scholarly journals Pemurnian Parsial dan Karakterisasi Urease dari Biji Kacang Panjang (Vigna unguiculata subsp sesquipedalis L.)

2018 ◽  
Vol 14 (1) ◽  
pp. 72
Author(s):  
Zusfahair Zusfahair ◽  
Dian Riana Ningsih ◽  
Amin Fatoni ◽  
Darul Santri Pertiwi
Keyword(s):  
Vigna Unguiculata ◽  
Crude Extract ◽  
Metal Ion ◽  
Urease Activity ◽  
Specific Activity ◽  
Partial Purification ◽  
Isolation And Purification ◽  
Molecular Weights ◽  
Sds Page ◽  
Analytical Result

<p>Urease merupakam enzim yang digunakan dalam hidrolisis urea menjadi amoniak dan asam bikarbonat dan telah banyak digunakan dalam proses industri. Tujuan penelitian adalah isolasi dan pemurnian urease dari kacang panjang serta karakterisasinya. Penelitian dimulai dengan melakukan perkecambahan biji kacang panjang selama 8 hari. Kecambah biji kacang panjang selanjutnya diekstraksi dengan menggunakan buffer fosfat pH 7 dan dipisahkan menggunakan sentrifugasi sehingga diperoleh ekstrak kasar urease. Ekstrak kasar urease selanjutnya difraksinasi menggunakan aseton pada tingkat konsentrasi 20, 40, 60 dan 80%. Fraksi yang mempunyai aktivitas spesifik paling tinggi selanjutnya dianalisis menggunakan metode SDS-PAGE untuk menentukan berat molekulnya dan dikarakterisasi lanjut meliputi: pengaruh suhu, pH, konsentrasi substrat dan penambahan ion logam terhadap aktivitas urease. Aktivitas urease ditentukan dengan metode Nessler. Hasil penelitian menunjukkan aktivitas spesifik urease dari kacang panjang paling tinggi ditemukan pada fraksi aseton (FA) 20. Hasil analisis berat molekul dengan metode SDS-PAGE diperoleh beberapa pita protein yang diduga berukuran sekitar 25 KDa dan 17 KDa. Kondisi optimum dari aktivitas urease diperoleh pada suhu 30 ºC, pH 7 dan konsentrasi urea 16,6 mM dengan nilai aktivitas 407,62 U/mL. EDTA dan ion logam dalam CaCl<sub>2</sub>, NaCl, NiCl<sub>2</sub> dan CuCl<sub>2 </sub>pada variasi konsentrasi 10<sup>-3</sup>, 10<sup>-4 </sup> dan 10<sup>-5 </sup>M merupakan inhibitor urease FA 20 dari kacang panjang.</p><p><strong><span lang="EN-US">Partial Purification and Characterization of Urease from Asparagus Bean (<em>Vigna unguiculata subsp sesquipedalis </em>L<em>.</em>). </span></strong><span lang="EN-US">Urease is an enzyme used in urea hydrolysis to ammonia and bicarbonate acid and has been widely used in industrial processes. The study focused on isolation and purification of urease from asparagus beans and its characterization. The study was started with germination of asparagus beans for 8 days. Germinated asparagus beans were further extracted using phosphate buffer pH 7 and separated by centrifugation to obtain a crude extract of urease. The crude extract of urease was further fractionated using acetone at concentrations of 20, 40, 60 and 80%. The fraction with highest specific activity was then </span><span>analyzed using SDS-PAGE method to determine its molecule weight and characterized further including the influence of </span><span lang="EN-US">temperature, pH, substrate concentration, and metal ion addition to urease activity. The urease activity was determined by the Nessler̕ s method. The results showed that the specific activity of urease from asparagus beans was found with highest activity in fraction of acetone (FA) 20. Analytical result using SDS-PAGE method was obtained some protein bands having molecular weights about 25 KD and 17 KDa. The optimum conditions of urease activity was obtained at 30 °C, pH 7, incubation time 20 min and urea concentration 16.6 mM with activity value 407.62 U/mL. EDTA and </span><span>metal ions contained in </span><span lang="EN-US">CaCl<sub>2</sub>, NaCl, NiCl<sub>2</sub> and CuCl<sub>2</sub> at concentrations of 10<sup>-3</sup>, 10<sup>-4</sup> and 10<sup>-5</sup> M were FA 20 urease inhibitors.</span></p>

