A Proteomic Approach to Identify Zein Proteins upon Eco-Friendly Ultrasound-Based Extraction

Zein is a type of prolamin storage protein that has a variety of biomedical and industrial applications. Due to the considerable genetic variability and polyploidity of the starting material, as well as the extraction methods used, the characterization of the protein composition of zein requires a combination of different analytical processes. Therefore, we combined modern analytical methods such as mass spectrometry (MS), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), atomic force microscopy (AFM), or Fourier transform infrared spectroscopy–attenuated total reflectance (FTIR-ATR) for a better characterization of the extracted zein. In this study, we present an enhanced eco-friendly extraction method, including grinding and sieving corn seeds, for prolamins proteins using an ultrasonic extraction methodology. The use of an ultrasonic homogenizer, 65% ethanol extraction buffer, and 710 µm maize granulation yielded the highest protein extraction from all experimental conditions we employed. An SDS PAGE analysis of the extracted zein protein mainly revealed two intense bands of approximatively 20 and 23 kDa, suggesting that the extracted zein was mostly α-zein monomer. Additionally, MS analysis revealed as a main component the α-zein PMS2 (Uniprot accession no. P24450) type protein in the maize flour extract. Moreover, AFM studies show that extracting zein with a 65% ethanol and a 710 µm granulation yields a homogeneous content that could allow these proteins to be employed in future medical applications. This research leads to a better understanding of zeins content critical for developing new applications of zein in food and pharmaceutical industries, such as biocompatible medical vehicles based on polyplexes complex nanoparticles of zein with antimicrobial or drug delivery properties.

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Study on cocoonase, sericin, and degumming of silk cocoon: computational and experimental

Journal of Genetic Engineering and Biotechnology ◽

10.1186/s43141-021-00125-2 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Preeti Anand ◽

Jay Prakash Pandey ◽

Dev Mani Pandey

Keyword(s):

San Francisco ◽

Tertiary Structure ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Evolutionary Genetics ◽

Imaging Analysis ◽

Silk Cocoon ◽

Natural Color ◽

Sds Page

Abstract Background Cocoonase is a proteolytic enzyme that helps in dissolving the silk cocoon shell and exit of silk moth. Chemicals like anhydrous Na2CO3, Marseille soap, soda, ethylene diamine and tartaric acid-based degumming of silk cocoon shell have been in practice. During this process, solubility of sericin protein increased resulting in the release of sericin from the fibroin protein of the silk. However, this process diminishes natural color and softness of the silk. Cocoonase enzyme digests the sericin protein of silk at the anterior portion of the cocoon without disturbing the silk fibroin. However, no thorough characterization of cocoonase and sericin protein as well as imaging analysis of chemical- and enzyme-treated silk sheets has been carried out so far. Therefore, present study aimed for detailed characterization of cocoonase and sericin proteins, phylogenetic analysis, secondary and tertiary structure prediction, and computational validation as well as their interaction with other proteins. Further, identification of tasar silkworm (Antheraea mylitta) pupa stage for cocoonase collection, its purification and effect on silk sheet degumming, scanning electron microscope (SEM)-based comparison of chemical- and enzyme-treated cocoon sheets, and its optical coherence tomography (OCT)-based imaging analysis have been investigated. Various computational tools like Molecular Evolutionary Genetics Analysis (MEGA) X and Figtree, Iterative Threading Assembly Refinement (I-TASSER), self-optimized predicted method with alignment (SOPMA), PROCHECK, University of California, San Francisco (UCSF) Chimera, and Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) were used for characterization of cocoonase and sericin proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein purification using Sephadex G 25-column, degumming of cocoon sheet using cocoonase enzyme and chemical Na2CO3, and SEM and OCT analysis of degummed cocoon sheet were performed. Results Predicted normalized B-factors of cocoonase and sericin with respect to α and β regions showed that these regions are structurally more stable in cocoonase while less stable in sericin. Conserved domain analysis revealed that B. mori cocoonase contains a trypsin-like serine protease with active site range 45 to 180 query sequences while substrate binding site from 175 to 200 query sequences. SDS-PAGE analysis of cocoonase indicated its molecular weight of 25–26 kDa. Na2CO3 treatment showed more degumming effect (i.e., cocoon sheet weight loss) as compared to degumming with cocoonase. However, cocoonase-treated silk cocoon sheet holds the natural color of tasar silk, smoothness, and luster compared with the cocoon sheet treated with Na2CO3. SEM-based analysis showed the noticeable variation on the surface of silk fiber treated with cocoonase and Na2CO3. OCT analysis also exemplified the variations in the cross-sectional view of the cocoonase and Na2CO3-treated silk sheets. Conclusions Present study enlightens on the detailed characteristics of cocoonase and sericin proteins, comparative degumming activity, and image analysis of cocoonase enzyme and Na2CO3 chemical-treated silk sheets. Obtained findings illustrated about use of cocoonase enzyme in the degumming of silk cocoon at larger scale that will be a boon to the silk industry.

