scholarly journals Effects of Tannin Extract fromGongronema latifoliumLeaves on LipoxygenaseCucumeropsis maniiSeeds

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Sabinus O. O. Eze ◽  
Bennett C. Nwanguma
Keyword(s):  
Ascorbic Acid ◽  
Propyl Gallate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Oxidative Enzymes ◽  
Kinetic Properties ◽  
Tannin Extract ◽  
Ammonium Sulphate Precipitation ◽  
Show Maximum Activity

Lipoxygenase (EC 1.13.11.12) was partially purified from germinated seeds ofCucumeropsis maniito a purification fold of 47.14, enzyme activity recovery of 72.18%, and specific activity of 326.25 units/mg protein, using a three-step process of centrifugation, ammonium sulphate precipitation and gel filtration. Kinetic properties show maximum activity at pH 6.0 and at optimum temperature of 40°C. Inhibitory effects of the extract fromGongronema latifoliumand two other known antioxidants: ascorbic acid and propyl gallate on lipoxygenase fromCucumeropsis maniiwere studied. Result shows presence of inhibition with IC50 of4.2×10−3±0.09×10−3 g/L,4.3×10−2±0.11×10−2 g/L and7.9×10−2±0.11×10−2 g/L for the extract from ascorbic acid and propyl gallate, respectively. The extract when compared to the other antioxidants exhibits a competitive mechanism of inhibition. This tannin extract could be included during food processing as preservative against food deterioration that might be caused by oxidative enzymes such as lipoxygenase.

scholarly journals Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

1996 ◽  
Vol 314 (2) ◽  
pp. 587-593 ◽  
Author(s):  
Antonio del CASTILLO-OLIVARES ◽  
Miguel A. MEDINA ◽  
Ignacio NÚÑEZ de CASTRO ◽  
Javier MÁRQUEZ
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Nadh Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumour Cells ◽  
Ammonium Sulphate Precipitation ◽  
Apparent Homogeneity

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis–Menten kinetics for the substrates, with apparent Km values of 4.3×10-5 M and 6.7×10-5 M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thiol-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

scholarly journals Partial purification and characterization of fatty acid esterase from pearl millet

2021 ◽  
Vol 42 (1) ◽  
pp. 144-153
Author(s):  
S. Bajaj ◽  
◽  
L.K. Chugh ◽  
P. Goyal ◽  
A. Kumar ◽  
...  
Keyword(s):  
Ascorbic Acid ◽  
Fatty Acid ◽  
Shelf Life ◽  
Pearl Millet ◽  
Specific Activity ◽  
Gel Filtration ◽  
Kinetic Properties ◽  
Lipolytic Enzyme ◽  
Processing Technologies ◽  
Km Value

Aim: The present study aimed to identify a negative modulator of lipolytic enzyme fatty acid esterase (FAE) for exploring possibilities of increasing shelf life of pearl millet flour through arresting in-situ hydrolysis of lipids. Methodology: FAE was partially purified from flour of pearl millet hybrid HHB 234 by (NH4)2SO4 fractionation (30-60% saturation) and gel filtration chromatography using Sephadex G-75. The enzyme was characterized for physico-chemical properties viz., molecular mass, optimum pH, optimum temperature and thermal stability and kinetic properties viz., Km value and effect of modulators. Results: Crude extract contained 1008 units of activity and 421 mg proteins resulting in to specific activity of 2.4 units mg-1 protein. The enzyme was purified 10.7 fold with a recovery of 21.52% and specific activity of 25.7 units mg-1 protein by ammonium sulphate fractionation followed by gel filtration. The molecular weight of purified enzyme preparation was 60 kDa, as determined by gel filtration through Sephadex G-75. The enzyme exhibited maximum activity at pH 8.2 and 45°C. The enzyme was stable up to 60°C for 20 min and showed Km value of 0.65 µM for p-NPB. At 10 mM concentration, Mg2+ and Zn2+ altered the activity positively by 54% and negatively by 42% whereas EDTA, DTT, PMSF, ascorbic acid inhibited the activity by 75, 68, 50 and 48%, respectively. Interpretation: Partially purified lipolytic enzyme FAE from pearl millet flour was strongly inhibited by ascorbic acid. This novel information might be useful in developing processing technologies for improving shelf life of flour. Key words: Characterization, Fatty acid esterase, Pearl millet, Purification, Shelf life

scholarly journals PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

2017 ◽  
Vol 18 (2) ◽  
pp. 1-10 ◽  
Author(s):  
Dzun Noraini Jimat ◽  
Intan Baizura Firda Mohamed ◽  
Azlin Suhaida Azmi ◽  
Parveen Jamal
Keyword(s):  
Specific Activity ◽  
Hot Spring ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Bacillus Sp ◽  
Sds Page ◽  
Fast Flow ◽  
Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60°C.