scholarly journals Purificación Parcial y Caracterización de Alfa Amilasa de granos germinados de Chenopodium quinoa (Quinua)

2018 ◽  
pp. 52-58
Keyword(s):  
Specific Activity ◽  
Chenopodium Quinoa ◽  
Maximum Activity ◽  
Alpha Amylase ◽  
Partial Purification ◽  
Chemical Hydrolysis ◽  
Germination Process ◽  
Sds Page ◽  
Global Population

Purificación Parcial y Caracterización de Alfa Amilasa de granos germinados de Chenopodium quinoa (Quinua) Partial Purification and Characterization of Alpha Amylase from germinated grains from Chenopopdium quinoa (Quinua) Melissa Bedón Gómez, Oscar Nolasco Cárdenas, Carlos Santa Cruz C. y Ana I. F. Gutiérrez Román Universidad Nacional Federico Villarreal, Facultad de Ciencias Naturales y Matemática, Laboratorio de Bioquímica y Biología Molecular, Jr. Río Chepén S/N, El Agustino. Telefax: 362 - 3388 DOI: https://doi.org/10.33017/RevECIPeru2013.0007/ Resumen Las alfa amilasas son las enzimas más estudiadas e importantes en el campo biotecnológico e industrial; ya que han reemplazado por completo la hidrólisis química del almidón. Estas enzimas son imprescindibles en la elaboración de productos alimenticios, combustibles, medicamentos y detergentes con la finalidad de optimizar procesos y conservar el medio ambiente. La α-amilasa puede ser purificada de diferentes organismos como plantas, animales, hongos y bacterias; actualmente un gran número de α-amilasas bacterianas en especial del género Bacillus están disponibles comercialmente y son las más utilizadas en las industrias. Sin embargo, la producción de éstas no satisfacen los requerimientos industriales en el mundo; ya que, la demanda de esta enzima se ha incrementado en los últimos dos años y el empleo de α-amilasas bacterianas ha provocado alergias afectando al 15% de la población a nivel mundial. . En este estudio, como fuente de α-amilasa se emplearon semillas de Chenopodium quinoa (quinua) var hualhuas blanca durante el proceso de germinación; esta enzima fue parcialmente purificada por precipitación con sulfato de amonio obteniendo una actividad específica final de 35.60U/mg y un grado de purificación de 5 veces. La purificación fue confirmada por SDS-PAGE, encontrando un peso molecular de 44kDa. La actividad enzimática se evaluó mediante el método de Miller mostrando máxima actividad a pH 7 y a temperatura de 37ºC. La linealización de Lineweaver-Burk nos dio un Km de 16mg/mL y Vmax de 100µM de maltosa/min. Por lo tanto, esta caracterización reúne los pre-requisitos necesarios para la aplicación en la industria. Descriptores: Chenopodium quinoa, alfa amilasa, germinación, purificación parcial. Abstract The alpha amylases are the enzymes most studied and important in biotechnology and industry; because they have completely replaced the starch’s chemical hydrolysis. These enzymes are essential in the food production, medicines and detergents in order to optimize processes and conserve the environment. The α-amylase can be isolated from different organisms such as plants, animals, fungi and bacteria, now a large number of bacterial α-amylases especially from genus Bacillus are commercially available and they are the most used in industry. However, the production of these do not meet industry requirements in the world, because the demand for this enzyme has increased in the last two years and the use of bacterial α-amilase has caused allergies affecting the 15% of the global population. In this study, as a source of α-amylase used the seeds from Chenopodium quinoa (quinoa). Var. white hualhuas during the germination process, this enzyme was partially purified by ammonium sulfate precipitation to obtain a final specific activity of 35.60U/mg, and a grade of purification of 5 times. The purification was confirmed by SDS-PAGE, where the molecular weight was 44kDa. The enzyme activity was evaluated by Miller method showing maximum activity at pH 7 and 37ºC. The Lineweaver-Burk linearization shows a Km of 16mg/mL and Vmax of 100μM the maltose / min. Therefore, these characterizations meet the prerequisites need for industry. Keywords: Chenopodium quinoa; alpha amylase; germination; partial purification

