This document summarizes research on purifying and characterizing manganese peroxidase (MnP) enzyme produced by the white-rot fungus Irpex lacteus. The MnP enzyme was purified using ion exchange and size exclusion chromatography. Characterization found the molecular mass to be 38.3 kDa by mass spectrometry and 53 kDa by SDS-PAGE. The isoelectric point was 3.7. Spectroscopy showed it is a heme-containing glycoprotein. Mass spectrometry identified tryptic peptides. The purified MnP effectively decolorized textile dye wastewater and oxidized Mn2+ to Mn3+, with optimal activity at pH 6 and 35°C.
Proteins play crucial roles in nearly all biological processes. These many functions of proteins are a result of the folding of proteins into many distinct 3D structures.
Protein analysis tries to explore how amino acid sequences specify the structure of proteins and how these proteins bind to substrates and other molecules to perform their functions.
Protein analysis allows us to understand the function of the protein based on its structure.
Metabolomic Profiling of Spent Biomass Of Marine Microalgae, Chlorella vulgarispriyanka raviraj
OBJECTIVE:
To evaluate the presence of any high value added compounds in the spent biomass of C. vulgaris
To identify the biological activity of the extracted compounds
To evaluate the structure and nature of the compounds using Nuclear Magnetic Resonance Spectroscopy and other analytical techniques.
Development of economically viable methodologies for the simultaneous extraction of by-products from a single set of biomass.
biological activities performed -Total antioxidant capacity, Anti bacterial activity, Anti-tuberculosis activity, Anti proliferative assay
This document provides an overview of several common methods for analyzing proteins including the Kjeldahl method, Dumas method, Biuret assay, Lowry assay, BCA assay, UV-Vis spectroscopy, and dye-binding methods. The Kjeldahl method involves digestion, distillation, and titration to determine total nitrogen as a measure of protein. The Dumas method uses combustion and gas chromatography to measure total nitrogen. Colorimetric assays like Biuret, Lowry, and BCA use reactions between proteins and reagents to produce colors measured spectrophotometrically. UV-Vis spectroscopy measures protein absorbance at 280nm. Dye-binding methods quantify protein by measuring unbound dye after binding
This document summarizes a study that synthesized homopolymers of 2,6-dimethyl-phenol (DMP) and 2,6-diphenyl-phenol (DPP) as well as copolymers with varying compositions of DMP and DPP. Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (NMR) spectroscopy were used to characterize the polymers and determine their compositions. FTIR provided more accurate composition measurements for copolymers with low DPP content, while both methods showed the final copolymer compositions were lower than the initial monomer ratios. Glass transition temperatures from dynamic mechanical thermal analysis correlated with the calculated copolymer compositions.
This document discusses lipid composition and analysis. It introduces that lipids are insoluble in water but soluble in organic solvents. They include fatty acids, neutral fats, and waxes. Lipids serve important functions as an energy source and in cell structure. Common methods to analyze lipids include fatty acid methyl ester analysis using gas chromatography and mass spectrometry to characterize bacteria based on their lipid profiles.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research paper publishing, where to publish research paper, journal publishing, how to publish research paper, Call for research paper, international journal, publishing a paper, call for paper 2012, journal of pharmacy, how to get a research paper published, publishing a paper, publishing of journal, research and review articles, Pharmacy journal, International Journal of Pharmacy, hard copy of journal, hard copy of certificates, online Submission, where to publish research paper, journal publishing, international journal, publishing a paper
The document discusses various methods for determining enzyme activity through kinetic assays. It explains that enzyme activity is measured by how quickly an enzyme catalyzes the conversion of substrates to products under set conditions. There are two main types of assays discussed: single enzyme assays where the reaction is directly monitored, and coupled assays where the reaction is linked to a secondary reaction that allows for detection. Key factors discussed include using saturating substrate concentrations, fixed temperatures and pH, and ensuring sufficient amounts of indicator enzymes in coupled assays to quickly reach steady state levels.
Proteins play crucial roles in nearly all biological processes. These many functions of proteins are a result of the folding of proteins into many distinct 3D structures.
Protein analysis tries to explore how amino acid sequences specify the structure of proteins and how these proteins bind to substrates and other molecules to perform their functions.
Protein analysis allows us to understand the function of the protein based on its structure.
Metabolomic Profiling of Spent Biomass Of Marine Microalgae, Chlorella vulgarispriyanka raviraj
OBJECTIVE:
To evaluate the presence of any high value added compounds in the spent biomass of C. vulgaris
To identify the biological activity of the extracted compounds
To evaluate the structure and nature of the compounds using Nuclear Magnetic Resonance Spectroscopy and other analytical techniques.
Development of economically viable methodologies for the simultaneous extraction of by-products from a single set of biomass.
biological activities performed -Total antioxidant capacity, Anti bacterial activity, Anti-tuberculosis activity, Anti proliferative assay
This document provides an overview of several common methods for analyzing proteins including the Kjeldahl method, Dumas method, Biuret assay, Lowry assay, BCA assay, UV-Vis spectroscopy, and dye-binding methods. The Kjeldahl method involves digestion, distillation, and titration to determine total nitrogen as a measure of protein. The Dumas method uses combustion and gas chromatography to measure total nitrogen. Colorimetric assays like Biuret, Lowry, and BCA use reactions between proteins and reagents to produce colors measured spectrophotometrically. UV-Vis spectroscopy measures protein absorbance at 280nm. Dye-binding methods quantify protein by measuring unbound dye after binding
This document summarizes a study that synthesized homopolymers of 2,6-dimethyl-phenol (DMP) and 2,6-diphenyl-phenol (DPP) as well as copolymers with varying compositions of DMP and DPP. Fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (NMR) spectroscopy were used to characterize the polymers and determine their compositions. FTIR provided more accurate composition measurements for copolymers with low DPP content, while both methods showed the final copolymer compositions were lower than the initial monomer ratios. Glass transition temperatures from dynamic mechanical thermal analysis correlated with the calculated copolymer compositions.
