cerevisiae yeasts. Samples were obtained from fermentation runs and prepared in the laboratory to span relevant concentration ranges. Reference methods were used to determine glucose, ethanol, biomass, glycerol, and acidity levels. Partial least squares models were developed using NIR spectra and reference values. The models effectively predicted glucose, ethanol, and biomass concentrations and allowed real-time monitoring of fermentation progress.
This document describes the development of a novel fluorescent protein-based sensor for detecting 2-oxoglutarate (2OG) levels in living cells. The sensor, termed mOGsor, was created by inserting the 2OG-binding domain GAF from the NifA protein into yellow fluorescent protein (YFP). mOGsor exhibits increased fluorescence intensity upon binding to 2OG in a concentration-dependent manner. Testing showed mOGsor has high specificity for 2OG and fast kinetics. Using mOGsor, the authors were able to monitor real-time changes in 2OG levels in E. coli cells under different nutrient conditions. mOGsor represents an improvement over previous FRET-based 2OG sensors by providing a
Comparative Study of Free Radical Scavenging and Lipid Peroxidation Inhibitio...ijtsrd
Oxytenanthera abyssinica rhizomes have been used in the treatment of aliments varying from colics, diabetes, polyuria and albuminiura, these ethanomedicinal potencies have been attributed to its rich phytochemical contents and free radical scavenging capacity. This work is aimed at investigating the effect of crude oil pollution on total phenolic, flavonoids and free radical scavenging potentials of methanol extract of Oxytenanthera abyssinica rhizomes. The ability of the plant extracts to scavenge 2,2 diphenyl 1 picylhydrazyl DPPH , hydroxyl radical .OH , superoxide radical O2 , nitric oxide radical NO. and ability to inhibit lipid peroxidation was assayed following standard methods, spectrophotometric method was used to assay for total phenolic and flavonoid content. Non crude oil polluted O. abyssinica extract NCOPOAE and crude oil polluted O. abyssinica extract COPOAE in a concentration dependent manner showed a potent antioxidant activity in DPPH radical scavenging assay EC50= 1.03 ±0.01µg ml and EC50 = 1.04 ±1.01µg ml , inhibited hydroxyl radical EC50 = 1.17 ±1.02µg ml and EC50 = 1.46 ±1.21µg ml , superoxide anion radical EC50 =1.30 ±1.21µg ml and EC50 =1.51 ±1.42µg ml and nitric oxide EC50 = 1.40 ±1.23µg ml and EC50= 1.53 ±1.42µg ml also inhibited non enzymatic lipid peroxidation EC50 = 1.58 ±1.42µg ml and EC50 = 2.47 ±2.13µg ml and had more of phenolic and flavonoid contents than COPOAE. This result indicates that NCOPOAE contains more antioxidant compounds than COPOAE, which could make it more potent in ethanomedicinal use. Jonathan Chinenye Ifemeje | Kizito Ifeanyi Amaefule | Charles Tochukwu Nwachukwu "Comparative Study of Free Radical Scavenging and Lipid Peroxidation Inhibition Potentials of Oxythenanthera Abyssinica (Rhizomes) Sourced from South Eastern Nigeria" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-3 | Issue-6 , October 2019, URL: https://www.ijtsrd.com/papers/ijtsrd29226.pdf Paper URL: https://www.ijtsrd.com/biological-science/biochemistry/29226/comparative-study-of-free-radical-scavenging-and-lipid-peroxidation-inhibition-potentials-of-oxythenanthera-abyssinica-rhizomes-sourced-from-south-eastern-nigeria/jonathan-chinenye-ifemeje
The document compares different solid phase extraction (SPE) sorbents for extracting eight nonsteroidal anti-inflammatory drugs (NSAIDs) from human plasma samples. Five commercially available SPE cartridges with different sorbent materials were tested for their ability to extract and retain the NSAID analytes. The extracted samples were then analyzed using high performance liquid chromatography coupled with diode array detection (HPLC-DAD). Oasis HLB, Bond-Elut and Strata X cartridges showed the best extraction recoveries for the NSAID analytes from human plasma samples according to the experiments.