Cysteine proteinase activities in the fish pathogenPhilasterides dicentrarchi(Ciliophora: Scuticociliatida)

Parasitology ◽  
2004 ◽  
Vol 128 (5) ◽  
pp. 541-548 ◽  
Author(s):  
A. PARAMÁ ◽  
R. IGLESIAS ◽  
M. F. ÁLVAREZ ◽  
J. LEIRO ◽  
F. M. UBEIRA ◽  
...  
Keyword(s):  
Crude Extract ◽  
Type I Collagen ◽  
Cysteine Proteinase ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Type I ◽  
Molecular Weights ◽  
Reducing Conditions ◽  
Sds Page

This study investigated protease activities in a crude extract andin vitroexcretion/secretion (E/S) products ofPhilasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS–PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30–63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptidep-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobinin vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology ofPhilasterides dicentrarchi.

scholarly journals Isolation and partial purification of anticoagulant fractions from the venom of the Iranian snake Echis carinatus.

2012 ◽  
Vol 60 (1) ◽  
Author(s):  
Mahdi Babaie ◽  
Hossein Zolfagharian ◽  
Hossein Salmanizadeh ◽  
Abbas Zare Mirakabadi ◽  
Hafezeh Alizadeh
Keyword(s):  
Blood Coagulation ◽  
Gel Filtration ◽  
Partial Purification ◽  
Crude Venom ◽  
Molecular Weights ◽  
Time Value ◽  
Sds Page ◽  
Echis Carinatus ◽  
The Many ◽  
Coagulation Pathway

Many snake venoms comprise different factors, which can either promote or inhibit the blood coagulation pathway. Coagulation disorders and hemorrhage belong to the most prominent features of bites of the many vipers. A number of these factors interact with components of the human blood coagulation. This study is focused on the effect of Echis carinatus snake venom on blood coagulation pathway. Anticoagulant factors were purified from the Iranian Echis carinatus venom by two steps: gel filtration (Sephadex G-75) and ion-exchange (DEAE-Sephadex) chromatography, in order to study the anticoagulant effect of crude venom and their fractions. The prothrombin time was estimated on human plasma for each fraction. Our results showed that protrombin time value was increase from 13.4 s to 170 s for F2C and to 280 s for F2D. Our study showed that these fractions of the venom delay the prothrombine time and thus can be considered as anticoagulant factors. They were shown to exhibit proteolytic activity. The molecular weights of these anticoagulants (F2C, F2D) were estimated by SDS/PAGE electrophoresis. F2C comprises two protein bands with molecular weights of 50 and 79 kDa and F2D a single band with a molecular weight of 42 kDa.

scholarly journals Partial purification and characterization of a superoxide distimutase (SOD1) from black tiger shrimps Penaeus monodon

2020 ◽  
Vol 42 (1) ◽  
Author(s):  
Tran Minh Hien ◽  
Nguyen Thi Hong Loan ◽  
Trinh Dinh Quynh ◽  
Ngo Thi Trang ◽  
Dang Thi Lua ◽  
...  
Keyword(s):  
Specific Activity ◽  
Penaeus Monodon ◽  
Protein Band ◽  
Partial Purification ◽  
Sod Activity ◽  
Black Tiger Shrimp ◽  
Tiger Shrimp ◽  
Sds Page ◽  
Muscular Tissues

