scholarly journals Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain

Marine Drugs ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. 197
Author(s):  
Diana R. Baidamshina ◽  
Victoria A. Koroleva ◽  
Svetlana S. Olshannikova ◽  
Elena Yu. Trizna ◽  
Mikhail I. Bogachev ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Treatment Effectiveness ◽  
Specific Activity ◽  
Life Time ◽  
Total Activity ◽  
Biochemical Properties ◽  
Antimicrobial Treatment ◽  
Papaya Latex ◽  
High Cytotoxicity ◽  
Dressing Materials

Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan−1) and specific activity (U mg protein−1), leading to the preservation of more than 90% of the initial total activity (U mL−1). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6–7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.

scholarly journals PEMURNIAN PARSIAL DAN KRISTALISASI PAPAIN DARI GETAH Carica papaya

2019 ◽  
Vol 4 (2) ◽  
pp. 152
Author(s):  
Dwi Putri Mashfufatur Rohmah ◽  
Sofijan Hadi ◽  
Afaf Baktir
Keyword(s):  
Ammonium Sulphate ◽  
Carica Papaya ◽  
Specific Activity ◽  
Total Activity ◽  
Partial Purification ◽  
Purification Factor ◽  
Papaya Latex ◽  
Carica Papaya Latex

AbstractThis research has done partial purification by fractionation and optimization crystallization of papain from Carica papaya latex with the addition of ammonium sulphate. The enhancement of purification factor was determined by its specific activity. The fractionation results show that papain fraction of Carica papaya can be obtained by adding 40-80% saturated ammonium sulphate, with the highest specific activity, i.e. 2,0819 U/µg and the factor purification increase of 6,27 fold than papain extract. Meanwhile, the highest total activity, i.e. 10,7780 U of papain crystals can be obtained by presipitant agent addition of ammonium sulphate in the level / concentration 80% saturated at 15 °C. Microscopycally papain crystals profile in this condition have cube and tetragonal shape.Key words: crystallization, fractionation, ammonium sulphate, papain

Optimization of a Method for Ficin Immobilization Using Glutaraldehyde

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).

Isolation and Partial Characterization of Adenylate Cyclase-Enriched Membranes from Human Platelets

1986 ◽  
Vol 55 (02) ◽  
pp. 178-183
Author(s):  
N Shimada ◽  
M Tsubokura ◽  
N Kimura
Keyword(s):  
Molecular Weight ◽  
Adenylate Cyclase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
High Specific Activity ◽  
Total Activity ◽  
Human Platelets ◽  
Protein Profiles

SummaryIsolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/ 34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes.Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.

scholarly journals Biochemical Properties of Rhodanese from Almond (Prunus amygdalus) Nuts

2018 ◽  
Vol 1 (1) ◽  
pp. 17-24
Author(s):  
Adeola F. Ehigie ◽  
Mohammed A. Abdulrasak ◽  
Ona L. Ehigie
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Biochemical Properties ◽  
Prunus Amygdalus ◽  
Cyanide Detoxification ◽  
Km Value

Study of the characteristic pattern of enzymes are useful in the understanding of certain physiological and biochemical process-es. Thiosulfate: cyanide sulfurtransferase (rhodanese) is a ubiquitous multifunctional enzyme, that is believed to function in cyanide detoxification. The present study was conducted to determine the activity of rhodanese in almonds (Prunus amygdalus) that belong to the rose family, rosaceae. Rhodanese from the almond nuts was purified by ammonium sulphate precipitation, ion exchange and affinity chromatography. The molecular weight of the enzyme was determined by sodium dodecyl sulphate poly-acrylamide gel electrophoresis. The purified rhodanese from the almond nuts had a specific activity of 5.09 RU/mg with yield of 0.06%. A Km value of 11.14 mM with Vmax 0.46 RU/ml/min were obtained from KCN while a Km value of 13.95 mM with Vmax of 0.48 RU/ml/min was obtained from Na2S2O3. The substrate specificity studied indicated that Mercapto-ethanol (MCPE), Ammonium per sulfate ((NH2)2S2O8, Ammonium sulfate ((NH2)2SO4, Sodium sulfate (Na2SO4) and Sodium metabi-sulfate (Na2S2O5) cannot be substituted for sodium thiosulphate (Na2S2O3) as sulphur donor for rhodanese catalytic reaction. The optimum activity of the enzyme was observed at 50oC and an optimum pH of 8. The effect of metals on rhodanese from Almond nut showed that at 1 mM concentration of the metals used did not pronouncedly affect the activity of the enzyme metals except that of HgCl2 and MnCl2. However, the divalent metals including MnCl2 HgCl2, CaCl2, and BaCl2 inhibited the enzyme at 10 mM concentration. The molecular weight obtained from sodium dodecyl sulphate polyacrylamide gel electrophoresis was estimated to be 35 kDa. The study validates the expression of rhodanese activity in almond nut. The characteristic property of rhodanese in the plant may be exploited for bioremediation of cyanide polluted soil.