This document discusses lipid composition and analysis. It introduces that lipids are insoluble in water but soluble in organic solvents. They include fatty acids, neutral fats, and waxes. Lipids serve important functions as an energy source and in cell structure. Common methods to analyze lipids include fatty acid methyl ester analysis using gas chromatography and mass spectrometry to characterize bacteria based on their lipid profiles.
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research...iosrphr_editor
IOSR Journal of Pharmacy (IOSRPHR), www.iosrphr.org, call for paper, research paper publishing, where to publish research paper, journal publishing, how to publish research paper, Call for research paper, international journal, publishing a paper, call for paper 2012, journal of pharmacy, how to get a research paper published, publishing a paper, publishing of journal, research and review articles, Pharmacy journal, International Journal of Pharmacy, hard copy of journal, hard copy of certificates, online Submission, where to publish research paper, journal publishing, international journal, publishing a paper
The document discusses various methods for determining enzyme activity through kinetic assays. It explains that enzyme activity is measured by how quickly an enzyme catalyzes the conversion of substrates to products under set conditions. There are two main types of assays discussed: single enzyme assays where the reaction is directly monitored, and coupled assays where the reaction is linked to a secondary reaction that allows for detection. Key factors discussed include using saturating substrate concentrations, fixed temperatures and pH, and ensuring sufficient amounts of indicator enzymes in coupled assays to quickly reach steady state levels.
1) The first protein sequencing was achieved in 1953 by Frederic Sanger who determined the amino acid sequence of bovine insulin.
2) Common strategies for protein sequencing include determining the number of subunits, disulfide bonds, amino acid composition, and sequencing fragments using Edman degradation or enzymatic cleavage.
3) Techniques like end-group analysis, solid-phase support, exopeptidases, and mass spectrometry help overcome challenges and complete the protein sequence.
The determination of amino acid sequences presentation autumne 2015Richard Trinh
1. The document outlines common strategies used for protein sequencing, including Edman degradation. Edman degradation involves breaking the peptide bonds of a protein one by one to determine the amino acid sequence.
2. Other strategies discussed include peptide mass fingerprinting, tandem mass spectrometry, and de novo sequencing methods. Peptide mass fingerprinting involves breaking a protein into peptides and using mass spectrometry to determine the peptide masses and compare them to databases. Tandem mass spectrometry further breaks peptides into fragment ions for sequencing.
3. The strategies each have advantages and limitations. While Edman degradation was previously standard, mass spectrometry techniques are increasingly used due to their high-throughput capabilities and ability to sequence smaller protein amounts. The
This document summarizes a study investigating the initialization behavior of reversible addition-fragmentation chain transfer (RAFT)-mediated styrene-maleic anhydride copolymerizations using in situ 1H NMR spectroscopy. The results indicate specificity of addition of the RAFT agent leaving groups for either styrene or maleic anhydride. Analysis of the NMR spectra also showed that monomers are added individually, favoring the penultimate unit model of polymer propagation over other proposed mechanisms. Stereoselectivity was observed during monomer addition to the RAFT agent.
This document provides an overview of the topics that will be covered in an enzyme assays and metabolism course over several weeks in November and December. The course will cover topics like enzyme assay fundamentals, mass spectrometry techniques, primary metabolism, glycogen metabolism, metabolic networks in humans, flux analysis, and metabolic databases and modeling tools. It includes the reading assignments and discussion sessions for each class. The readings will cover developing effective enzyme assays, factors that influence enzyme activity, coupled enzyme assays and potential errors, and new techniques for measuring enzyme activity and metabolic states.
This document discusses peptide mapping, which involves enzymatically or chemically cleaving a protein into peptides and analyzing the peptides to identify the protein's primary structure. Peptide mapping is used to confirm identity, detect alterations, and ensure consistency for biopharmaceutical characterization. It involves four main steps - selectively cleaving peptide bonds, separating peptides chromatographically (often via RP-HPLC), analyzing and identifying peptides (usually via mass spectrometry), and comparing results to a reference standard to confirm identity. The document provides details on reagents, conditions, and parameters for each step of peptide mapping.
- The document describes efforts to optimize the solid-phase synthesis of peptide nucleic acids (PNAs) for use as tags to encode small molecule libraries. Two solid supports - Rink amide resin and 2-chlorotrityl chloride (CTC) resin - were tested.
- While the Rink amide resin approach yielded poor results due to difficulties cleaving the Fmoc protecting group, the CTC resin approach successfully produced three PNA trimers that were identified using mass spectrometry, demonstrating the potential of this method for producing encoding elements.
- Going forward, the CTC resin approach will be used to generate larger PNA oligomer libraries that can be efficiently coupled to encode and track collections of
Structure-Function Relationships in the MppP FamilyOURUWM
This document summarizes research characterizing a mutant form of the enzyme MppP. The researchers induced a single amino acid mutation (A87S) in MppP from Pseudomonas to investigate structural differences that lead to different catalytic activities between bacterial species. They purified the mutant enzyme and used NMR spectroscopy and oxygen probe analysis to show the mutation eliminated catalytic activity, with oxygen turnover only occurring once. While product identification requires further mass spectrometry analysis, the research provides insights into structure-function relationships in MppP enzymes.
Protein sequencing is a technique to determine the amino acid sequence of proteins. It involves hydrolyzing proteins into amino acids, separating the amino acids, and using techniques like chromatography to identify the order of the amino acids. There are chemical methods like Edman degradation and physical methods like mass spectrometry to sequentially determine the order of amino acids from the N-terminal to C-terminal end of the protein. Understanding amino acid sequences provides insight into protein structure and function.