Immobilization of two endoglucanases from different sourcesIJEAB
Cellulases are a important family of hydrolytic enzymes which catalyze the bond of cellulose and other related cello-oligosaccharide derivates. Industrial applications require enzymes highly stable and economically viable in terms of reusability. These costs can be reduced by immobilizing the cellulases, offering a potential solution through enzyme recycling and easy recovery. The covalent immobilization of enzymes is reported here: one is commercial cellulase from Aspergillus niger and other one is recombinant enzyme, named CelStrep it because was isolated from a new cellulolytic strain, Streptomyces sp. G12,. The optimal pH for binding is 4.6 for both cellulases and the optimal enzyme concentrations are 1 mg/mL and 5 mg/mL respectively. The support for immobilization is a poliacrylic matrix. Experiments carried out in this work show positive results of enzyme immobilization in terms of efficiency and stability and confirm the economic and biotechnical advantages of enzyme immobilization for a wide range of industrial applications.
In vitro conservation of Centella asiatica (Linn.)Urban. and Bacopa monnieri ...Sugandika Weerasinghe
1) The document describes an in vitro study of Centella asiatica and Bacopa monnieri, two medicinal plants known to have antioxidant properties.
2) Explants from both plants were cultured on MS medium and multiple shoots were successfully induced from nodal explants and leaves.
3) Extracts from Centella asiatica were evaluated for antioxidant activity using DPPH and FRAP assays, and for antifungal activity against Aspergillus niger. The extracts showed free radical scavenging and ferric ion reducing abilities.
Antioxidant and antiproliferative effects on human liver hepg2epithelial cell...Alexander Decker
This document summarizes a study that evaluated the antioxidant and antiproliferative effects of artichoke (Cynara scolymus L.) and its byproducts on human liver (HePG2) epithelial cells. The study found that the free phenolic extract of artichoke bracts showed significant antioxidant activity in DPPH radical scavenging and iron chelating assays. This extract also exhibited the highest antiproliferative effects against HePG2 cells, reducing viability by 78.3%, higher than the heart extract's effect of 36.7%. Therefore, artichoke byproducts contain compounds with antioxidant and anticancer properties and may provide nutritional supplements.
Antibody Glycan Analysis with Normal Phase PhyTip ColumnsChris Suh
1) Glycans are complex carbohydrates that are attached to proteins and lipids and play important roles in cellular functions and disease.
2) This study presents methods to fluorescently label and purify glycans from glycoproteins using PhyTip columns containing Sephadex G10 or normal phase resin, enabling their analysis by capillary electrophoresis.
3) Glycoproteins were digested with enzymes to release glycans, which were then labeled with a fluorescent tag and purified using the PhyTip columns to remove excess label prior to profiling and quantification by capillary electrophoresis.
Similar to 35.Isolation and purification of endopolygalacturonase produced by Alternaria cepulae (20)
This document lists the research interests of an individual including medicinal plants, production and purification of industrial enzymes, bioprocess technology, biofuels, biochemical engineering, prediction of enzyme structures, genetically modified organisms, food biotechnology, food microbiology, food enzymology, aerobiology, plant communications, signal transduction, pectins and pectinases, dairy technology, virology of tuber crops, medicinal characteristics of plants, and bioactive compounds.
The document outlines the author's teaching interests which include a wide range of topics in biotechnology, food technology, plant biology, microbiology, biochemistry, and related fields. Some key courses taught include plant physiology, biotechnology, tissue culture technology, general plant biology, biodiversity, cell and molecular biology, and courses specific to various degrees in biotechnology, bioinformatics, and more. The author's research interests involve the production, purification, and kinetics of various enzymes as well as their gene sequences, with a focus on pectinases, amylases, arabinases, xylanases, proteinases, and laccases. Other research areas covered include aerobiology, plant-animal interactions, water analysis
This letter certifies that B. Annadurai completed his Ph.D. studies under the supervision of Professor J. Mohanac. The title of his thesis was "Studies on Homopolygalacturonase in Leaf Blight Disease of Rice (Pyricularia oryzae Cav.) caused by Magnaporthe grisea (Hebert) Barr and its interaction with salicylic acid". The letter states that during his research studies, B. Annadurai was found to be intelligent, industrious, and sincere in his work. He showed great effort to complete his research studies. The professor wishes B. Annadurai success in his future efforts and progress.
