EXTRACTION AND  PURIFICATION OF BETA-GALACTOSIDASE FROM LOCAL ALMOND AND  ITS  USE FOR LACTOSE INTOLERANCE TREATMENT

This study was aimed, extraction and purification of beta-Galactosidase from local almond(Amygdalus communis) for lactose intolerance treatment. The best one among 10 methods method of the extraction was using sodium phosphate buffer at 0.2 molar. Which was achieves the highest specific activity amounted to 3.66unit/mg protein. Then, partial purification of enzyme was done using five methods. The highest specific activity was obtained using the method of precipitation with ammonium sulphate at 30-70% since the specific activity was 15.85units/mg protein. Which represented the best way to precipitation the enzyme. Three iso enzymes were obtained. One of them was taken for its high specific activity(20.10units/mg protein) and ion exchange chromatography was used and followed by gel filtration technique using sephadex-100 column to increase purification. The specific activity was increased to 21.95units/mg protein. Lactose hydrolysis efficiency test was performed and the purified enzyme showed high efficiency in standard lactose hydrolysis test.

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Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

Bayero Journal of Pure and Applied Sciences ◽

10.4314/bajopas.v13i2.14 ◽

2021 ◽

Vol 13 (2) ◽

pp. 107-112

Author(s):

C.F. Okechukwu ◽

P.L. Shamsudeen ◽

R.K. Bala ◽

B.G. Kurfi ◽

A.M. Abdulazeez

Keyword(s):

Ion Exchange ◽

Phospholipase A2 ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Partial Purification ◽

Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km of 1.47mg/ml and Vmax of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

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Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19821139 ◽

1982 ◽

Vol 47 (4) ◽

pp. 1139-1148 ◽

Cited By ~ 6

Author(s):

Karel Hauzer ◽

Linda Servítová ◽

Tomislav Barth ◽

Karel Jošt

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Peptide Bond ◽

Exchange Chromatography ◽

Partial Purification ◽

Purification And Characterization ◽

Optimum Ph ◽

Hydrolysis Of ◽

Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

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

Jurnal Agritech ◽

10.22146/agritech.17004 ◽

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.

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Distribution and partial purification of a liver membrane protein capable of inactivating cytosol enzymes

Biochemical Journal ◽

10.1042/bj1860571 ◽

1980 ◽

Vol 186 (2) ◽

pp. 571-579 ◽

Cited By ~ 15

Author(s):

G L Francis ◽

F J Ballard

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Plasma Membranes ◽

Specific Activity ◽

Gel Filtration ◽

Exchange Chromatography ◽

Partial Purification ◽

Major Protein ◽

Major Protein Band ◽

Glucose 6 Phosphate Dehydrogenase

1. The inactivation of cytosol enzymes in liver extracts was carried out by several subcellular fractions, with plasma membranes having the highest specific activity. Rough and smooth microsomal fractions were both active, whereas lysosmal inactivation capacity appeared to be derived entirely from contaminating plasma-membrane fragments. 2. Inactivation capacity in liver fractions was derived from parenchymal cells. Of the non-liver cells tested, plasma membranes from H35 hepatoma cells were able to inactivate glucose 6-phosphate dehydrogenase (EC 1.1.1.49), adipocyte “ghosts” showed slight activity and erythrocyte and reticulocyte “ghosts” were inactive. 3. Liposomes prepared from pure lipids with net negative, positive or neutral charge did not possess inactivation capacity. 4. Liver plasma-membrane inactivation capacity was destroyed by heating at 50 degrees C. 5. Inactivation factor solubilized from membranes by trypsin plus Triton X-100 treatment was partially purified by (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and hydroxyapatite chromatography. 6. Partially purified inactivation factor analysed by gel electrophoresis gave a major protein band that co-migrated with capacity for inactivation of glucose 6-phosphate dehydrogenase. 7. It is concluded that inactivation factor is a membrane protein whose intracellular distribution and other properties are consistent with a possible role for this activity in the initial step of protein degradation.

