scholarly journals Extraction, Purification and Characterization of Transglutaminase From Some Plants

2021 ◽  
Vol 910 (1) ◽  
pp. 012061
Author(s):  
Hala Abdel Wahed Alhasani ◽  
Zena Kadhim Al-Younis
Keyword(s):  
Enzyme Activity ◽  
Sodium Chloride ◽  
Sodium Phosphate ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Phosphate Solution ◽  
Distilled Water ◽  
Optimum Temperature ◽  
Practical Applications ◽  
Hcl Solution

Abstract The present study aimed to Isolate trans-glutaminase EC:2.3.2.13 from some plants sources and Purified it and Studied it’s Charctarestics as well as it’s practical applications in the production of sausage.The enzyme was extracted from four types of plants (rosemary, chard, radish, arugula) using nine extraction solutions that included distilled water,Sodium chloride 3% solution, sodium chloride 5% solution, sodium phosphate solution 0.1 M and an pH 6.5, sodium phosphate 0.1 M and an pH of 7.5, Tris - Hcl solution 0.2 M and pH 7, Tris - Hcl solution 0.2 M and an pH 8, Tris - Hcl solution 0.1 M and an pH 7 and Tris - Hcl solution 0.1 M and an pH 8 in order to find out the best source of enzyme and the best extraction solution. chard was the best source of enzyme compared with other sources, Tirs -HCl 0.1M, pH 8 solution was the best extraction solution which gave the highest specific activity 8.104 unit/mg.Protein content for the crude enzyme extracts were concentrated using saturated ammonium sulfate in arrange 20-60%, Dialysis was done using distilled water. Then, the purification steps of the enzyme were completed using the gel filtration in the Sephadex G-100 Purification Folds 13.91 time and the yield was 20.04% %. Electrophoresise process using poly acryl amid gel in the absence of SDS observe the presence of one protein band which indicates the complete purification of transglutaminase. transglutaminase molecular weight was 42,660 Dalton when it was evaluated using poly acryl amide electrophoresis in the presence of SDS. the optimum pH for enzyme activity and enzyme stability was 7, while the optimum temperature for enzyme activity was 55°C, and the optimum temperature for enzyme stability was between 25-45°C.

scholarly journals Enzyme Stability in Nanoparticle Preparations Part 1: Bovine Serum Albumin Improves Enzyme Function

Molecules ◽  
2020 ◽  
Vol 25 (20) ◽  
pp. 4593
Author(s):  
Jason Thomas Duskey ◽  
Federica da Ros ◽  
Ilaria Ottonelli ◽  
Barbara Zambelli ◽  
Maria Angela Vandelli ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Polymeric Nanoparticles ◽  
Release Kinetics ◽  
Titration Calorimetry ◽  
Therapeutic Effects

Enzymes have gained attention for their role in numerous disease states, calling for research for their efficient delivery. Loading enzymes into polymeric nanoparticles to improve biodistribution, stability, and targeting in vivo has led the field with promising results, but these enzymes still suffer from a degradation effect during the formulation process that leads to lower kinetics and specific activity leading to a loss of therapeutic potential. Stabilizers, such as bovine serum albumin (BSA), can be beneficial, but the knowledge and understanding of their interaction with enzymes are not fully elucidated. To this end, the interaction of BSA with a model enzyme B-Glu, part of the hydrolase class and linked to Gaucher disease, was analyzed. To quantify the natural interaction of beta-glucosidase (B-Glu,) and BSA in solution, isothermal titration calorimetry (ITC) analysis was performed. Afterwards, polymeric nanoparticles encapsulating these complexes were fully characterized, and the encapsulation efficiency, activity of the encapsulated enzyme, and release kinetics of the enzyme were compared. ITC results showed that a natural binding of 1:1 was seen between B-Glu and BSA. Complex concentrations did not affect nanoparticle characteristics which maintained a size between 250 and 350 nm, but increased loading capacity (from 6% to 30%), enzyme activity, and extended-release kinetics (from less than one day to six days) were observed for particles containing higher B-Glu:BSA ratios. These results highlight the importance of understanding enzyme:stabilizer interactions in various nanoparticle systems to improve not only enzyme activity but also biodistribution and release kinetics for improved therapeutic effects. These results will be critical to fully characterize and compare the effect of stabilizers, such as BSA with other, more relevant therapeutic enzymes for central nervous system (CNS) disease treatments.