This document is a letter of recommendation from the Principal of G. Abdul Hakeem College in Melvisharam, India, certifying the qualifications and experience of Dr. B. Annadurai, an Associate Professor and Head of the Department of Botany at the college. It details that Dr. Annadurai has 34 years of teaching experience in subjects like plant biology, biochemistry, microbiology, and biotechnology. He has also worked as a researcher, editor, and examiner. The principal recommends him highly as a sincere teacher, innovative researcher, and able administrator.
This document lists over 45 publications by the author, including 20 popular articles, 40 articles published in Tamil encyclopedias, 5 books, and over 30 scientific papers. The publications cover a wide range of topics in biology and related fields, such as plant quarantine, recombinant DNA, molecular biology techniques, immunology, bioinformatics, biochemistry, forest science, microbiology, and biotechnology. Many of the publications focus on research regarding plant pathogens like Alternaria cepulae and their cell wall degrading enzymes.
58.Comparative modelling of cellulase from Aspergillus terreusAnnadurai B
The document discusses homology modeling of the cellulase enzyme in Aspergillus terreus. It begins with an abstract that describes cellulase as a widely used hydrolytic enzyme involved in converting biomass to simpler sugars. It then provides details on homology modeling and the steps involved, which include template recognition, alignment, backbone and loop modeling, and model validation. The document discusses modeling of the cellulase protein from Aspergillus terreus using templates from the PDB and visualization software. It evaluates the modeled cellulase structure using validation servers to check accuracy.
57.insilico studies of cellulase from Aspergillus terreusAnnadurai B
This document describes various in silico studies performed on cellulases from Aspergillus terreus. The physicochemical properties of the cellulases were analyzed using tools from the ExPASy bioinformatics server. It was found that the molecular weights ranged from 40,927 to 100,058 Daltons and the isoelectric points were acidic. Secondary structure prediction using SOPMA showed that random coils dominated. Multiple sequence alignments and phylogenetic analysis were performed using CLC Workbench. 3D structures were obtained from ESyPred 3D server. The analyses provide insight into cellulase properties that can aid in purification and industrial applications.
56.Synthesis, Characterization and Antibacterial activity of iron oxide Nanop...Annadurai B
This document summarizes the synthesis, characterization, and antibacterial activity of iron oxide nanoparticles. Key points:
- Iron oxide nanoparticles were synthesized using a co-precipitation method by adding mixtures of metal salts to a sodium hydroxide solution, producing particles between 14-68 nm in size.
- Characterization using XRD, FTIR, VSM, and SEM confirmed the crystalline cubic spinel structure and magnetic properties of the nanoparticles. Particle size decreased with increased manganese substitution.
- Magnetic measurements showed saturation magnetization and coercivity decreased with increased manganese content due to changes in exchange interactions between metal sites.
- Antibacterial tests showed the nanoparticles had moderate antibacterial effects against E
55.Extended spectral Beta Lactamase activity of selected Bacteria and analysi...Annadurai B
This document summarizes a study on Extended-spectrum beta-lactamase (ESBL) producing bacteria isolated from different environmental samples. The study isolated bacteria from soil, water, and air samples collected near hospitals and dense population areas. The isolated bacteria - Staphylococcus aureus, Escherichia coli, Bacillus subtilis, and Pseudomonas aeruginosa - were identified using morphological, biochemical, and cultural characterization tests. Antibiotic susceptibility testing showed resistance to penicillin and cephalosporin derivatives. However, sulbactam and clavulanic acid were effective against the beta-lactamase activity.
53.Effect of different nuitritive sources for enhancing cellulase production ...Annadurai B
1. The document analyzes the effect of different carbon sources, nitrogen sources, metal ions, vitamins, and substrates like groundnut meal and mouldy bran on cellulase production by Aspergillus terreus.
2. It finds that certain carbon sources like lactose, glutamic acid, and ascorbic acid; nitrogen sources like ammonium sulfate and gelatin; and adding all vitamins together increased cellulase production and mycelial growth the most.
3. Groundnut meal in concentrations from 0.05-0.5% and mouldy bran extract supported increased cellulase activity.
52.Screeing of industrial production of CellulaseAnnadurai B
1) The study screened 73 microorganism species for their ability to produce cellulase enzymes. Alternaria, Bacillus, Aspergillus, Trichoderma, Fusarium and Penicillium showed good cellulase production.