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PURIFIKASI PARSIAL DAN KARAKTERISASI ß-GALAKTOSIDASE Lactobacillus plantarum B123 INDIGENOS DAN HIDROLISIS LAKTOSA UNTUK PRODUKSI SUSU ULTRA HIGH TEMPERATURE RENDAH LAKTOSA

Jurnal Kimia Terapan Indonesia ◽

10.14203/jkti.v17i2.31 ◽

2015 ◽

Vol 17 (2) ◽

pp. 147-161

Author(s):

Tatik Khusniati ◽

Neny Mariyani ◽

Hanifah Nuryani Lioe ◽

Didah Nur Faridah ◽

Abdul Choliq ◽

...

Keyword(s):

High Temperature ◽

Lactobacillus Plantarum ◽

Lactose Intolerance ◽

Specific Activity ◽

Partial Purification ◽

Lactose Hydrolysis ◽

Galactosidase Activity ◽

Purification And Characterization ◽

Uht Milk

β-Galactosidase is enzyme which hidrolyze lactose to glucose and galactose. This enzyme is used in production low lactose milk for consumption human which have lactose intolerance. Partial purification of β-galactosidase is important to be conducted to increase β-galactosidase activity in order to its hydrolysis potency on UHT milk lactose increased.This research was aimed to production by partially purification and characterization indigenous β-galactosidase from Lactobacillus plantarum B123, and lactose hydrolysis for production low lactose UHT milk. Partially purification were precipitation following dialysis. Characterization included optimazion and stabilization of enzyme, while lactose hydrolisis for production low lactose UHT milk was detected by enzymatic GOD-POD kit. The results showed that production of β-galactosidase by using partial purification increased from 21.51 ± 0.23 U/mL (crude) to 106.34 ± 0.56 U/mL (dialysis). The optimum crude β-galactosidase activity was reached in precipitation by using 60 % ammonium sulphate. The purity of crude β-galactosidase increased 3.71 times after precipitation, and 14.28 times after dialysis. Characterization of β-galactosidase showed that optimum activities of crude and dialyzed β-galactosidase were at pH 6.5 and 50 oC, respectively. Stability of crude β-galactosidase incubated for 1 h were at pH: 5.0-8.5 and 25-50 °C. Specific activity of crude β-galactosidase was 15.05 U/mg protein, while that dialyzed β-galactosidase was 109.58 U/mg protein. Lactose hidrolysis to produce low lactose UHT milk showed that glucose concentration increased with the increase of hidrolysis time. Time needed to hidrolyze lactose 50 % with 4.8 U/mL β-galactosidase at 50°C was 6.08 h. In conclusion that indigenous β-galactosidase from Lactobacillus plantarum B123 purified partially can be used as lactose hidrolyzer in production of low lactose UHT milk.Key words : b-galactosidase, indigenous Lactobacillus plantarum B123, purification, lactose hidrolysis, UHT Milk

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Extraction, and Purification of Peroxidase Enzyme from Peganum harmala Seeds

International Journal of Drug Delivery Technology ◽

10.25258/ijddt.9.4.30 ◽

2019 ◽

Vol 9 (04) ◽

pp. 689-692

Author(s):

Hayder Nasser Al-Mentafji ◽

Mahmoud Hamid Al-Fahdawi ◽

Albab Fawwaz Al-farras

Keyword(s):