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Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.252-256.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 252-256 ◽

Cited By ~ 19

Author(s):

Katsuichi Saito ◽

Kazuya Kondo ◽

Ichiro Kojima ◽

Atsushi Yokota ◽

Fusao Tomita

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Extracellular Enzyme ◽

Molecular Weights ◽

Sds Page ◽

Monomeric Structure ◽

Ph Range ◽

Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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Evaluation of effective protein extraction procedure to profile petiole of Moringa oleifera

Plant Science Today ◽

10.14719/pst.2020.7.2.730 ◽

2020 ◽

Vol 7 (2) ◽

pp. 214

Author(s):

Zetty Amirah Zulkifli ◽

Zaidah Rahmat

Keyword(s):

Moringa Oleifera ◽

Protein Extraction ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Extraction Procedure ◽

Electrophoretic Pattern ◽

Antioxidant Property ◽

Extraction Methods ◽

Protein Profiling ◽

Sds Page

Moringa oleifera is widely known as multipurpose tree since all of its parts confer multiple functions. The leaf is highly favourable among consumers while the petiole is mostly wasted. There are numerous studies on the flavonoid and antioxidant property of the stem and twig. However, study on the petiole has never been done. There-upon, this study was conducted to develop protein profiling of the petiole. In this study, 6 different protein extraction methods were tested on the fresh petiole before its protein quantity and quality were checked via Bradford assay and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) respectively. The in-solution digestion was then done prior to LC-MS/MS analysis. The protein electrophoretic pattern from the SDS-PAGE proves that method 6 using Tris HCl buffer with incorporation of dithiothreitol (DTT) and phenylmethylsulfonyl fluoride (PMSF) confers the best quality of protein. It produced the highest number of visible individual bands compared to other methods. Meanwhile, 93 proteins were successfully identified via LCMS analysis where the protein, signal response and carbohydrate metabolism categories confer the highest percentage. High quality and content of the protein extracted from the petiole including the antioxidant, anticancer and antidiabetic protein identified suggested that consuming this part of the plant could enhance nutrients of human body.

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Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

Journal of Bacteriology ◽

10.1128/jb.181.1.91-99.1999 ◽

1999 ◽

Vol 181 (1) ◽

pp. 91-99 ◽

Cited By ~ 111

Author(s):

Hisayo Ono ◽

Kazuhisa Sawada ◽

Nonpanga Khunajakr ◽

Tao Tao ◽

Mihoko Yamamoto ◽

...

Keyword(s):

Molecular Mass ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Apparent Molecular Mass ◽

Optimum Temperature ◽

Sds Page ◽

Halomonas Elongata ◽

Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

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Changes in total and individual proteins during drying and ruminal fermentation of alfalfa

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200011091 ◽

2005 ◽

Vol 2005 ◽

pp. 198-198

Author(s):

A. A. Sadeghi ◽

P. Shawrang ◽

M. Moradi ◽

A. Nikkhah

Keyword(s):

Crude Protein ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Composition ◽

Plant Proteins ◽

Ruminal Fermentation ◽

Protein Nitrogen ◽

Sds Page ◽

Total Crude Protein ◽

Hydrolysis Of

Proteolysis within plant cells occurs during wilting and drying. Changes in plant proteins during those periods usually are monitored by measurement of total crude protein and non protein nitrogen. Alternatively, changes in concentrations of individual proteins can be measured. Plants are composed of an array of different proteins. Electrophoresis can be used to separate these proteins and has been used to study effects of wilting and ensiling on proteins of some forages (Grum et al., 1991). Electrophoresis also has been used in the study of ruminal hydrolysis of oilseed meals proteins (Sadeghi et al., 2004). Most of the experiments designed to use electrophoresis to study protein metabolism in forages and ruminants have been qualitative. The main objective of this study was to determine whether sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry could be used to monitor quantitatively the changes in alfalfa protein composition during wilting, drying and ruminal exposure.