2) Trichoderma viride and Aspergillus terreus produced the highest amounts of cellulase. Maximum enzyme production of 1.76 U/ml was achieved by A. terreus at 30°C, pH 5.0 over 6 days of incubation.
3) Supplementing the culture medium with plant waste materials like banana leaves, rice husk, and wheat bran further improved cellulase production, with 6% supplementation
51.Biological control of Macrophomina phaseolina(Tassi)goid root root in Vign...Annadurai B
This document summarizes research on using Trichoderma fungi to biologically control the root rot pathogen Macrophomina phaseolina in black gram (Vigna mungo). The research found that:
1) Trichoderma viride most strongly inhibited the growth of M. phaseolina in vitro, reducing its growth by 77.77%.
2) Seed treatment with T. viride resulted in the lowest root rot incidence (21.4%) among treatments. It also led to the highest seed germination, shoot length, and root length.
3) Soil application of T. viride resulted in the lowest root rot incidence in black gram.
50.Isolation and identification of proteolytic bacteria from raw milk samplesAnnadurai B
- Twenty five raw milk samples were collected from the Kancheepuram area of India and tested for bacterial contamination.
- A variety of bacteria were isolated from the samples including Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Micrococcus luteus and Serratia marcescens.
- Five of the isolated bacteria (Bacillus cereus, Pseudomonas aeruginosa, Proteus mirabilis, Micrococcus luteus and Serratia marcescens) showed proteolytic activity, which can negatively impact the nutritional value of milk.
49.Antitumour and Radioprotective activity of Alloe veraAnnadurai B
1) The study examined the anti-tumor and radioprotective effects of Aloe vera in mice.
2) In solid tumor models, Aloe vera significantly reduced tumor volume compared to the control group.
3) In radio protective studies, Aloe vera did not significantly affect body weight or hemoglobin levels in irradiated animals but did slightly increase total white blood cell count compared to radiation alone.
48.Effect of R394 on Ecdysteroid titre of haemolymph after fourth moult of Si...Annadurai B
This document summarizes a study that investigated the effect of the juvenile hormone mimic R394 on ecdysteroid titre levels in the haemolymph of Bombyx mori silkworms after their fourth moult. The study applied different doses of R394 topically to larvae at different time points during the fifth instar. Results found that applying 0.031 nl of R394 to one day old fifth instar larvae improved cocoon shell weight by 8% without prolonging the instar duration. Applying the same dose at 48, 72 and 96 hours prolonged the larval period by one day and improved cocoon and shell weight by 3-10%. Daily haemolymph ecdysteroid levels in treated larvae did not
47.Aerobiology of Mulberry fields in Vellore districtAnnadurai B
This document discusses an investigation into the aerobiology of mulberry fields in Vellore District, India. It aims to identify airborne microflora and causative organisms involved in mulberry diseases. Samples were collected from mulberry fields using an Andersen sampler and cultured on growth media to identify organisms. Weather data on rainfall, humidity and temperature was also collected, as these factors influence microflora growth. A variety of fungi, bacteria and other microbes were identified from the samples. The results provide information on mulberry pathogens and environmental conditions supporting their growth in Vellore District. This data can help with managing mulberry diseases and improving silk production.
45.Isolation, Purification and kinetics of deoxy Ribonucleic acid at differen...Annadurai B
This document summarizes the isolation, purification, and kinetics of deoxyribonucleic acid (DNA) at different stages of feeding in Bombyx mori (silkworm). It begins with an abstract describing the study and an introduction on silk production. It then details the isolation of DNA from silkworm samples using lysis and precipitation. Tests for DNA like the diphenylamine reaction are also described. The aim was to estimate and separate DNA and RNA from silkworms at different stages using gel electrophoresis to study protein and nucleic acid interactions during silk production.
44.Antimicrobial activity in leaf extract of Neem(Azadirachta indica Linn.)Annadurai B
1) The study investigated the antimicrobial activity of extracts from neem (Azadirachta indica) leaves against several human pathogenic bacteria.
2) Methanol and ethanol extracts of neem leaves were tested using disc diffusion and microdilution assays at concentrations from 200-25 mg/ml.