Ion Exchange ◽

Specific Activity ◽

Gel Filtration ◽

Deae Cellulose ◽

Exchange Chromatography ◽

Peganum Harmala ◽

Enzyme Recovery ◽

Extraction And Purification ◽

Peroxidase Enzyme ◽

Filtration Step

The aim of this study was to extract peroxides enzyme from Peganum harmala seeds; peroxides was extracted by using different extraction buffer solutions, then it was purified by three steps of purification includes precipitation with ammonium sulfate in a saturation ratio of 70 %, ion exchange chromatography through DEAE-Cellulose, and gel filtration chromatography throughout Sephadex G-100, and determine the optimum condition for extraction. This was performed by controlling the type and concentration of buffer, pH of the buffer used, and the ratio of extraction. The Sodium acetate buffer with 0.2mM and pH 5.0 was found to be the best buffer for the extraction of peroxidase. By using the extraction ratio for a plant of 1:3 (W/V), the specific activity was 195 U/mg protein. These three purification steps raised the specific activity to 235U/mg protein in the precipitation step with purification fold 2.3 and enzyme recovery 69%; the specific activity was increased to 243U/mg protein in Ion exchange step with purification fold 2.4 and enzyme recovery 23%, also the specific activity doubled after gel filtration step to 447U/mg protein with purification fold 4.4 and enzyme recovery 15%.

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Studies on an Inhibitor of Plasminogen Activation in Human Serum

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649120 ◽

1973 ◽

Vol 30 (02) ◽

pp. 414-424 ◽

Cited By ~ 20

Author(s):

Ulla Hedner

Keyword(s):

Ion Exchange ◽

Human Serum ◽

Antithrombin Iii ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Specific Adsorption ◽

Partial Purification ◽

Plasminogen Activation ◽

Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

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Kinetic and Thermodynamic Characterization of Lignin Peroxidase Isolated from Agaricus bitorqus A66

International Journal of Chemical Reactor Engineering ◽

10.1515/1542-6580.2837 ◽

2012 ◽

Vol 10 (1) ◽

Author(s):

Ismat Bibi ◽

Haq Nawaz Bhatti

Keyword(s):

Lignin Peroxidase ◽

Thermal Inactivation ◽

Specific Activity ◽

Gel Filtration ◽

Veratryl Alcohol ◽

Exchange Chromatography ◽

Different Temperatures ◽

Scale Experiment ◽

Menten Kinetic

This study deals with purification and characterization of lignin peroxidase (LiP) isolated from Agaricus bitorqus A66 during decolorization of NOVASOL Direct Black dye. A laboratory scale experiment was conducted for maximum LiP production under optimal conditions. Purification & fractionation of LiP was performed on DEAE-Sepharose ion exchange chromatography followed by Sephadex G-50 gel filtration. The purified LiP has a specific activity of 519 U/mg with 6.73% activity recover. The optimum pH and temperature of purified LiP for the oxidation of veratryl alcohol were 6.8 and 45 °C, respectively. Michaelis-Menten kinetic constants (Vmax and Km) were determined using different concentrations of veratryl alcohol (1-35 mM). The Km and Vmax were 16.67 mM and 179.2 U/mL respectively, for veratryl alcohol oxidation as determined from the Lineweaver-Burk plot. Thermal inactivation studies were carried out at different temperatures to check the thermal stability of the enzyme. Enthalpy of activation decreased where Free energy of activation for thermal denaturation increased at higher temperatures. A possible explanation for the thermal inactivation of LiP at higher temperatures is also discussed.

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Gastric cancer is the world's second-largest death cause. Peptides can be used to deliver radiation or other fatal chemicals to tumors

10.31219/osf.io/eu5mj ◽

2021 ◽

Author(s):

Moataz Dowaidar

Keyword(s):