There are several key steps to sequencing a protein, including:
1) Separating individual protein chains and breaking disulfide bonds. This can involve reagents like mercaptoethanol or performic acid.
2) Purifying individual protein chains using techniques like electrophoresis or chromatography.
3) Cleaving the purified protein into fragments using enzymes like trypsin or chemicals like cyanogen bromide.
4) Determining the amino acid sequence of each fragment, then combining the overlapping sequences to deduce the full protein sequence.
Evaluation of antioxidant and antiradical properties of pomegranate (punica g...Pritam Kolge
Evaluation of antioxidant and antiradical properties
of Pomegranate (Punica granatum L.) seed and defatted
seed extracts
This is Journal Club activity Presentation with the reference of various research papers.
This Presentation Contain following...
#Info about Paper
#Abstract
#Materials
#Methods
#Results
#Discussion
#Conclusion
#References
*Important Methods used
#Moisture content
#Fat content
#Acid value
#Peroxide value
#Oxidative stability index
#Total phenols content
#Preparation of Pomegranate seed extracts and calculate extract yield
#Evaluation of antioxidant properties of Pomegranate seed extracts using
-DPPH radicals scavenging activity
-FRAP assay
#Antioxidant efficiency of seed extract (Oxidative stability extract)
#Statistical analysis
Journal Club Presentation at Bharati Vidyapeeth College of Pharmacy, Kolhapur.
Thanks for Help and Guidance of Dr. P. B. Choudhari (Assistant Professor, Pharmaceutical Chemistry) and Dr. A. J. Shinde (Assistant Professor, Department of Pharmaceutics)
This document discusses protein sequencing techniques. There are three main methods for determining a protein's amino acid sequence: N-terminal sequencing, C-terminal sequencing, and predicting the sequence from DNA sequencing. N-terminal sequencing involves using methods like Edman degradation to sequentially remove amino acids from the N-terminus and identify them. C-terminal sequencing uses carboxypeptidases to sequentially remove amino acids from the C-terminus over time. DNA sequencing allows predicting the protein sequence by first sequencing the gene that encodes the protein. Protein sequencing is important for understanding cellular processes and developing targeted drugs.
This document discusses various methods for determining the amino acid sequence of proteins, including:
- Edman degradation, which sequentially removes amino acids from the N-terminus. Up to 60 amino acids can typically be determined.
- Mass spectrometry techniques like MALDI that help determine the mass and sequence of protein fragments.
- Enzymatic cleavage techniques using enzymes like trypsin to break proteins into smaller fragments that can then be sequenced.
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.
Determination of protein structure by Dr. Anurag YadavDr Anurag Yadav
Proteins perform important roles in organisms and their structure and function can be studied by determining their amino acid sequence. There are several steps to determine the sequence, including isolating the protein, determining the number of peptide chains, identifying the N- and C-terminals, and Edman degradation which sequentially removes amino acids from the N-terminal for identification. Mass spectrometry can also be used to analyze protein structure. Higher levels of structure can be analyzed using techniques like X-ray crystallography and NMR spectroscopy.
Lectin-functionalized carboxymethylated kappa-carrageenan microparticles for ...ankit
This document describes a study on the development of lectin-functionalized carboxymethylated kappa-carrageenan microparticles for oral insulin delivery. The microparticles were prepared using an ionotropic gelation technique and characterized. Insulin was encapsulated within the microparticles and lectin was conjugated to the surface. In vitro studies showed the lectin functionalization enhanced insulin release and microparticle interactions with intestinal tissue. In vivo studies in diabetic rats demonstrated the lectin-functionalized microparticles provided prolonged glycemic control for 12-24 hours following oral administration.
The document discusses protein sequencing techniques. It begins with an introduction to proteins and protein sequencing. The history of protein sequencing is then outlined, including early work by Edman and Sanger. Methods for determining amino acid composition through hydrolysis, separation, and analysis are described. The mechanisms of Sanger's method using dinitrophenyl reagents and Edman degradation using phenylisothiocyanate are explained. Applications of protein sequencing include determining protein structure and function. The document concludes with locations of protein sequencing institutes in India and abroad.
Frederick Sanger developed two important methods for protein sequencing: 1) using fluorodinitrobenzene to determine the N-terminal amino acid, and 2) cleaving proteins into fragments and piecing their sequences together. The standard strategy involves separating chains, identifying terminal residues, cleaving the protein, sequencing fragments, and reconstructing the full sequence. The Edman degradation method allows sequential removal of residues from the N-terminus. Mass spectrometry techniques like peptide mass fingerprinting and tandem mass spectrometry now dominate protein sequencing.
The document discusses applications of liquid chromatography-mass spectrometry (LCMS). It describes how LCMS combines liquid chromatography separation with mass spectrometry detection. Several studies using LCMS are summarized, including analyzing phytochemicals in Solanum plants and identifying compounds in Archidendron bubalinum seed shell extracts. Key components detected include chlorogenic acids, flavonoids, alkaloids, and the compounds phlorizin and astilbin.
plain 4 SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLETAndrew Apals
This document outlines Andrew Apals' thesis defense presentation on the synthesis, purification, and stability study of the islet neogenesis-associated protein peptide (INGAP-P) and analogs. The presentation covers solid phase peptide synthesis of INGAP-P, linear and cyclic analogs. It also discusses reverse-phase HPLC method development for the separation and quantification of peptides in synthetic mixtures, including degradation studies of INGAP-P standards. The goal of the research is to develop purification methods to resolve INGAP-P from crude synthesis mixtures using analytical HPLC instrumentation.