scholarly journals Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

2005 ◽  
Vol 37 (6) ◽  
pp. 363-370 ◽  
Author(s):  
Ye-Yun Li ◽  
Chang-Jun Jiang ◽  
Xiao-Chun Wan ◽  
Zheng-Zhu Zhang ◽  
Da-Xiang Li
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Optimum Activity ◽  
Development Of Resistance ◽  
Sds Page ◽  
C 50 ◽  
Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

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

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 

scholarly journals Purification and Characterization of Melanogenic Enzyme Tyrosinase from Button Mushroom

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Kamal Uddin Zaidi ◽  
Ayesha S. Ali ◽  
Sharique A. Ali
Keyword(s):  
Agaricus Bisporus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Edible Mushroom ◽  
Total Activity ◽  
Protein Band ◽  
Exchange Chromatography ◽  
Ammonium Sulphate Precipitation ◽  
Key Enzyme

Melanogenesis is a biosynthetic pathway for the formation of the pigment melanin in human skin. A key enzyme, tyrosinase, catalyzes the first and only rate-limiting steps in melanogenesis. Since the discovery of its melanogenic properties, tyrosinase has been in prime focus and microbial sources of the enzyme are sought. Agaricus bisporus widely known as the common edible mushroom, it’s taking place in high amounts of proteins, enzyme, carbohydrates, fibers, and low fat contents are frequently cited in the literature in relation to their nutritional value. In the present study tyrosinase from Agaricus bisporus was purified by ammonium sulphate precipitation, dialysis followed by gel filtration chromatography on Sephadex G-100, and ion exchange chromatography on DEAE-Cellulose; the enzyme was purified, 16.36-fold to give 26.6% yield on total activity in the crude extract and final specific activity of 52.19 U/mg. The SDS-PAGE electrophoresis showed a migrating protein band molecular weight of 95 kDa. The purified tyrosinase was optimized and the results revealed that the optimum values are pH 7.0 and temperature 35°C. The highest activity was reported towards its natural substrate, L-DOPA, with an apparent Km value of 0.933 mM. This indicated that tyrosinase purified from Agaricus bisporus is a potential source for medical applications.

Purification and Partial Characterization of Cellulase Produced by Aspergillus niger Cultured on Vitellaria paradoxa shells

2017 ◽  
Vol 6 (2) ◽  
Author(s):  
Abdulhakeem Olarewaju Sulyman ◽  
Yusuf A Iyanda ◽  
Afolabi Olaniyi Opasola ◽  
OtunOla Adedayo ◽  
Raliat Abimbola Aladodo
Keyword(s):  
Aspergillus Niger ◽  
Specific Activity ◽  
Gel Filtration ◽  
Inoculum Size ◽  
Partial Characterization ◽  
Effect Of Temperature ◽  
Vitellaria Paradoxa ◽  
Endoglucanase Activity ◽  
Ammonium Sulphate Precipitation

This research investigated the purification and partial characterization of cellulase produced by Aspergillus niger cultured on Vitellaria paradoxa shells. Cellulase (endoglucanase) from A. niger was produced under optimum fermentation conditions at 35 °C, pH 4.7, V. paradoxa, 4 g/L, inoculum size of 10 mm and the fermentation media incubated for 120 hours. The crude endoglucanase obtained were partially purified by subjecting to ammonium sulphate precipitation, dialysis and gel filtration chromatography for further purification. The effect of temperature and pH on the activity of purified endoglucanase was determined. Cellulase was purified to 734.33 folds by Sephadex G-100 column chromatography with a specific activity and yield of 4.406 U/mg and 63.03% respectively. Fractions 4 and 7 contained the highest endoglucanase activity out of 18 fractions collected and the two fractions were pooled for further analysis. The activity of purified endoglucanase was optimum at a temperature of 40 °C and pH 5. Therefore, the purified endoglucanase produced may be explored in detergent industry.

scholarly journals Isolation and characterization of three distinct forms of lipases from Candida rugosa produced in solid state fermentation

2001 ◽  
Vol 44 (2) ◽  
pp. 213-221 ◽  
Author(s):  
Sailas Benjamin ◽  
Ashok Pandey
Keyword(s):  
Solid State ◽  
Solid State Fermentation ◽  
Specific Activity ◽  
Candida Rugosa ◽  
Gel Filtration ◽  
Coconut Oil ◽  
Apparent Molecular Weight ◽  
Ammonium Sulphate Precipitation ◽  
Isolation And Characterization ◽  
Ultra Filtration