3) The results showed that both extracts inhibited the growth of Bacillus pumilus, Pseudomonas aeruginosa, and Staphylococcus aureus, with the methanol extract demonstrating the highest level of inhibition against Bacillus pumilus and the ethanol extract showing the strongest effect against Pseudomonas aeruginosa.
43.Studies on chlorophyll conttent, soluble protein, carbohydrates and moistu...Annadurai B
This study analyzed the chlorophyll content, soluble proteins, carbohydrates, and moisture content of Morus alba leaves from Vellore district in Tamil Nadu, India. The study found that MR2 mulberry variety had higher levels of these nutrients compared to the commonly used Mysore local variety. Specifically, MR2 variety leaves contained more chlorophyll, proteins, carbohydrates, and moisture. The study recommends farmers in the district cultivate MR2 variety over Mysore local to improve leaf quality and silk production. Proper cultivation techniques like irrigation, spacing of silkworms, and sanitation measures can also increase cocoon and silk yields. The nutrient composition of mulberry leaves impacts silkworm growth
43.Studies on chlorophyll conttent, soluble protein, carbohydrates and moistu...
35.Isolation and purification of endopolygalacturonase produced by Alternaria cepulae
1. I.J.A.B.R., VOL. 2(3) 2012: 436-440 ISSN 2250 - 3579
436
ISOLATION AND PURIFICATION OF ENDO POLYGALACTURONASE
PRODUCED BY ALTERNARIA CEPULAE
a
Brindha, S., b
Maragathavalli, S., & c
Gangwar, S. K. & c
Annadurai, B.
a
Department of Bioinformatics, New Prince Arts and Science College, Chennai,
b
Department of Biochemistry, King Nandi Varman College of Arts and Science, Thellar,
c
Biotechnology Team, College of Dryland Agriculture and Natural Resources, Mekelle University, Mekelle, Ethiopia.
ABSTRACT
The extracellular endopolygalacturonase from Alternaria cepulae causing leafblight disease in Onion was isolated and
purified. The crude enzyme extract obtained in onion medium was precipitated with 50-95% ammonium sulphate at pH
5.0. The isoenzymes were separated on DEAE - Cellulose column with 0.01 M Phosphate buffer at pH 7.0. Further
purification was achieved in ultrogel ACA 44 (LKB) column. The endo PG I and endo PG II have migrated as a single
band on PAGE & SDS - PAGE. These isoenzymes have an isoelectric point of 6.7 and 7.1 and give single
immunopreciptin line.
KEY WORDS: endopolygalacturonase, isoenzymes, DEAE, PAGE & SDS - PAGE etc.
INTRODUCTION
Pectic enzymes play an integral part in pathogenesis of a
wide spectrum of plant diseasesAmong these enzymes,
the occurrence and production of polygalacturonase ( poly
x 1-4 D galacturonideglycano hydolase EC3.2.1.15 )by
many fungi during disease development has been widely
ascertained ( Bateman and Millar, 1966 Shiro,etal,1984,
Bashan, etal,1985 ). Alternaria cepulae is known to
produce endopolygalacturonase ( Ponnappa et al 1977 ). A
study was therefore planned to purify the enzyme.
MATERIALS AND METHODS
Fungi
The fungus Alternaria cepulae ( Ponnappa ) employed in
the present study was isolated from the infected onions.
The isolate was maintained on PDA at 32 2C. This was
subcultured at intervals of 3 months and the stock cultures
were maintained at 4C.
Enzyme preparation
One kg samba onions were cut into small pieces. This was
taken along with500ml of water in 5lit haffkins flask. The
flask was sterilized with the contents at 15 lb pressure in
Barnslead autoclave (Boston) for 20 minutes. Then the
flask was cooled to room temperature (321C) and a
slant of A. cepulae was inoculated into the onion medium
.After 16 days the contents along with 0.25 M NaCl were
blended in a waring blender for 5 seconds at 4C. The
blended juice was filtered and centrifuged at 20.000 rpm
for 20 minutes at 4C. The clear supernatant was dialysed
against 2 lit of 0.01 M phosphate buffer atpH 7.0 for 48
hours with 2 changes at 4o
C. Ammonium sulphate
fractionation. To 1 lit of dialysed supernatant solution,
ammonium sulphate (AR) was added slowly with a
constant stirring in a magnetic stirrer for 4 to 6 hours at 4
C to give 0-40%, 40-80%, 80-95% saturation. After the
desired percentage of saturation the contents of each step
was centrifuged at 15,000 rpm for 20 minutes at 4C. The
precipitate obtained was dissolved and dialysed against
0.01 M phosphate buffers at pH 7.0 containing 0.1 M
NaCl with 3 changes of buffer.