1) The first protein sequencing was achieved in 1953 by Frederic Sanger who determined the amino acid sequence of bovine insulin.
2) Common strategies for protein sequencing include determining the number of subunits, disulfide bonds, amino acid composition, and sequencing fragments using Edman degradation or enzymatic cleavage.
3) Techniques like end-group analysis, solid-phase support, exopeptidases, and mass spectrometry help overcome challenges and complete the protein sequence.
The determination of amino acid sequences presentation autumne 2015Richard Trinh
1. The document outlines common strategies used for protein sequencing, including Edman degradation. Edman degradation involves breaking the peptide bonds of a protein one by one to determine the amino acid sequence.
2. Other strategies discussed include peptide mass fingerprinting, tandem mass spectrometry, and de novo sequencing methods. Peptide mass fingerprinting involves breaking a protein into peptides and using mass spectrometry to determine the peptide masses and compare them to databases. Tandem mass spectrometry further breaks peptides into fragment ions for sequencing.
3. The strategies each have advantages and limitations. While Edman degradation was previously standard, mass spectrometry techniques are increasingly used due to their high-throughput capabilities and ability to sequence smaller protein amounts. The
This document summarizes a study investigating the initialization behavior of reversible addition-fragmentation chain transfer (RAFT)-mediated styrene-maleic anhydride copolymerizations using in situ 1H NMR spectroscopy. The results indicate specificity of addition of the RAFT agent leaving groups for either styrene or maleic anhydride. Analysis of the NMR spectra also showed that monomers are added individually, favoring the penultimate unit model of polymer propagation over other proposed mechanisms. Stereoselectivity was observed during monomer addition to the RAFT agent.
This document provides an overview of the topics that will be covered in an enzyme assays and metabolism course over several weeks in November and December. The course will cover topics like enzyme assay fundamentals, mass spectrometry techniques, primary metabolism, glycogen metabolism, metabolic networks in humans, flux analysis, and metabolic databases and modeling tools. It includes the reading assignments and discussion sessions for each class. The readings will cover developing effective enzyme assays, factors that influence enzyme activity, coupled enzyme assays and potential errors, and new techniques for measuring enzyme activity and metabolic states.
This document discusses peptide mapping, which involves enzymatically or chemically cleaving a protein into peptides and analyzing the peptides to identify the protein's primary structure. Peptide mapping is used to confirm identity, detect alterations, and ensure consistency for biopharmaceutical characterization. It involves four main steps - selectively cleaving peptide bonds, separating peptides chromatographically (often via RP-HPLC), analyzing and identifying peptides (usually via mass spectrometry), and comparing results to a reference standard to confirm identity. The document provides details on reagents, conditions, and parameters for each step of peptide mapping.
- The document describes efforts to optimize the solid-phase synthesis of peptide nucleic acids (PNAs) for use as tags to encode small molecule libraries. Two solid supports - Rink amide resin and 2-chlorotrityl chloride (CTC) resin - were tested.
- While the Rink amide resin approach yielded poor results due to difficulties cleaving the Fmoc protecting group, the CTC resin approach successfully produced three PNA trimers that were identified using mass spectrometry, demonstrating the potential of this method for producing encoding elements.
- Going forward, the CTC resin approach will be used to generate larger PNA oligomer libraries that can be efficiently coupled to encode and track collections of
Structure-Function Relationships in the MppP FamilyOURUWM
This document summarizes research characterizing a mutant form of the enzyme MppP. The researchers induced a single amino acid mutation (A87S) in MppP from Pseudomonas to investigate structural differences that lead to different catalytic activities between bacterial species. They purified the mutant enzyme and used NMR spectroscopy and oxygen probe analysis to show the mutation eliminated catalytic activity, with oxygen turnover only occurring once. While product identification requires further mass spectrometry analysis, the research provides insights into structure-function relationships in MppP enzymes.
Protein sequencing is a technique to determine the amino acid sequence of proteins. It involves hydrolyzing proteins into amino acids, separating the amino acids, and using techniques like chromatography to identify the order of the amino acids. There are chemical methods like Edman degradation and physical methods like mass spectrometry to sequentially determine the order of amino acids from the N-terminal to C-terminal end of the protein. Understanding amino acid sequences provides insight into protein structure and function.
There are several key steps to sequencing a protein, including:
1) Separating individual protein chains and breaking disulfide bonds. This can involve reagents like mercaptoethanol or performic acid.
2) Purifying individual protein chains using techniques like electrophoresis or chromatography.
3) Cleaving the purified protein into fragments using enzymes like trypsin or chemicals like cyanogen bromide.
4) Determining the amino acid sequence of each fragment, then combining the overlapping sequences to deduce the full protein sequence.
Evaluation of antioxidant and antiradical properties of pomegranate (punica g...Pritam Kolge
Evaluation of antioxidant and antiradical properties
of Pomegranate (Punica granatum L.) seed and defatted
seed extracts
This is Journal Club activity Presentation with the reference of various research papers.
This Presentation Contain following...
#Info about Paper
#Abstract
#Materials
#Methods
#Results
#Discussion
#Conclusion
#References
*Important Methods used
#Moisture content
#Fat content
#Acid value
#Peroxide value
#Oxidative stability index
#Total phenols content
#Preparation of Pomegranate seed extracts and calculate extract yield
#Evaluation of antioxidant properties of Pomegranate seed extracts using
-DPPH radicals scavenging activity
-FRAP assay
#Antioxidant efficiency of seed extract (Oxidative stability extract)
#Statistical analysis
Journal Club Presentation at Bharati Vidyapeeth College of Pharmacy, Kolhapur.
