Purification, Characterization and N-terminal Protein Sequencing of the Enzyme Dextransucrase Produced by Leuconostoc mesenteroides

Background: The wide use of dextran in many different applications, makes its industrial production a challenge and, hence, to obtain a control branched structure of this enzyme research is in progress. Objectives: In the present paper, the enzyme dextransucrase, produced by cultivation of the bacterium Leuconostoc mesenteroides CMG713, was purified and characterized. Methods: The produced dextransucrase was partially purified by PEG400 obtaining a purification factor of 29.4-fold and an overall yield of 18.3% from the initial crude enzymatic extract. Results: The partially purified dextransucrase had a specific activity of 24.0 U/mg and presented a molecular weight of about 200 kDa. In addition, the produced dextransucrase was stable at 30ºC and pH 5.5 for 3 days and led to a highly soluble dextran with wide potential industrial applications. The current study has successfully partial purification, characterization and conformation of dextransucrase produced by fermentation of the bacterium Leuconostoc mesenteroides CMG713.

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Amylase from bacillus thuringiensis isolated from tapioca waste: isolation, partial purification and characterization

Malaysian Journal of Fundamental and Applied Sciences ◽

10.11113/mjfas.v12n1.404 ◽

2016 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Zusfahair Zusfahair ◽

Dian Riana Ningsih ◽

Dwi Kartika ◽

Amin Fatoni

Keyword(s):

Molecular Weight ◽

Bacillus Thuringiensis ◽

Specific Activity ◽

Production Condition ◽

Liquid Waste ◽

Industrial Applications ◽

Partial Purification ◽

Production Time ◽

Optimal Production ◽

Sds Page

Microorganism enzymes are the most widely used in industrial applications. Tapioca liquid waste could be a great source of amylase-producing bacteria. The aim of this research was to isolate the amylase-producing bacteria form the tapioca waste, to produce amylase and to purify the resulted amylase. The screening, identification, and the optimal production condition of the amylase‒producing bacteria were studied. The optimization of bacteria production growth phase and the amylase production time were investigated. The crude amylase was purified using ammonium sulfate fractionation followed by SDS PAGE electrophoresis to identify the molecular weight and to purity of the amylase. The amylase activity assay used was based on the measuring of resulted reducing sugar by Somogyi-Nelson method. The result showed that the amylase producing bacteria was identified as Bacillus thuringiensis. The exponential phase of the bacteria growth for bacteria adaptation before production was 18 h and the optimal production time of amylase enzyme was 24 h. The highest specific activity of the purified amylase was fraction (FHD) 40% with specific activity of 37.56 ± 0.38U/mg. The SDS PAGE of FHD 40% profile showed two clear bands with molecular weight of 32 kDa and 35 kDa respectively

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Immunoaffinity Chromatography of Canine High-Molecular-Weight Renin: Partial Purification and Characterization

Clinical Science ◽

10.1042/cs0650117 ◽

1983 ◽

Vol 65 (2) ◽

pp. 117-120 ◽

Cited By ~ 2

Author(s):

Fumihiko Ikemoto ◽

Victor J. Dzau ◽

Edgar Haber ◽

Kazuo Takaori ◽

Kenjiro Yamamoto

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Specific Activity ◽

Immunoaffinity Chromatography ◽

Partial Purification ◽

Low Molecular Weight ◽

Specific Method ◽

Disulphide Bonds ◽

Binding Substance ◽

Molecular Weight Form

1. Canine high-molecular-weight renin (mol. wt. 60 000) is believed to be a complex of renin (low-molecular-weight form, mol. wt. 40 000) and renin-binding substance. The immunocross-reactivity of high-molecular-weight renin and low-molecular-weight renin was demonstrated by using antibodies specific to low-molecular-weight renin. 2. Immunoaffinity chromatography with renin-specific antibodies coupled to Sepharose provided a simple and specific method for isolation of high-molecular-weight renin. High-molecular-weight renin with a specific activity of 137 600 ng of ANG I h−1 mg−1 of protein (19.6 Goldblatt units/mg of protein) was obtained. 3. This high-molecular-weight renin was stable in dithiothreitol (25 mmol/l), suggesting that disulphide bonds may not be involved in the binding mechanism between low-molecular-weight renin and renin-binding substance. 4. However, exposure to low pH (3.0) resulted in conversion of high-molecular-weight renin into the low-molecular-weight form.