Ion exchange column chromatography
50 ml of the dialysed samples from 80-95% (NH4 )2 SO4
precipitate were applied to a DEAE cellulose column( 3 x
25 cm ) equilibrated with 0.01 M phosphate buffer (pH
7.0). They were eluted with the same buffer containing
stepwise gradient of 0.05 M NaC1 at the flow rate of 9ml
h-1. 3 ml fractions were collected. The individual fractions
were read at 280 nm and EPG activity was estimated.
EPG Assay
Endo polygalacturonase activity was estimated by the
method of Nelson somogyi (1944 and 1952).
Estimation of protein
Protein content was determined by the method of Lowry et
al (1951) using crystalline bovine serum albumin (BSA)
as standard.
Tests for homogeneity
PAGE was carried out according to the methods of
DAVIS(1964). SDS-PAGE was done by adopting the
procedure of Laemmli (1970). Doubleimmunodiffusion
was carried out by the method of Ouchterlony (1973).
Immuno diffusion electrophoresis was performed
according to the method of Graber and Burtin (1964)
Isoelectricfocussing was carried out in a
semipreparativemanner based on the method of Wrigley
(1971)
RESULTS
Fig 1: shows theelution profileofDEAE - Cellulose cloumn
chromatography of EPG. The precipitate obtained
2. Problems of urbanization in developing countries
437
between 80-95% (NH4)2 SO4 fraction was dialysed and
applied on DEAE - Cellulose column. The stepwise
gradient elution shows that the first fraction of EPG
activity (designated as EPG x) eluted at the
concentration of 0.1 M NaC1 and the second fraction of
EPG activity (Designated as EPG II) eluted at the
concentration of 0.2 M NaCl.
Elution profile of EPG I from DEAE - Cellulose column
after pooling and dialysis against 0.01 M phosphate buffer
(pH 7.0) was lyophilized. A small amount was dissolved
in the same buffer and loaded on ultrogel column (Fig2)
shows two protein fractions but EPG activity is seen only
in 1st fraction, ie from 36th to 52nd fraction.
The second fraction of DEAE column after conducting the
same procedure as described above was loaded to the
ultrogel column. The elution profile (Fig 3) shows only
the one peak which contains both protein and EPG
activity. The EPG II activity is seen from 48th to 56th
fraction.
A summary of purification presented in Table 4 reveals
that EPGI is purifiedto 245 fold with 26% yield. EPG II is
purified to 270 fold and with 22% yield.
From each step of purification the enzyme samples
weretested for homogeneityusing PAGE and SDS-PAGE.
After final purification EPGI shows only one band in
PAGE and SDS - PAGE (Fig 6). Similarly EPG II also
shows single band in PAGE (Fig 7) and SDS - PAGE (Fig
8).