Effect of High Sodium Chloride Concentrations in the Growth Medium on the Activity of Glucose-6-Phosphate Dehydrogenase From Pea Roots

1974 ◽  
Vol 1 (4) ◽  
pp. 483 ◽  
Author(s):  
A Poljakoff-Mayber ◽  
H Greenway
Keyword(s):  
Enzyme Activity ◽  
Sodium Chloride ◽  
Growth Medium ◽  
Vascular Plants ◽  
Specific Activity ◽  
High Sodium ◽  
Brown Discoloration ◽  
Pea Roots ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Chloride Concentrations

The experiments reported in this paper examined discrepant results, obtained in earlier investigations, concerning the effects of NaCl in the growth medium of vascular plants on the specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) extracted from the plants. NaCl at 120 mM or higher concentrations increased the specific activity of the enzyme in extracts from roots of germinating peas. However, this effect of NaCl was only clearly expressed when 0.1M phosphate was the buffer used to extract the enzyme. When organic buffers were used the observed increase in enzyme activity was either much smaller or absent. Increases in specific activity of glucose-6-phosphate dehydrogenase were only found in extracts from roots that showed severely retarded growth and some brown discoloration. No increase in activity was found either when 120 mM NaCl was added to the growth medium after seedling establishment, or if seeds were germinated in 75 mM NaCl. These observations account for the discrepancies reported in the literature.

scholarly journals Purification, Characterization and De-Staining Potentials of a Thermotolerant Protease Produced by Fusarium oxysporum

2019 ◽  
Vol 64 (4) ◽  
pp. 539-547
Author(s):  
Mohammed Inuwa Ja’afaru ◽  
Konjerimam Ishaku Chimbekujwo ◽  
Obinna Markraphael Ajunwa
Keyword(s):  
Enzyme Activity ◽  
Fusarium Oxysporum ◽  
Treatment Time ◽  
Specific Activity ◽  
Total Yield ◽  
Tween 20 ◽  
Optimum Temperature ◽  
Industrial Enzymes ◽  
Sds Page ◽  
Optimum Ph

Proteases are important industrial enzymes and fungi prove to be good sources of such enzymes. Purification techniques are however necessary for increased specificity in activity and better industrial value. Based on this, a protease produced by a Fusarium oxysporum was purified to homogeneity by Sephadex G-200 column and α–casein agarose chromatography. The enzyme had a molecular weight of 70 kDa in SDS-PAGE. Purified Fusarium oxysporum protease had a specific activity of 93.88 U/mg protein. The purification magnitude was 7.7 and the total yield was 20 %. Purified protease had an optimum pH of 5.0 while the optimum temperature was 40 °C. The enzyme was also thermotolerant (approximately 100 % at 40 °C for 2 h). The enzyme activity was stimulated by surfactants and metal ions like, Tween-20 and Mg2+. Enzyme activity was inhibited in presence of PMSF and EDTA. Casein was found to be the best substrate for protease activity of Fusarium oxysporum FWT1. Protease were tested upon blood stain for de-clotting of blood and was found to exhibit good de-clotting and de-staining activity after 15 minutes treatment time.

Molecular characterization of an endo-type chitosanase from the fish pathogenRenibacteriumsp. QD1

2014 ◽  
Vol 94 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Peichuan Xing ◽  
Dan Liu ◽  
Wen-Gong Yu ◽  
Xinzhi Lu
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Maximal Activity ◽  
Optimum Temperature ◽  
Sds Page ◽  
Ph Range ◽  
Tlc Analysis

Renibacteriumsp. QD1, a bacteria strain capable of hydrolysing chitosan, was isolated from the homogenate of small crabs. An extracellular chitosanase, Csn-A, was purified from the QD1 fermentation broth. The enzyme was purified to homogeneity, with a yield of eight-fold, 67% recovery and a specific activity of 1575 U/mg proteins. The molecular weight of Csn-A was estimated to be 26.1 kDa by SDS-PAGE. Unlike other chitosanases, the purified Csn-A displayed maximal activity at a pH range of 5.3–6.5, and it was stable in a broad pH range of 5.0–10.0. The optimum temperature for chitosanlytic activity was 55°C. The enzyme activity was strongly stimulated by Mn2+but inhibited by Fe3+, Cu2+, Al3+, Zn2+and SDS. TLC analysis demonstrated that Csn-A hydrolysed N-deacetylated polymeric glucosamines into chito-biose and -triose in an endo-type manner. The amino acid seuquence of Csn-A showed close identity with an uncharacterized chitosanase of strain ATCC33209.

scholarly journals The study of angiotensin converting enzyme isolation from crossbred rabbit lung and enzyme storage capacity in frozen conditions

Food Research ◽  
2020 ◽  
Vol 4 (4) ◽  
pp. 1082-1088
Author(s):  
V.M. Nguyen ◽  
T.T. Tran ◽  
H.N. Vo
Keyword(s):  
Enzyme Activity ◽  
Angiotensin Converting Enzyme ◽  
Ammonium Sulfate ◽  
G Protein ◽  
Specific Activity ◽  
Distilled Water ◽  
Converting Enzyme ◽  
Angiotensin Converting Enzyme Activity ◽  
Rabbit Lung ◽  
Extraction Solvent