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Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

Tikrit Journal of Pure Science ◽

10.25130/j.v23i10.756 ◽

2019 ◽

Vol 23 (10) ◽

pp. 46

Author(s):

Saif M. Hasan ◽

Firas T. Maher ◽

Nagham Q. Kadhim

Keyword(s):

Molecular Weight ◽

Specific Activity ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Maximum Velocity ◽

Partial Purification ◽

Diabetic Patients ◽

Kinetic Properties ◽

Topoisomerase Ib ◽

Electrophoresis Method

This study was done to partially purification of topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD). http://dx.doi.org/10.25130/tjps.23.2018.168

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Production, purification and characterization of a thermostable β-1,3-glucanase (laminarinase) produced by Moniliophthora perniciosa

Anais da Academia Brasileira de Ciências ◽

10.1590/s0001-37652011005000007 ◽

2011 ◽

Vol 83 (2) ◽

pp. 599-609 ◽

Cited By ~ 7

Author(s):

Amanda R. Sena ◽

Gildomar L.V. Júnior ◽

Aristóteles Góes Neto ◽

Alex G. Taranto ◽

Carlos P. Pirovani ◽

...

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Industrial Applications ◽

Enzymatic Extract ◽

Multivariate Statistical ◽

Moniliophthora Perniciosa ◽

Fermentation Time ◽

Optimum Ph ◽

Multivariate Statistical Approach

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology - RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L-1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40°C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90°C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.

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Study of some hormones and Partial purification of prolidase from serum of women with polycystic ovary syndrome

Tikrit Journal of Pure Science ◽

10.25130/j.v24i7.913 ◽

2019 ◽

Vol 24 (7) ◽

pp. 59

Author(s):

Reemy M. Mohamed Saleh ◽

Firas T . Maher ◽

Nagham Q. Kadhim

Keyword(s):

Molecular Weight ◽

Polycystic Ovary Syndrome ◽

Specific Activity ◽

Polycystic Ovary ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Partial Purification ◽

Ovary Syndrome ◽

Electrophoresis Method ◽

High Level

This study was done by partially purification of prolidase from serum of patients with polycystic ovary syndrome by Gel filtration technique, and using sephadex G100 gel as a stationary phase. The degree of purification (15.1) fold, enzyme yield (95.5%) and specific activity (0.00176 IU/I), were carried out .Kinetics studies for the partial purified enzyme technique showed optimal concentration of substrate which was 5 mmol/l Km = 0.66ng and Vmax =0.80 mM, while optimum Temperature was (35C°) and optimum pH was (8). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method , in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS_PAGE) which showed that the approximates molecular weight was (54KD),we found high level of prolactin in the patient with polycystic ovary syndrome which was(24.03) when in the control was( 10.09),the value of TSH in the patient was( 17.08) which is high value and in the control was( 1.49), the value of T4 in the patient was (100.2) and in control was (118.4),the value of T3 in the patient was (0.3)and in control was (1.3).Testosterone in patient was (0.391) and in control was (0.206). http://dx.doi.org/10.25130/tjps.24.2019.130

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Optimization of a Method for Ficin Immobilization Using Glutaraldehyde

Biotekhnologiya ◽

10.21519/0234-2758-2020-36-5-81-88 ◽

2020 ◽

Vol 36 (5) ◽

pp. 81-88

Author(s):

M.G. Holyavka ◽

V.G. Artyukhov

Keyword(s):

Molecular Weight ◽

Protein Content ◽

Russian Federation ◽

Specific Activity ◽

Total Activity ◽

Industrial Applications ◽

Covalent Immobilization ◽

Protein Film ◽

The Russian Federation ◽

Enzyme Molecules

Ficin was covalently immobilized on an acid-soluble matrix of medium (200 kDa) and high molecular weight (350 kDa) chitosans. The enzyme molecules were immobilized in a protein film and copolymerized with glutaraldehyde. The optimal ratio of protein content, total activity and specific activity was observed as a result of ficin covalent immobilization on a matrix of medium-molecular chitosan with 15% glutaraldehyde and high-molecular chitosan with 10% glutaraldehyde. The obtained biocatalysts are promising for industrial applications. ficin, chitosan, glutaraldehyde, covalent immobilization. This work was financially supported by a grant from the President of the Russian Federation for state support to young Russian scientists-doctors of sciences MD-1982.2020.4. Agreement 075-15-2020-325).

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Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from the Gut of Chanos chanos

BioMed Research International ◽

10.1155/2021/7190152 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Muhammad A. Kurnianto ◽

Harsi D. Kusumaningrum ◽

Hanifah N. Lioe ◽

Ekowati Chasanah

Keyword(s):