Purification and Biochemical Properties of Acid Phosphatase Enzyme from Seedlings of Rumex dentatus (Curly dock)

2019 ◽  
Vol 41 (6) ◽  
pp. 1065-1065
Author(s):  
Umber Zaman Umber Zaman ◽  
Rubina Naz Rubina Naz ◽  
Asma Saeed Asma Saeed ◽  
Jaweria Gul Jaweria Gul ◽  
Muhammad Zeeshan Bhatti and Ahmad Saeed Muhammad Zeeshan Bhatti and Ahmad Saeed
Keyword(s):  
Molecular Weight ◽  
Acid Phosphatase ◽  
Specific Activity ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Biochemical Properties ◽  
Wheat Crop ◽  
Experimental Conditions

The study was designed to purify enzyme from weed plant in association with wheat crop to assess and compare the purification and biochemical properties with acid phosphatases of seedlings of various plants. Acid phosphatase from seedlings of Rumex dentatus (Curly dock) was purified by different chromatography and chromatofocusing techniques with specific activity of 63U/mg of protein. The yield was about 3%. Single band was detected on SDS-polyacrylamide gel electrophoresis confirming the enzyme was homogeneous. Apparent molecular weight of 48 kDa was obtained. Gel filtration experiment indicated that native enzyme had a approximately molecular weight of 96 kDa, suggesting that this enzyme is homodimer. The enzyme showed Km value of 0.5 mM and Vmax of 60 and#181;M of p-nitrophenyl phosphate hydrolysed/min/mg of protein under experimental conditions. The optimum pH of 5.0 and temperature of 50and#176;C were obtained. Divalent cations such as copper and zinc ions caused acid phosphatase inhibition but the presence of 20 mM EDTA in the enzyme-metal ions incubation mixtures reversed the enzyme inhibition to some extent. The activity of 54 % and 63% were recovered back, respectively. Ca2+ and Mg2+ had very small activating effect on activity in the absence or presence of EDTA. The reaction of enzyme with iodoacetic acid or N-ethyl-maleimide had no inhibitory effect, pointing to a non-involvement of cysteine residues in enzyme action. Further, β-mercaptoethanol or dithiothreitol at low concentrations had very little activating effect revealing that SH-group containing amino acid in the enzyme may not be significant for its catalytic activity. The pH dependent variation of Km study showed that histidine may constitute a part of the active site. Acid phosphatase was competitively inhibited by phosphate and vanadate. Fluoride and Zn2+ acted as non-competitive inhibitors while molybdate showed mixed type of inhibition.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
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.

Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

1983 ◽  
Vol 49 (02) ◽  
pp. 109-115 ◽  
Author(s):  
M Hoylaerts ◽  
E Holmer ◽  
M de Mol ◽  
D Collen
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Life Time ◽  
Low Molecular Weight ◽  
High Affinity ◽  
Plasma Half Life ◽  
Covalent Complex

SummaryTwo high affinity heparin fragments (A/r 4,300 and M, 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401-3408) with an apparent 1:1 stoichiometry and a 30-35% yield.The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin.The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t'/j) of both low Afr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 ≃ 7.7 hr), approaching that of free antithrombin III (t1/2 ≃ 11 ± 0.4 hr) and resulting in a 30fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 ≃ 0.25 ± 0.04 hr).

Simultaneous optimization of activity and stability of xylose reductase from D. nepalensis NCYC 3413 using statistical experimental design

2020 ◽  
Vol 27 ◽  
Author(s):  
Shwethashree Malla ◽  
Sathyanarayana N. Gummadi
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Reductase Activity ◽  
Xylose Reductase ◽  
Life Time ◽  
Economic Feasibility ◽  
Simultaneous Optimization ◽  
Physical Parameters ◽  
Statistical Experimental Design

Background: Physical parameters like pH and temperature play a major role in the design of an industrial enzymatic process. Enzyme stability and activity are greatly influenced by these parameters; hence optimization and control of these parameters becomes a key point in determining the economic feasibility of the process. Objective: This study was taken up with the objective to optimize physical parameters for maximum stability and activity of xylose reductase from D. nepalensis NCYC 3413 through separate and simultaneous optimization studies and comparison thereof. Method: Effects of pH and temperature on the activity and stability of xylose reductase from Debaryomyces nepalensis NCYC 3413 were investigated by enzyme assays and independent variables were optimised using surface response methodology. Enzyme activity and stability were optimised separately and concurrently to decipher the appropriate conditions. Results: Optimized conditions of pH and temperature for xylose reductase activity were determined to be 7.1 and 27 ℃ respectively, with predicted responses of specific activity (72.3 U/mg) and half-life time (566 min). The experimental values (specific activity 50.2 U/mg, half-life time 818 min) were on par with predicted values indicating the significance of the model. Conclusion: Simultaneous optimization of xylose reductase activity and stability using statistical methods is effective as compared to optimisation of the parameters separately.

scholarly journals Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Peixian Bai ◽  
Liyuan Wang ◽  
Kang Wei ◽  
Li Ruan ◽  
Liyun Wu ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Camellia Sinensis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Sequences ◽  
Biochemical Properties ◽  
High Similarity ◽  
New Gene ◽  
Tea Plants

Abstract Background Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. Results The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0–8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5′-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). Conclusions Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

1992 ◽  
Vol 12 (1) ◽  
pp. 15-21
Author(s):  
S. Kojima ◽  
K. Nara ◽  
Y. Inada ◽  
S. Hirose ◽  
Y. Saito
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
Specific Activity ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Lipid Vesicles ◽  
Specific Factor ◽  
Cell Lysate ◽  
Aggregation Activity ◽  
Molecular Weight Fractions

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [3H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [3H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.

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