Three distinct forms (Lip A, Lip B and Lip C) of extra-cellular lipases (EC- 3.1.1.3), produced by Candida rugosa in solid state fermentation (SSF) were purified and characterised. SSF was carried out in glass columns using coconut oil cake and wheat bran. The enzyme was purified from the aqueous extract of fermented matter by ammonium sulphate precipitation, dialysis, ultra-filtration and gel filtration using Sephadex-200 to a 43-fold purification and 64.35-mg/ml specific activity. SDS-PAGE of purified enzyme revealed three distinct bands, indicating the existence of three iso-forms, Lip A, Lip B and Lip C with apparent molecular weight about 64,000, 62,000 and 60,000 Da, respectively. All the three iso-forms were optimally active at 35-40°C and pH 7-8. They showed marked differences in their Km values with different saturated and unsaturated triacyl glycerols. Ag++ and Hg++ strongly inhibited enzyme activity of all the iso-forms, Mn++ has no effect and Ca++ and Mg++ enhanced the activity. EDTA also strongly inhibited the enzyme activities of iso-forms. However, activities of all the three lipases were completely inhibited by serine protease inhibitors such as 3,4-dichloroisocoumarin, pefabloc and partially by phenylmethanesulphonyl fluoride. To the best of our knowledge, this is the first report describing the purification and characterisation of C. rugosa lipase iso-forms from solid cultures. These lipase iso-forms with diverse characteristics produced in solid cultures may find potential application in biomedical field.

scholarly journals New Lipase for Biodiesel Production: Partial Purification and Characterization of LipSB 25-4

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Aysel Ugur ◽  
Nurdan Sarac ◽  
Rukiye Boran ◽  
Berk Ayaz ◽  
Ozgur Ceylan ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Nitrogen Sources ◽  
Biodiesel Production ◽  
Partial Purification ◽  
Period Effect ◽  
Carbon And Nitrogen ◽  
Ammonium Sulphate Precipitation ◽  
Thermophilic Conditions

The lipolytic activities of 300 Streptomyces isolates were determined in Tributyrin and Rhodamine-B Agar. Lipase activities were also measured with p-nitrophenyl palmitate (p-NPP) as a substrate. The strain of Streptomyces bambergiensis OC 25-4 used in this study was selected among 300 strains of Streptomyces from MUCC as the best lipase producer. The incubation conditions were optimized and the inoculum amount, incubation period, effect of carbon and nitrogen sources, and rates of MgSO4 and CaCO3 were investigated. LipSB 25-4 (the lipase produced by S. bambergiensis OC 25-4 strain) was partially purified with ammonium sulphate precipitation, dialysis, and gel filtration chromatography 2.73-fold and with 92.12 U/mg specific activity. The optimal pH and temperature for LipSB 25-4 were determined as 8.0 and 50°C, respectively. The lipase has high stability in all pH and temperature values used in this study. While LipSB 25-4 was slightly activated in the presence of β-mercaptoethanol, it was slightly reduced by PMSF. The enzyme conserved approximately 75% of its activity at the end of 60 h, in the presence of methanol and ethanol. Since LipSB 25-4 displays high activity in the thermophilic conditions and stability in the presence of organic solvents, this lipase can catalyse the biodiesel production from olive oil by the transesterification reactions.

scholarly journals Biobleaching of Industrial Important Dyes with Peroxidase Partially Purified from Garlic

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Akudo Chigozirim Osuji ◽  
Sabinus Oscar O. Eze ◽  
Emmanuel Emeka Osayi ◽  
Ferdinand Chiemeka Chilaka
Keyword(s):  
Ammonium Sulphate ◽  
Specific Activity ◽  
Low Cost ◽  
Gel Filtration ◽  
Enzyme Concentration ◽  
Gel Filtration Chromatography ◽  
Vat Dyes ◽  
Ammonium Sulphate Precipitation ◽  
Ph Range ◽  
Better Than

An acidic peroxidase was extracted from garlic (Allium sativum) and was partially purified threefold by ammonium sulphate precipitation, dialysis, and gel filtration chromatography using sephadex G-200. The specific activity of the enzyme increased from 4.89 U/mg after ammonium sulphate precipitation to 25.26 U/mg after gel filtration chromatography. The optimum temperature and pH of the enzyme were 50°C and 5.0, respectively. The Km andVmaxfor H2O2and o-dianisidine were 0.026 mM and 0.8 U/min, and 25 mM and 0.75 U/min, respectively. Peroxidase from garlic was effective in decolourizing Vat Yellow 2, Vat Orange 11, and Vat Black 27 better than Vat Green 9 dye. For all the parameters monitored, the decolourization was more effective at a pH range, temperature, H2O2concentration, and enzyme concentration of 4.5–5.0, 50°C, 0.6 mM, and 0.20 U/mL, respectively. The observed properties of the enzyme together with its low cost of extraction (from local sources) show the potential of this enzyme for practical application in industrial wastewater treatment especially with hydrogen peroxide. These Vat dyes also exhibited potentials of acting as peroxidase inhibitors at alkaline pH range.

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