Thanks for Help and Guidance of Dr. P. B. Choudhari (Assistant Professor, Pharmaceutical Chemistry) and Dr. A. J. Shinde (Assistant Professor, Department of Pharmaceutics)
This document discusses protein sequencing techniques. There are three main methods for determining a protein's amino acid sequence: N-terminal sequencing, C-terminal sequencing, and predicting the sequence from DNA sequencing. N-terminal sequencing involves using methods like Edman degradation to sequentially remove amino acids from the N-terminus and identify them. C-terminal sequencing uses carboxypeptidases to sequentially remove amino acids from the C-terminus over time. DNA sequencing allows predicting the protein sequence by first sequencing the gene that encodes the protein. Protein sequencing is important for understanding cellular processes and developing targeted drugs.
This document discusses various methods for determining the amino acid sequence of proteins, including:
- Edman degradation, which sequentially removes amino acids from the N-terminus. Up to 60 amino acids can typically be determined.
- Mass spectrometry techniques like MALDI that help determine the mass and sequence of protein fragments.
- Enzymatic cleavage techniques using enzymes like trypsin to break proteins into smaller fragments that can then be sequenced.
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.
Determination of protein structure by Dr. Anurag YadavDr Anurag Yadav
Proteins perform important roles in organisms and their structure and function can be studied by determining their amino acid sequence. There are several steps to determine the sequence, including isolating the protein, determining the number of peptide chains, identifying the N- and C-terminals, and Edman degradation which sequentially removes amino acids from the N-terminal for identification. Mass spectrometry can also be used to analyze protein structure. Higher levels of structure can be analyzed using techniques like X-ray crystallography and NMR spectroscopy.
Lectin-functionalized carboxymethylated kappa-carrageenan microparticles for ...ankit
This document describes a study on the development of lectin-functionalized carboxymethylated kappa-carrageenan microparticles for oral insulin delivery. The microparticles were prepared using an ionotropic gelation technique and characterized. Insulin was encapsulated within the microparticles and lectin was conjugated to the surface. In vitro studies showed the lectin functionalization enhanced insulin release and microparticle interactions with intestinal tissue. In vivo studies in diabetic rats demonstrated the lectin-functionalized microparticles provided prolonged glycemic control for 12-24 hours following oral administration.
The document discusses protein sequencing techniques. It begins with an introduction to proteins and protein sequencing. The history of protein sequencing is then outlined, including early work by Edman and Sanger. Methods for determining amino acid composition through hydrolysis, separation, and analysis are described. The mechanisms of Sanger's method using dinitrophenyl reagents and Edman degradation using phenylisothiocyanate are explained. Applications of protein sequencing include determining protein structure and function. The document concludes with locations of protein sequencing institutes in India and abroad.
Frederick Sanger developed two important methods for protein sequencing: 1) using fluorodinitrobenzene to determine the N-terminal amino acid, and 2) cleaving proteins into fragments and piecing their sequences together. The standard strategy involves separating chains, identifying terminal residues, cleaving the protein, sequencing fragments, and reconstructing the full sequence. The Edman degradation method allows sequential removal of residues from the N-terminus. Mass spectrometry techniques like peptide mass fingerprinting and tandem mass spectrometry now dominate protein sequencing.
The document discusses applications of liquid chromatography-mass spectrometry (LCMS). It describes how LCMS combines liquid chromatography separation with mass spectrometry detection. Several studies using LCMS are summarized, including analyzing phytochemicals in Solanum plants and identifying compounds in Archidendron bubalinum seed shell extracts. Key components detected include chlorogenic acids, flavonoids, alkaloids, and the compounds phlorizin and astilbin.
plain 4 SYNTHESIS, PURIFICATION AND STABILITY STUDY OF ISLETAndrew Apals
This document outlines Andrew Apals' thesis defense presentation on the synthesis, purification, and stability study of the islet neogenesis-associated protein peptide (INGAP-P) and analogs. The presentation covers solid phase peptide synthesis of INGAP-P, linear and cyclic analogs. It also discusses reverse-phase HPLC method development for the separation and quantification of peptides in synthetic mixtures, including degradation studies of INGAP-P standards. The goal of the research is to develop purification methods to resolve INGAP-P from crude synthesis mixtures using analytical HPLC instrumentation.
Mass spectrometry is an analytical technique that ionizes samples and measures the mass-to-charge ratio of ions to determine molecular masses. It requires charged gaseous molecules for analysis. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry uses a laser to ionize analyte molecules embedded in a matrix, which are then accelerated through a flight tube based on their mass-to-charge ratios. MALDI-TOF is commonly used to analyze biomolecules like proteins and is applied in fields like proteomics, metabolomics, environmental analysis, and forensics.
In-vitro evaluation techniques of anticancer, anti oxidant, anti microbial ZakiyaUsmani
This document discusses various in vitro methods used to evaluate potential anti-cancer and antioxidant compounds, as well as antimicrobial activity. It describes cytotoxicity assays such as MTT, SRB, clonogenic assays and dye exclusion tests that are used to study anti-cancer activity against cell lines. Methods to evaluate antioxidant activity in vitro include DPPH radical scavenging, hydrogen peroxide and superoxide radical scavenging assays. Diffusion and dilution methods are discussed for determining antimicrobial activity of compounds in vitro prior to animal studies.
Introduction
What is Protein Sequencing?
History
Determination of amino acid composition
Sequencing methods
N terminal sequencing
C terminal sequencing
Mass spectrometer
Application
Reference
Research articles enzyme optimization studies.Salman Khan
The document summarizes research on optimizing the production of various enzymes through manipulation of culture and fermentation conditions. Key points discussed include:
1. Bacillus sp. was used to produce the enzyme pectinase, and production was optimized by varying incubation time, temperature, pH, carbon sources, and nitrogen sources. Highest production occurred at 96 hours, 35°C, pH 6 using glucose as the carbon source and yeast extract as the nitrogen source.