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Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

The Journal of Cell Biology ◽

10.1083/jcb.96.4.1030 ◽

1983 ◽

Vol 96 (4) ◽

pp. 1030-1039 ◽

Cited By ~ 6

Author(s):

W J Brown ◽

W A Shannon ◽

W J Snell

Keyword(s):

Membrane Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Protein Composition ◽

Granule Protein ◽

Isolation And Characterization ◽

Cytoplasmic Face ◽

Sds Page ◽

Granule Membrane ◽

Granule Membrane Proteins

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

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Serotypes of Neisseria meningitidis associated with an increased incidence of meningitis cases in the Hamilton area, Ontario, during 1978 and 1979

Canadian Journal of Microbiology ◽

10.1139/m83-020 ◽

1983 ◽

Vol 29 (1) ◽

pp. 129-136 ◽

Cited By ~ 8

Author(s):

Fraser E. Ashton ◽

J. Alan Ryan ◽

Colina Jones ◽

Bernard R. Brodeur ◽

Benito B. Diena

Keyword(s):

Neisseria Meningitidis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Age Groups ◽

Double Diffusion ◽

Sds Page ◽

Class 1 ◽

Group B

The distribution of serotypes among strains of Neisseria meningitidis responsible for a marked increase of meningitis cases in the Hamilton area, Ontario, in 1978 and 1979 was determined. Twenty-six serogroup B and two serogroup W135 strains isolated from cerebrospinal fluid, blood, and skin of 28 patients were serotyped by agar gel double diffusion. Twenty-one (81 %) of the group B strains were serotype 2b as judged by the formation of characteristic serotype precipitin bands with the specific anti-2996 (type 2b) serum. Fourteen of the serotype 2b strains also reacted with anti-77252 serum, which suggested that one strain or several closely related strains were mainly responsible for the increase in meningitis during the 2-year period. Examination of the outer membrane complexes (OMC) of the strains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS–PAGE) revealed that all 21 of the serotype 2b strains contained the class 2 protein (molecular weight 41 500) which is known to be the site of the serotype 2b determinant. Further characterization of the serotype 2b,77252 strains by enzyme-linked immunosorbent assays (ELISA) and SDS–PAGE suggested that the 77252 determinant was present in the class 1 proteins of these strains. The serotype 2b containing strains were isolated from 77.7 and 70% of males and females, respectively, from 81.8% of children less than 5 years of age, and from 75.0% of patients of all age groups. The study indicates the important role of serotype 2b meningococci in causing the increased incidence of meningitis and further substantiates the important association of the serotype 2b determinant with group B serotype 2 meningococcal disease in Canada.

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Acid and alkaline phosphatases of Capnocytophaga species. II. Isolation, purification, and characterization of the enzymes from Capnocytophaga ochracea

Canadian Journal of Microbiology ◽

10.1139/m83-211 ◽

1983 ◽

Vol 29 (10) ◽

pp. 1361-1368 ◽

Cited By ~ 9

Author(s):

Thomas P. Poirier ◽

Stanley C. Holt

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Alkaline Phosphatases ◽

Purification And Characterization ◽

Molecular Weights ◽

Sds Page ◽

Kinetics Of

Capnocytophaga ochracea acid (AcP; EC 3.1.3.2) and alkaline (AlP; EC 3.1.3.1) phosphatase was isolated by Ribi cell disruption and purified by sodium dodecyl sulphate – polyacrylamide gel electrophoresis (SDS–PAGE.) Both phosphatases eluted from Sephadex G-150 consistent with molecular weights (migration) of 140 000 and 110 000. SDS–PAGE demonstrated a 72 000 and 55 000 subunit molecular migration for AcP and AlP, respectively. The kinetics of activity of purified AcP and AIP on p-nitrophenol phosphate and phosphoseryl residues of the phosphoproteins are presented.