TABLE 1: Ammonium Sulphate precipitation of crude enzyme preparation of EPG from natural onion medium
Sl
No
Percentage of (NH4)2
SO4 (%)
Volume(ml) EPG activity units up of gal. acid
rel/ml/30 min
Protein
content g/mi
Spcific activity
in units/ug
1
2
3
4
Crude enzyme
0-40
40-80
80-95
900
30
40
50
56.4
1.32
2.04
976.4
71.2
26.4
29.1
76.9
0.79
0.05
0.07
12.0
TABLE 2: Purification of endopolygalacturonase
3. I.J.A.B.R., VOL. 2(3) 2012: 436-440 ISSN 2250 - 3579
438
Steps Volume
(ml)
Total
Protein
(g)
Total endoPG
Activity units
(g of gal.acid
rel)
Specific activity
Units/g protein
Purifocation
fold
Yield
(%)
Culture filtrate(supernatant)
80-95% (NH4)2 SO4
Precipitation DEAE-column
EPG I-fraction
EPG II - fraction
Ultrogel column EPG 1-fraction
EPG II –fraction
900
50
138
40
34
16
64080
3845
201
162
68
52
50760
48820
21618
20825
13285
11226
0.79
12.7
107.55
128.54
195.36
215.88
1
16.0
135.5
161.34
245.18
270.93
100
96.18
42.58
41.01
26.16
22.11
Determination of isoeletric point in a semipreparative
manner of EPGI is shown in Fig. 9. It indicates a single
peak of EPG activity between 11th and 12th fractions at
isoelectric pH of 6.7. The semipreparative isoelectric
focussing of EPG II is shown in Fig10. It shows a single
of EPG activity between fractions 16 and 17 and its
isoelectric pH is 7.1.The ouchterlony's double immune
diffusion pattern indicatesthe homogeneity of the EPG I
and EPG II (Fig11). The highly purified enzyme gave a
single immunoprecipitin line in reflected light when
reacted with its rabbit antiserum showing the presence of
only one component.Graber & Burtin qualitative analysis
by immunoelectrophoresis also confirmed the
homogeneity of the enzyme as depicted in Figure 12.
4. Problems of urbanization in developing countries
439
DISCUSSION
Multiple formsof endopolygalacturonase have been
reported in leaf spots,blights,wilt disease of plants
(Endo,1964, Fielding et al., 1969, pressey et al., 1973,
Hislop et al., 1974, Bartheetal, 1981 Scala, 1983 and
Arinze, 1985) The DEAE Cellulose column treatment at
pH 5.0 selectively removed the brown pigments from the
crude enzyme extract giving appreciable increase in
specific activity. Similar affinity column was used for
thepurification by Pressay, 1973, Strand, et al, 1976 and
Takahashi, 1985. The DEAE - Cellulose column
chromatography at pH 7.0 selectively adsorbed the entire
EPG.
The adsorbed enzyme was eluted from the stepwise
column gradient as a major fraction (Fig1). Similar
elution pattern was reported for the purification of EPG
(Urbanek,et al, 1975, Magro et al., 1980, Hoffman, et al,
1982 and York, et al, 1986).When crude enzyme from A.
cepulae was subjected sequentially to six different
treatments, the purified EPG I having 245 fold increase in
specific acitvity with 26.16% yield & EPG II having 270
fold increase in activity with 22.11% yield were
obtained (Table 4)
The results of polyacrylamide gel electrophoresis &
SDS-PAGE (Fig 5, 6, 7, and 8) suggest that the purified
enzyme ishomogeneous. Isoelectricfocussing pattern (9 &
10) also confirmed the previous findings of the
homogeneity of the enzyme. The presence of a single
proteinband suggests that EPGIandIIconsists of a single
polypeptide chain (Cervons et al., 1977).The
immunodiffusion & immunoelectrophoresis results
showing single precipition line confirm the homogeneity
of the purified EPG
ACKNOWLEDGEMENT
The authors are grateful to Dr. S. C. Dhar and Dr. R.
Puvanakrishnan, Scientists at the Department of
Biotechnology, CLRI, Chennai for Laboratory facilities
and Useful suggestion and UGC, NewDelhi for research
grant.
REFERENCES
Arinze, A.E. (1985). The Adsorptionof a
polygalacturonase isoenzymeof Botryodiplodia
theobromae on plant tissues & the implication on the
pathogenicity of thefungus. Phytopathologiache zeit
schirft, 114. 13-19
Barthe,J.P. Cantenys, D. and Touze, A. (1981).
Purification & characterization of Two Potygalacturonase
secreted by Collectotrichum lindemuthianum. phytopath,
z. 100. 162-171.
Bashan, Y. Okon, Y. & Henis, Y. (1985) Detection of
cutinase & pectic enzymes during infection of Tomato
by Psendomonas svringae Pv tomato, phytopathology 75,
940-945.
Bateman, D.F.& Millar, R. L.(1966) pectic Enzymes in
Tissue Degradation Ann. Rev. phytopathol, 4. 119-146.