2. Lipase production by Staphylococcus sp. was optimized by varying oils, nitrogen sources, temperature, pH, incubation period, and agitation speed. Olive oil was used as the substrate.
3. Alkaline protease
This document summarizes a research article that studied the transport of the antibiotic tetracycline (Tet) in Escherichia coli. The key findings are:
1. Tet is transported into E. coli cells by three transporters: TetA, TetB, and TetH.
2. TetA is a primary transporter that uses ATP hydrolysis to actively transport Tet into cells. TetB and TetH are secondary transporters that harness gradients to transport Tet.
3. Experiments showed TetA is the major transporter and is responsible for most Tet uptake. TetB and TetH play lesser but still significant roles in Tet transport.
4. Understanding the roles and interactions of these
This document provides an overview of various protein purification techniques, including:
- Chromatography methods like affinity chromatography, ion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography.
- Key steps in protein purification like cell lysis, centrifugation, assaying fractions for protein and activity, and monitoring purity with SDS-PAGE gels.
- Considerations for each chromatography method like matrix selection, binding capacities, and driving forces.
- Emerging techniques like capillary electrochromatography and multi-dimensional separations.
JBEI Research Highlights - January 2022SaraHarmon4
The study investigated resistance to the parasitic plant field dodder (Cuscuta campestris) in some Heinz tomato cultivars. It was discovered that these resistant cultivars respond to dodder attachment by locally lignifying their stem cortex, preventing the parasite from entering the host plant. Three factors - LIF1, SlMYB55, and CuRLR1 - were identified as regulating this lignification-based resistance mechanism. This work demonstrates how lignification can be used by plants as a defense against parasitic plants like dodder, and could help develop resistance for other crops impacted by parasitic plants.
The document summarizes the biochemical and biophysical characterization of AnAEst, a novel SGNH hydrolase. Key points:
1) AnAEst is characterized as an arylesterase that hydrolyzes small chain fatty acid aryl esters, with optimal activity at pH 7.5 and temperatures of 25-45°C.
2) Active site residues serine 17, arginine 54, and leucine 86 were selected for mutagenesis studies. Mutants showed altered substrate specificity and kinetic parameters compared to the wild-type.
3) Biophysical analysis found that AnAEst is most stable at pH 5.5 and temperatures of 25-45°C, with half
IRJET- Understanding the cDNA isolation and antimitogenic property in plant l...IRJET Journal
This document summarizes research on the isolation and characterization of cDNA from plant lectins and their anti-mitogenic properties. Key points include:
- Plant lectins are proteins found in many plants that bind carbohydrates and can agglutinate red blood cells. Their structure gives therapeutic and biotechnological potential.
- Researchers have isolated cDNA from various plants like Glechoma hederacea and Salvia stenophylla to study lectin genes and properties. Methods included RNA isolation, cDNA library construction, sequencing, and molecular modeling.
- Studies found that isolated lectins showed anti-mitogenic effects, inhibiting the growth and killing of some cancer cell lines, utilizing their carbohydrate-binding and toxic
This study investigated the polymerization of lactic acid as a model for prebiotic peptide formation via ester-amide exchange. Lactic acid was polymerized in a closed system at 85°C over various time points. HPLC and 1H-NMR were used to analyze the polymers and determine degree of polymerization (DP) and total lactic acid units. DP was found to increase with time while total units decreased, showing polymer regeneration. Methods showed consistent results within 10-15% error. Further studies will compare kinetics to a computer simulation to determine rate constants and model polymerization from various monomers.
MASS SPECTROMETRY IN THE FIELD OF FOOD INDUSTRYErin Davis
Mass spectrometry is an analytical technique that identifies chemicals in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions. It produces a mass spectrum plot showing ion abundance versus mass-to-charge ratio. Mass spectrometry is used to quantify known materials, identify unknown compounds, and elucidate molecular structure and properties. The technique involves converting the sample to gaseous ions, optionally with fragmentation, and then separating and detecting the ions.
The document describes methods to separate and sequence isomeric oligonucleotide adducts using ion-pair reversed-phase liquid chromatography electrospray ionization tandem mass spectrometry (IP-RP-LC-ESI-MS/MS). A model 17-mer oligonucleotide adducted with N-acetoxy-2-acetylaminoflourene was used. Various ion pairing reagents and mobile phase conditions were evaluated to optimize the separation and structural identification of the adducted oligonucleotide isomers by MS/MS. The methodology aims to develop an analytical platform for characterizing DNA adducts to enable cancer risk assessment.
Amino acid sequencing determines the order of amino acids in a protein. Frederick Sanger determined the first protein sequence in 1953 using N-terminal analysis methods like Edman degradation. Large proteins are sequenced by first breaking them into smaller fragments using enzymes or chemicals, then determining the sequence of individual fragments and combining sequences to deduce the full protein sequence. Modern techniques like mass spectrometry have made sequencing faster and applicable to modified proteins.
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Purification and Characterization of Manganese Peroxidase of the White Rot Fungus
1. Name: Sugandhi Hansini
Admission ID :LUC000000259093052021
MSc Biotechnology
Lincoln University College
Purification & Characterization of
Manganese Peroxidase of the White-
Rot Fungus Irpex lacteus
3. White-Rot Fungus - Irpex lacteus
Belong to
Basidiomycota
Usually whitish in color
and fibrous in texture.