Superroxide dismutase (SOD, EC.1.15.1.1) is the enzyme which dismutates superoxide radicals and plays an important role in protection of living cells against oxidative stress. SOD is also involved in immune response in shrimps. In this study, it was found that the total SOD activity of black tiger shrimp muscular tissues is 10 fold higher than that of the haemolymph, however, the specific activity of SOD in the shrimp haemolymph is 9.2 fold higher than that of muscular tissues. By using active gel electrophoresis, 2 different SOD forms were found in black tiger shrimps (one in muscular tissues and two in haemolymph).Using DE-52 cellulose and Q-Sepharose ion exchange column chromatography, one SOD (SOD1) from black tiger shrimp haemolymph was partially purified, and its purity was 31.2 times higher than that of the starting haemolymph. The SOD1 was shown to have mainly one protein band of approximately 24 kDa on SDS-PAGE. SOD1 was most active at 45oC and pH of 5.5. At a concentration of 5 mM, Mn2+ strongly activated SOD1 (up 200% activity), Ca2+ và Zn2+ could increase approximately 20% activity while Cu2+ inhibited more than 60% ativity of the enzyme. 

scholarly journals Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

2017 ◽  
Vol 37 (1) ◽  
pp. 31
Author(s):  
Fitria Fitria ◽  
Nanik Rahmani ◽  
Sri Pujiyanto ◽  
Budi Raharjo ◽  
Yopi Yopi
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Centrifugal Filter ◽  
Sds Page ◽  
Enzyme Specific Activity ◽  
Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial  fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to  conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the  xylooligosaccharide types from xylan hydrolysis by this enzyme.  Based on this research, the optimum time for enzyme production  occurred at 96 hours with the enzyme activity of 6.275 U/mL and  enzyme specific activity of 5.093 U/mg. The specific activities were  obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0  U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of  xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram  showed that the molecular weight of xylanase protein were about  25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan  (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian  ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji  hidrolisis untuk mengetahui jenis xilooligosakarida yang  dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96  dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil  pemekatan dengan amicon® ultra-15 centrifugal filter devices,  kromatografi filtrasi gel dan kromatografi penukar anion  mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas  tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang  dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

scholarly journals Partial Purification of a Catalase from an Improved Nigerian Sorghum Grain Variety

2020 ◽  
pp. 30-37
Author(s):  
C. I. Nnamchi ◽  
B. C. Nwanguma ◽  
O. C. Amadi
Keyword(s):  
Crude Protein ◽  
Specific Activity ◽  
Partial Purification ◽  
Optimum Activity ◽  
Preliminary Purification ◽  
Sds Page ◽  
Cellular Detoxification ◽  
Activity Temperature ◽  
Ph Range ◽  
Sorghum Grain

Catalases are key components of cellular detoxification pathways that prevent the formation of highly reactive hydroxyl radicals through catalyzing the decomposition of hydrogen peroxide into water and molecular oxygen. Their presence in brewery grains prevent the inactivation of important brewery enzymes and also stop lipid peroxidation. To determine their occurrence and establish some of its properties in sorghum, which has become as an important brewery grain similar to barley, crude catalase was obtained from a sorghum grain variety. Preliminary purification of catalase from the sorghum grain variety used, NRL-3, showed that the enzyme was purified 3.2-fold from the crude protein to give a 49% yield of the partially purified enzyme, with a final specific activity of 32 Umg-1 proteins. There was also a positive indication of sorghum catalase presence on SDS PAGE with positive bands occurring between the range of 48-62 kDa. Therefore, the molecular weight of sorghum catalase most likely falls within the two bands. The enzyme showed a narrow pH range with optimum activity occurring at pH 7. Similarly, its optimum activity temperature occurred at 40°C.  This work is the first reported attempt at purifying catalase from sorghum.