The study was carried out to obtain angiotensin converting enzyme (ACE) with high specific activity and to evaluate the ability to maintain enzyme activity in extract products as well as in rabbit lungs using frozen condition. In the scope of the content, the study conducted an evaluation to select the appropriate extraction solvent of four solvents including acetone, ethanol, Tris-HCl and distilled water. Initially, the research results have helped determine distilled water as the suitable extraction solvent and the difference is not statistically significant compared to Tris-HCl solvent. Angiotensin converting enzyme extract that was obtained by distilled water solvent has a specific activity of 10.35 U/g protein. In addition, the study investigated the ability to maintain angiotensin converting enzyme activity in rabbit lung and crude enzyme product during frozen storage (-18±2°C). The results of the study showed that angiotensin converting enzyme activity could be maintained for 3 months in rabbit lungs and 4 months in the crude product. Besides, the study also used ammonium sulfate with different concentrations to conduct angiotensin converting enzyme collection from extract product. The results of this content help determine the use of saturated ammonium sulfate with concentrations of 50% to 60% for the highest efficiency. The precipitation process helped obtain ACE products with a purity of 4.22 times, the specific activity of 42.64 U/g protein and the recovery rate of ACE up to 29.37%.

scholarly journals Purification and Characterization of protease from Zahdi dates plam seeds (Phoenix dactylifera L.)

2009 ◽  
Vol 6 (4) ◽  
pp. 633-639
Author(s):  
Baghdad Science Journal
Keyword(s):  
Phosphate Buffer ◽  
Sodium Phosphate ◽  
Specific Activity ◽  
Enzyme Stability ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Phoenix Dactylifera ◽  
Sodium Phosphate Buffer ◽  
Phoenix Dactylifera L ◽  
Enzyme Specific Activity

Proteinases (E.C.3.4.21) family are widely distributed in the nature; it was present in animals tissues , plants and microbial cell . Protease was purified from Zahdi seed (Phoenix dactylifera L.) by several steps included ammonium sulphite ppt (75%) saturation and dialyzed against the 80mM sodium phosphate buffer at pH 7.5 . The enzyme specific activity was 407.62 unit/mg protein. The obtained extract was purified by DEAE-Cellulose column followed by gel filtration through Sephacyl S-200 column .The enzyme specific activity ,yield and purification fold were 1873.49 unit/mg protein, 22.99 and 58.42% respectively. The results of protease characterization showed that the molecular weight was 25118 daltons as determined by gel filtration. The optimum temperature of the enzyme activity 35 C for 15 minutes and that for stability was 45 C for 15 minutes, using sodium phosphate buffer at pH 7.5, The optimum pH for the enzyme stability and activity were 8.5 for 15 minute and 7.5 respectively.

scholarly journals Characterization of the Recombinant Thermostable Lipase (Pf2001) from Pyrococcus furiosus: Effects of Thioredoxin Fusion Tag and Triton X-100

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Sylvia Maria Campbell Alquéres ◽  
Roberta Vieira Branco ◽  
Denise Maria Guimarães Freire ◽  
Tito Lívio Moitinho Alves ◽  
Orlando Bonifácio Martins ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Optimum Temperature ◽  
Optimal Temperature ◽  
Thermostable Lipase ◽  
Fusion Tag ◽  
Triton X

In this work, the lipase from Pyrococcus furiosus encoded by ORF PF2001 was expressed with a fusion protein (thioredoxin) in Escherichia coli. The purified enzymes with the thioredoxin tag (TRX−PF2001Δ60) and without the thioredoxin tag (PF2001Δ60) were characterized, and various influences of Triton X-100 were determined. The optimal temperature for both enzymes was 80°C. Although the thioredoxin presence did not influence the optimum temperature, the TRX−PF2001Δ60 presented specific activity twice lower than the enzyme PF2001Δ60. The enzyme PF2001Δ60 was assayed using MUF-acetate, MUF-heptanoate, and MUF-palmitate. MUF-heptanoate was the preferred substrate of this enzyme. The chelators EDTA and EGTA increased the enzyme activity by 97 and 70%, respectively. The surfactant Triton X-100 reduced the enzyme activity by 50% and lowered the optimum temperature to 60°C. However, the thermostability of the enzyme PF2001Δ60 was enhanced with Triton X-100.

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 Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 325 ◽  
Author(s):  
He Chen ◽  
Jie Huang ◽  
Binyun Cao ◽  
Li Chen ◽  
Na Song ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Plantarum ◽  
Industrial Development ◽  
Extraction Efficiency ◽  
Specific Activity ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Kinetic Studies ◽  
Predicted Values ◽  
Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

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