Decolorization of textile
industry waste water
Secretion of extracellular
ligninolytic enzymes
(Laccase/ Lignin peroxidase/
Manganese peroxidase)
Colonize plants or
plant residues (e.g.,
wood)
Promising
bioremediation
agents
Most efficient lignin
degraders in nature
Introduction
4. Manganese Peroxidase (MnP)
Belongs to the family
of oxidoreductases
Glycosylated heme
containing enzyme
Produced by
Ligninolytic fungi
Degradation of Lignin,
Polycyclic aromatic
hydrocarbons, Polychlorinated
biphenyls
Catalyzes the oxidation of
Mn2+ to Mn3+ in the presence of
hydrogen peroxide (H2O2)
Decolorization of
Textile industry waste
water
5. Purpose of the Study
• To purify the Manganese Peroxidase (MnP) enzyme produced by White-Rot
Fungus, Irpex lacteus
• To determine the characterization of MnP enzyme
6. Methodology
• Culture of the Organism
• Enzyme Purification
• Enzyme Characterization
Molecular mass
Isoelectric point
Spectroscopic properties
MS characterization
• Enzyme Assay
Catalytic properties of the enzyme
Effect of pH and Temperature
7. Culture of the organism
Irpex lacteus
Strain- KR 35W
Maintained on
100ml MGPY Agar slant (solid)
(1% malt extract,1% glucose, 0.5% peptone,
0.5% yeast extract)
For 7 days at 4°C
Four Mycelial agar
discs (0.9cm) for use as
inocula
Fungus + MGPY liquid
inocula (homogenized)
10ml of
inocula
90ml MGPY
broth
10% v/v diluted
inocula culture
fluid
8. Enzyme Purification
Culture fluid
Filtration
Whatman No 1
filter paper
Culture
filtrate
Cold Acetone (-
10°C) Precipitated
proteins
Remove
acetone Precipitate dissolve in
20mM sodium phosphate
buffer at pH 8
Protein
sample
mixture
9. Enzyme Purification
Protein
sample
mixture
Anion Exchange
Chromatography
(HiPrep 16/10 Q XL
column)
Enzyme fractions
containing MnP
collected and
concentrated
Size Exclusion
Chromatography/ Gel
Filtration (HiPrep 26/60
Sephacryl S-200)
Homogeneity of
the enzyme
confirmed by SDS
-PAGE
Figure 1: Ion Exchange Chromatography Figure 2: Size Exclusion Chromatography
10. Enzyme Characterization
Molecular mass
Estimated with SDS-PAGE 12.5% polyacrylamide
gel –
An analytical technique to separate proteins
based on their molecular weight
Estimated with MALDI-TOF mass
spectrometry –
Mass spectrometry is an analytical technique
in which samples are ionized into charged
molecules and ratio of their mass-to-charge
(m/z) can be measured
Estimated by Gel filtration
chromatography on a Superose
12 HR 10/30 column mounted
with FPLC system
Figure 3: FPLC System Figure 4: MALDI-TOF mass spectrometry Figure 5: SDS-PAGE Technique
11. Enzyme Characterization
Isoelectric point
Determined using isoelectric focusing polyacrylamide gel
electrophoresis (IEF-PAGE)
A method of separating proteins according to their Isoelectric
points in a pH gradient.
Stain – Coomassie brilliant blue G-250
Figure 6: IEF-PAGE Technique
12. Enzyme Characterization
Spectroscopic properties and Mass Spectrometry (MS) characterization
• An absorption spectrum of the purified enzyme MnP, was observed with the addition of hydrogen
peroxide
• MS characterization was determined by using MALDI-MS and ESI Q-TOF MS/MS after the digestion
of the purified enzyme with trypsin.
• Resulted protein mixture was analyzed using MASCOT peptide Mass Fingerprint software.
13. Enzyme Assay
Laccase
Activity
Lignin
Peroxidase
Activity
Manganese
peroxidase
Activity
Determined via the
oxidation of ABST
Assayed using veratryl alcohol
as substrate
Assayed by measuring the oxidation of
Mn(II) to Mn(III) at 270nm.
Assay mixture contained : 1mM
MnSO4 in 50mM sodium malonate(pH
4.5), 0.2mM Hydrogen peroxide
Purified
Protein
One enzyme unit:
Amount of enzyme
required to oxidize
1µmol of substrate per
min, under the assay
conditions
14. Enzyme Assay Continue…
MnP Catalytic Properties- Textile Industry Waste Water Decolorization
Culture filtrate
20ml of Textile
industry waste
water at 28ºC
Decolorized water
Figure 7: Catalytic cycle of MnP
With the
presence of
Hydrogen
peroxide
15. Results
• Molecular mass
• Isoelectric point
• Spectroscopic properties
• MS characterization
• Catalytic properties of the enzyme
• Effect of pH and Temperature
16. Molecular mass
Gel filtration chromatography on a
Superose 12 HR 10/30 column
mounted with FPLC system
Estimated with MALDI-TOF
mass spectrometry –
Estimated with SDS-PAGE 12.5%
polyacrylamide gel
Apparent
molecular mass of
the purified
enzyme was
observed as 53
kDa
Figure 8: MALDI-TOF mass spectrum of the native MnP.
Molecular mass was observed as 38.3kDa
Figure 9: SDS-PAGE of the purified MnP.
Molecular mass was observed as 53.2kDa
standard protein-bovine serum albumin
17. Isoelectric point
Figure 10: Isoelectric focusing of the purified MnP.
Isoelectric point of the enzyme was observed as 3.7
18. Spectroscopic properties
Figure 11: Absorption spectra of the MnP
Absorption spectrum of the purified enzyme
showed the peaks at about 407, 500 and
640nm.