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Characterization of metallothionein protein from hepatopancreas organ of Pilsbryoconcha exilis collected from Cikaniki River, Western Java, Indonesia

Nusantara Bioscience ◽

10.13057/nusbiosci/n120101 ◽

2019 ◽

Vol 12 (1) ◽

Author(s):

SATA YOSHIDA SRIE RAHAYU ◽

WAHYU PRIHATINI

Keyword(s):

Stress Proteins ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

The Body ◽

Freshwater Environment ◽

Sds Page ◽

Freshwater Biota ◽

Aas Method ◽

Endogenous Proteins

Abstract. Rahayu SYS, Prihatini W. 2020. Characterization of metallothionein protein from hepatopancreas organ of Pilsbryoconcha exilis collected from Cikaniki River, Western Java, Indonesia. Nusantara Bioscience 12: 1-5. Freshwater environment, undergoing various changes due to the presence of dangerous toxic anthropogenic waste. It causes pressure on the freshwater biota that lives in it, such as Pilsbryoconcha exilis mussel at the bottom of freshwater. This pressure is controlled by the body through the synthesis of a set of stress proteins. Endogenous proteins, metallothionein (MT), in the body of freshwater biota absorb heavy metals in the body of biota, in the form of stress control. This research identified MT protein on P. exilis from contaminated waters such as the Cikaniki river with the average of mercury levels in water, sediment, and hepatopancreas of mussels using AAS method were 0.001 mg/L, 0.120 mg/L, and 1.318 mg/L respectively. Hepatopancreas of P. exilis was extracted using a Tissue Extraction Reagent I kit (Invitrogen), with procedures following the factory manual. The extract was purified by filtration using Sephadec 50; then, the filtration results were migrated together with the PageRuler TM Unstained Low Range Protein Ladder (Fermentas) in Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS PAGE) gel medium on Biorad Protein II electrophoresis. After completion of electrophoresis, the gel was stained using Page Blue Protein Staining Solution (Fermentas), following the factory manual procedure. Characterization at this research has succeeded in obtaining the MT-I isoform protein measuring 5, 10, and 25 kDa from the hepatopancreas organ of P. exilis.

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MOLECULAR WEIGHT PROFILE OF PROTEASE OF PINEAPPLE (Ananas comosus L.Merr) AND PAPAYA (Carica papaya L.) USING SDS-PAGE METHOD

ALCHEMY Jurnal Penelitian Kimia ◽

10.20961/alchemy.v13i1.2540 ◽

2017 ◽

Vol 13 (1) ◽

pp. 52

Author(s):

Rizky Arcinthya Rachmania ◽

Priyo Wahyudi ◽

Aniza Mutia Wardani ◽

Dini Rohmatul Insani

Keyword(s):

Molecular Structure ◽

Molecular Weight ◽

Gel Electrophoresis ◽

Carica Papaya ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Ananas Comosus ◽

Sds Page ◽

Papaya Latex ◽

Pharmaceutical Industries

A group of protease enzymes such as papain and bromelain is able to decipher the molecular structure of the protein into amino acids which will be very useful in many fields, especially in food and pharmaceutical industries. The objective of this study to determine the molecular weight profile of enzyme bromelain from pineapple bark (Ananas comosus L. Merr) and papain (Carica papaya L.) from papaya latex with different varieties using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The precipitation was performed with ammonium sulfate ((NH4)2SO4)) 60 % addition and dialysized using a cellophane tubing with a pore size of 12,000 Dalton. The molecular weight of enzyme solution were determined using SDS-PAGE. The analysis results of molecular weight of enzyme bromelain of Subang and Bogor varieties were not different and were about 30.654 kDa, as well as the molecular weight of enzyme papain and Sukma California varieties were also not different and were about 23.485 kDa. It can be concluded that the different varieties of fruit of pineapple and papaya had no effect on the molecular weight of enzyme papain and bromelain.

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