Isolation and purification of the D(–)β-hydroxybutyric dehydrogenase of Azotobacter vinelandii

1968 ◽  
Vol 14 (7) ◽  
pp. 775-783 ◽  
Author(s):  
Peter Jurtshuk ◽  
S. Manning ◽  
C. R. Barrera
Keyword(s):  
Ammonium Sulfate ◽  
Metal Ion ◽  
Azotobacter Vinelandii ◽  
Specific Activity ◽  
Alkaline Ph ◽  
Membranous Structure ◽  
Isolation And Purification ◽  
Resting Cell ◽  
Direct Use ◽  
Marked Stability

It has been possible to isolate and purify the D(–)β-hydroxybutyric dehydrogenase from cell-free extracts of Azotobacter vinelandii. Standardized resting cell suspensions were disrupted by sonic oscillation, and purification of the enzyme was achieved by use of protamine sulfate, ammonium sulfate, and hydroxyapatite or by direct use of a Svensson-Porath preparatory column electrophoretic unit. The D(–)β-hydroxybutyric dehydrogenase was found to be a classical, soluble, NAD+-dependent dehydrogenase and neither bound nor associated with any intracellular membranous structure. The highest specific activity achieved was 17 μmoles NAD+ reduced per minute per milligram protein at 37°. Two unusual features were noted with this dehydrogenase: (a) loss of activity occurred when the enzyme was dialyzed in the absence of metal ion; and (b) marked stability was observed when the enzyme was exposed to alkaline pH even at 40 °C for hours.

scholarly journals Partial Purification and Characterisation of Pectinase Produced by Aspergillus niger LFP-1 Grown on Pomelo Peels as a Substrate

2021 ◽  
Vol 32 (1) ◽  
pp. 1-22
Author(s):  
Mohd Taufiq Mat Jalil ◽  
◽  
Darah Ibrahim ◽  
◽  
Keyword(s):  
Aspergillus Niger ◽  
Column Chromatography ◽  
Metal Ion ◽  
Specific Activity ◽  
Gel Filtration ◽  
Citrus Fruit ◽  
Crude Enzyme ◽  
Partial Purification ◽  
Anion Exchange Column ◽  
C 50

In the present study, pectinase was produced by local fungal isolate, Aspergillus niger LFP-1 grown on pomelo peels as a sole carbon source under solid-state fermentation (SSF). The purification process begins with the concentration of crude enzyme using ammonium sulfate precipitation and followed by purification using anion-exchange column chromatography (DEAE-Sephadex) and subsequently using gel filtration column chromatography (Sephadex G-100). On the other hand, the molecular weight of the purified enzyme was determined through SDS-PAGE. The findings revealed the crude enzyme was purified up to 75.89 folds with a specific activity of 61.54 U/mg and the final yield obtained was 0.01%. The molecular mass of the purified pectinase was 48 kDa. The optimum pH and temperature were 3.5 and 50°C, respectively. This enzyme was stable at a range of pH 3.5 to 4.5 and a relatively high temperature (40°C–50°C) for 100 min. The Km and Vmax were found to be 3.89 mg/mL and 1701 U/mg, respectively. Meanwhile, pectin from citrus fruit and the metal ion (Co2+) were the best substrate and inducer to enhance pectinase yield, respectively.

scholarly journals Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity

2018 ◽  
Vol 22 (2) ◽  
pp. 47
Author(s):  
Akhmad Solikhin ◽  
Apon Zaenal Mustopa ◽  
Suharsono Suharsono ◽  
Wendry Setiyadi Putranto
Keyword(s):  
Enzyme Activity ◽  
Metal Ion ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Iodoacetic Acid ◽  
Aspartic Protease ◽  
Partial Purification ◽  
Antioxidant Agents

   Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

Sign in / Sign up

Export Citation Format

Share Document