Addition of Hydrogen peroxide to the enzyme,
Resulted in the shift from 407 and 500 to 409
and 525nm with decreased absorbance
19. Mass Spectrometry (MS) characterization
Figure 12: MALDI-TOF MS spectrum of tryptic digest of MnP
Figure 13: Identification of tryptic peptides from I. lacteus MnP by
MASCOT PEPTIDE Mass Fingerprint software
Three peptides m/z 1039.6,1631.9 and 1854.0 were further
analyzed with ESI Q-TOF MS/MS
Amino acid sequences for each peptide were obtained
20. Catalytic properties of the enzyme
Figure 14: MnP and Laccase activity on textile industry waste water
20ml of Textile
industry waste
water at 28ºC
With the
presence of
Hydrogen
peroxide
Purified protein
solution
decolorization.
Decolorization rate was determined by measuring the
absorbance at 600nm
Decolorized water
Graph indicates that the MnP activity of the culture
filtrates was shown correlate with the decolorization
of textile industry waste water
21. Catalytic properties of the enzyme
Figure 16: Production of Mn(III) malonate from the oxidation of Mn (II) by the MnP
Reaction mixture contains 0.5mM MnSO4, 0.5mM sodium
malonate(pH 4.5), 0.2mM Hydrogen peroxide
(spectra recorded every 2 min)
Figure 15: Decolorization of various dyes by MnP
The azo dye, methyl orange was the most rapidly
decolorized
22. Effect of pH and Temperature
Figure 17: Effect of pH and Temperature on the MnP activity
Enzyme Activity was assayed at various pHs
and Temperature under the standard
conditions
Optimum pH for the enzyme activity was
estimated to be around 6.0
Optimum temperature for the enzyme activity
was about 35°C.
23. Discussion
Enzyme purification
Purification of MnP from the culture filtrate of Irpex lacteus
Molecular Mass
• Mass spectrometry showed 38.3kDa of molecular mass while gel filtration and SDS-PAGE showed
the molecular mass of 53kDa.
• It was suggested that, since the MnP is a glycoprotein, carbohydrate content of the enzyme affected
by its migration in gel filtration.
• These data indicate that the enzyme contains about 28% total carbohydrate and it is monomeric,
similar to other fungal MnP
• Enzyme had been purified 11.0 fold with a yield of 24.3%
24. Discussion
Isoelectric point ( Net charge =0)
• Depending on the point at which the sample is loaded,
protein starts to migrate.
• If a protein is at a pH range which is below its isoelectric
point, protein is (+) charged and move towards the cathode.
• If a protein is at a pH range which is higher than its
isoelectric point , protein is (-) charged and move towards
the anode
• Isoelectric point of the enzyme was 3.7. Usual
feature among fungal MnPs, which exhibit acidic isoelectric
point
25. Discussion
Spectroscopic properties
• After the addition of hydrogen peroxide to MnP, decreased
absorbance and red shift of wavelength was observed.
• This suggested that, with the presence of hydrogen
peroxide, MnP enzyme is oxidized and it is a heme protein
with iron protoporphyrin ix as the prosthetic group
Absorption spectra of the MnP
26. Discussion
Mass Spectrometry (MS) characterization
• Peptide1039.6 showed 78% similarity to AY217015
• Peptide1631.9 showed 67% similarity to AY677128
• Peptide 1854.0, no significant similarity found with any other
white rot fungal peroxidase
• This results suggested that amino acid sequences of
the tryptic peptides of purified MnP were shown to
have little similarity to those of other fungal MnPS
MALDI-TOF MS spectrum of tryptic digest of MnP Identification of tryptic peptides from I. lacteus MnP
27. Discussion
Catalytic properties of the enzyme
• LiP activity was negligible
• Laccase enzyme activity was not significantly related to
decolorization of waste water
• The MnP activity of the culture filtrate was shown to
correlate with the decolorization of textile industry waste
water
MnP and Laccase activity on textile industry waste water decolorization
28. • The azo dye, methyl orange was the most rapidly decolorized
Catalytic properties of the enzyme
Discussion
• Reaction mixture contains 0.5mM MnSO4, 0.5mM sodium
malonate(pH 4.5), 0.2mM Hydrogen peroxide
• This suggests that, In the presence of Hydrogen peroxide, enzyme
readily oxidized Mn(II) and form Mn(III) malonate (267nm)
• Absorbance increases with the time
Decolorization of various dyes by MnP Production of Mn(III) malonate from the oxidation of Mn (II) by the MnP (spectra
recorded in evry 2min)
30. References
Baborová, P., Möder, M., Baldrian, P., Cajthamlová, K., & Cajthaml, T. (2006). Purification of a new manganese
peroxidase of the white-rot fungus Irpex lacteus, and degradation of polycyclic aromatic hydrocarbons by the
enzyme. Research in Microbiology, 157(3), 248-253.
Chowdhary, P., Shukla, G., Raj, G., Ferreira, L. F. R., & Bharagava, R. N. (2019). Microbial manganese peroxidase: a
ligninolytic enzyme and its ample opportunities in research. SN Applied Sciences, 1(1), 1-12.
Shin, K. S., Kim, Y. H., & Lim, J. S. (2005). Purification and characterization of manganese peroxidase of the white-rot
fungus Irpex lacteus. Journal of microbiology, 43(6), 503-509.
Tripathi, A., & Dixit, S. (2016). Bioremediation of phenolic compounds by higher fungi—a review. Int J Adv Res, 4(7), 14-
35.
Wesenberg, D., Kyriakides, I., & Agathos, S. N. (2003). White-rot fungi and their enzymes for the treatment of industrial
dye effluents. Biotechnology advances, 22(1-2), 161-187.
Xu, H., Guo, M. Y., Gao, Y. H., Bai, X. H., & Zhou, X. W. (2017). Expression and characteristics of manganese peroxidase
from Ganoderma lucidum in Pichia pastoris and its application in the degradation of four dyes and phenol. BMC
biotechnology, 17(1), 1-12.