Cervone,F. Scala, A. Foresti, M. Cacacea, M. G. &
Noviello, C. (1977) Endopolygalacturonasefrom
Rhizoctoniz frgariae. Purificationand characterization of
twoisoenzymes. Biochim Biphys acta 482, 379-385
Davis, B.J. (1964) Disc electrophoresis II Mthod
&application to human serum proteins.Ann. N.Y. Acad
Sci, 121. 404-427
Endo, A.(1964) A studies on Pectolytic enzymes of molds
XI, purificationandproperties of exo-polygalacturonase.
Agric. Biol.chem. 28, 535-542.
Fielding, A.H. & Byrde, R. J. W. (1969) The partial
purification and properties of endo polyglalcturonase and
L-Arabino furanosidase secreted by sclerotinia fructigena
J.Gen.Microbiol, 58,73-84.
Graber,P & Burtin, P. (1964) immuno electrophoretic
analysis, Elsevier, Amsterdam, 337.
Hislop,E.C. Shellis, C. Fielding, A.H. Bourne,F.J. &
Chilow, J.W. (1974) Antisera produced to purified
Extracellular Pectolytic enzyme from sclerotina
fructigena.J.Gen. Microbiol 83. 135-143.
Hoffman, R. M.& Turner, J.G. (1982) Partial Purification
of Proteinsfrom pea leaflets that inhibit Ascochyta endo
polygalacturonase physiol, Pl pathol 20. 173-187
Laemmli, U.K. (1970) cleavage of structural proteins
during the Assembly of the Head of Bacteriophage.
Nature, 227. 680-685
5. I.J.A.B.R., VOL. 2(3) 2012: 436-440 ISSN 2250 - 3579
440
Lowry. O. H. Rosebrough, N. J., Farr, A.L. & Randall, R.
J. (1951) Proteinmeasurment with Folin phonol Reagent J.
Biolchem 193. 265-275
Magro, P., Dilenna, P., Marciano, P. & Pallavicini, C.
(1980) variability of polygalacturonase & protein
isloectric focussing patterns in Botrytis cinerea isolates
J. Gen. Microbobiol, 120. 105-109
Nelson, N. (1944) A Photometric Adaptation of the
Somogyi Method for the determination of Glucose. J. Biol.
chem 153. 375-380
Ouchterlony, O. and Nilsson, L. A. (1973)
immunodiffusion and immunoelectrophoresis in D.W.
Weir (ed) Handbook ofexp.tal immunology 2nd ed
Blackwell Oxford Edinburgh (1973). Chap19, 39.
Ponnappa,K.M. Anilkumar, T. B. Sulladmath, V.V. &
Hiremath P.C. (1977) Polygalacturonaseproduction by
Alternaria cepulae the causal gaent of Leaf blight of onion
Ind. J. Mycol and Pl. Pathol, 1. 67
Presey, R. and Avants, J. K., (1973) Two forms of
polygalacturonase in Tomatoes. Bio Chem. Biophys.
Acta,309. 363-369
Scala.F.and Zoina, A. (1983) ProductionofPectolytic and
cellulolytic enzymes by cornebacterium michiganese
(E.F.Smith) tensen. Ann. Fac. Sci. Arrar Univ. Studi.
Napoliportici, 17. 172-178. (Itali).
Shiro, I.Yasubori, N & Chitoshi, I. (1984) Agric, Biol.
Chem., 48. 633-640
Somogyi, M. (1952) Notes on Sugar determination J.
Biol.Chem, 195. 19-23
Strand. L.L. Corden, M.E. and MC. Donald, D.L. (1976)
Characterization of two endopolygalacluronase isozymes
produced by Fusaium oxysporim f-sp hycopersic. Bio
Chem. Biophysica Acta., 429. 870-883
Takahashi, T. and Doke, N. (1985) purification and partial
characterization of an agglutinin in citrusleaves against
extracellular polysaccharides of Xanthomonas
campestria pv citri Physiol. p1. Pathol 27. 1.3.
Wrigley, C.W. (1971) inmathods in Enzymology
(Jackoby, W.B ed)22, P 55 9. Academic Press, New York.
York, W.S. Darvill, A.G. Mc. Neil, M. Stevenson T.T. and
Albersheim. P. (1986) isolation &characteriation of plant
cellwallsand cellwall components in methods of
enzymology, Vol 118, Plant molecular biology edited by
Arthur Weissbach, Hesbert Weissbach, Academic Press
inc. 1986. p 3-40.