Molecular Weight ◽

Antibacterial Activity ◽

Antimicrobial Agents ◽

Specific Activity ◽

Proteolytic Enzymes ◽

Proteinase K ◽

Partial Purification ◽

Chanos Chanos ◽

Extracellular Metabolites ◽

Wide Range

Bacteriocin-like inhibitory substances (BLIS) have sparked great interest because of their promising use in food as natural antimicrobial agents. In this work, six Streptomyces isolates obtained from the gut of Chanos chanos demonstrated their ability to produce extracellular metabolites with inhibitory activity against Salmonella enterica serovar Typhimurium, Escherichia coli, Listeria monocytogenes, and Staphylococcus aureus. Exposure of the extracellular metabolites to proteolytic enzymes (i.e., proteinase-K, trypsin, and pepsin) revealed high sensitivity and confirmed their proteinaceous nature. The metabolites were stable at high temperatures (up to 100°C for 30 min) and a wide range of pH (pH 2.0–7.0). Fractionation of the crude BLIS by filtration yielded three fractions based on molecular weight: <3 kDa, 3–10 kDa, and >10 kDa. Analysis of the antibacterial activity of these fractions showed increased specific activity, especially in the fraction with a molecular weight (MW) of <3 kDa, relative to the crude sample. The fraction with MW < 3 kDa had minimum inhibitory and bactericidal concentrations in ranges 0.04–0.62 mg·mL−1 and 0.08–1.25 mg·mL−1, respectively. This fraction also showed better temperature and pH stability compared with crude BLIS. Brine shrimp toxicity assay revealed that this fraction has moderate toxicity with a 50% lethal concentration of 226.975 μg·mL−1 (i.e., moderate toxicity) to Artemia salina. Identification of the peptide sequences of this fraction by liquid chromatography–tandem mass spectrometry yielded 130 proteins with retention times of 15.21–19.57 min. Eleven proteins with MWs of 1345.66–2908.35 Da and composed of less than 30 amino acid residues with high hydrophobicity (15.34–26.22 kcal·mol−1) appeared to be responsible for the antibacterial activity of the fraction. This study revealed the potential application of BLIS from Streptomyces, especially BLIS SCA-8, as antibacterial agents.

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Secretion of Plasminogen Activators by Cultured Bovine Endothelial Cells: Partial Purification, Characterization and Evidence for Multiple Forms

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650174 ◽

1981 ◽

Vol 45 (03) ◽

pp. 219-224 ◽

Cited By ~ 19

Author(s):

W E Laug

Keyword(s):

Molecular Weight ◽

Endothelial Cells ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Plasminogen Activators ◽

Maximum Activity ◽

Partial Purification ◽

Diisopropyl Fluorophosphate

SummaryEndothelial cells were obtained from the aortae of newborn calves and cloned. High plasminogen activator (PA) activity was detected in the supernatant medium and the cell lysates of confluent cultures. The PA activity in the growth medium increased steadily during 12 hrs of incubation, indicating active enzyme secretion by these cells. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the concentrated medium demonstrated the presence of four plasminogen activators with apparent molecular weights of 77,000 (±3000), 43,000 (±2000), 26,000 (±1500) and 14,500 (±1500) respectively. The 43,000, 26,000 and 14,500 molecular weight forms could be converted to radioactive derivates by active site labeling with 3H diisopropyl fluorophosphate (3H DFP) while the 77,000 Dalton form took up only traces of this radioactively labeled compound. The 43,000 molecular weight form was partially purified by means of salt precipitation and gel filtration. This enzyme preparation activated plasminogen by proteolytic cleavage with maximum activity at pH 7.5-8.5 and demonstrated a specific activity of 80,000 CTA (Committee on Thrombolytic Agents) units/mg protein when tested on 125I-fibrin in the presence of plasminogen. This PA was rapidly and irreversibly inhibited by diisopropyl fluorophosphate (DFP), suggesting that it was a serine protease. The partially purified enzyme was extremely labile at temperatures from 0-60° C, but could be stabilized by lowering the pH to 3 or by the addition of albumin.

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Preparation and Properties of Human Prothrombin Complex

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654240 ◽

1970 ◽

Vol 24 (03/04) ◽

pp. 325-333 ◽

Cited By ~ 4

Author(s):

G. H Tishkoff ◽

L. C Williams ◽

D. M Brown

Keyword(s):

Molecular Weight ◽

Proteolytic Enzyme ◽

Enzyme Inhibitor ◽

Specific Activity ◽

Crystalline Form ◽

Sedimentation Equilibrium ◽

Crystalline Complex ◽

Interaction Product ◽

Proteolytic Enzyme Inhibitor ◽

Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

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Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

Scientific Research Journal ◽

10.24191/srj.v10i2.9403 ◽

2013 ◽

Vol 10 (2) ◽

pp. 29

Author(s):

Normah Ismail ◽

Nur' Ain Mohamad Kharoe

Keyword(s):

Molecular Weight ◽

Protein Content ◽

Ammonium Sulfate ◽

Molecular Weight Distribution ◽

Weight Distribution ◽

Protease Activity ◽

Protein Concentration ◽

Temperature Stability ◽

Partial Purification ◽

Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage.

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