Gastric Cancer ◽

High Efficiency ◽

Specific Activity ◽

High Specific Activity ◽

Systemic Circulation ◽

Efficient Delivery ◽

Liver And Kidney ◽

Targeting Ability ◽

Therapeutic Research

Gastric cancer is the world's second-largest death cause. Developing suitable medical therapies can help individuals live longer. So far, GC treatment has depended on several pharmaceutical techniques. Chemotherapy and surgery are GC patients' most frequent treatment choices. The most major hurdles to effective GC therapy are chemotherapeutic resistance and non-selective targeting. Recent GC-targeted therapeutic research has focused on building more selective and effective anti-GC pharmacological approaches. Because molecular focused therapy can greatly exacerbate the current inefficacy of normal GC therapy procedures, peptide base synthesis can be used as a carrier to deliver radiation or other fatal chemicals to tumor locations with precise protein overexpression. Different types of peptides with special binding affinity to GC overexpressed receptors have been identified for targeted therapy and imaging. Although some of these peptides have excellent GC targeting ability, they also need great GC penetration capacity and no systemic in vivo toxicity before they can be employed in clinical studies. One of these peptides' most notable limitations is their short plasma half-life, limiting their efficient delivery to tumor locations. Sluggish binding pharmacokinetics, along with in vivo instability, can produce targeted treatment failure. Using an appropriate modification strategy to boost blood circulation time may be advantageous.The key to producing successful, innovative anti-cancer targeting drugs with specific targeting capabilities is to mark the peptide with distinct diagnostic and therapeutic radioisotopes. Although a peptide's radiolabeling or enzymatic degradation may not affect its targeting capabilities, the radiation dose delivery impact on it is obvious. Selecting an appropriate type of radionuclide to achieve high-specific activity, using a simple and high-efficiency radiolabeling process, and selecting an adequate spacer and chelator to manage peptide biodistribution are all important considerations when designing a peptide-based radiopharmaceutical. High internalization and significant systemic circulation washout are other essential tumor targeting needs. Many of the peptides described in this work lack these critical features. The radiolabeled peptide should also remain intact and have a short blood washout period, allowing targeted imaging and therapy. SPECT and PET are the most extensively used technologies in nuclear medicine. Although PET has a greater resolution, SPECT technology gives a comparable sensitivity at a lesser cost. Combining fast binding pharmacokinetics with suitable stability in vivo can result in efficient tumor contrast. Non-target liver and kidney accumulation is required when employing radiolabeled peptides to target GC. When a radiolabeled peptide accumulates more in the liver and intestine than in the GC tumor, the image quality degrades. However, using the proper chelator and spacer can assist decrease non-target accumulation in the kidneys. Finally, considering all these conditions and being positive, it is conceivable to produce a unique peptide with avid binding to GC cells.

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Cardioactive Peptides from the CNS of a Caterpillar, the Tobacco Hornworm, Manduca Sexta

Journal of Experimental Biology ◽

10.1242/jeb.114.1.397 ◽

1985 ◽

Vol 114 (1) ◽

pp. 397-414

Author(s):

Nicholas Platt ◽

Stuart E. Reynolds

Keyword(s):

Manduca Sexta ◽

Specific Activity ◽

Gel Filtration ◽

Corpora Allata ◽

Performic Acid ◽

Partial Purification ◽

Relative Molecular Mass ◽

Two Factors ◽

Frontal Ganglion ◽

Manduca Sexta Larvae

1. A semi-isolated caterpillar heart bioassay was used to detect the presence of endogenous cardioactive material in the CNS of Manduca sexta larvae. 2. Cardioactivity was detected in all nervous tissue examined. Most activity (about 70% of the total in the CNS) was in the ganglia of the abdominal nerve cord (ANC). Cardioactivity was also detected in the abdominal transverse nerves, the proctodeal nerves and the corpora cardiaca/corpora allata. The source with the highest specific activity was the frontal ganglion. 3. Two factors, separable by Sephadex gel filtration, were distinguished in extracts of ANC: CAF 1, which has an estimated relative molecular mass (Mr) of about 4000, and CAF2 for which Mr is probably less than 1000. Both factors are apparently peptides. Neither is similar to any known insect cardioaccelerator. 4. Both CAF 1 and CAF 2 are able to cause cardioacceleration when injected into tetrodotoxin-paralysed caterpillars. 5. CAF 2 is present in both larvae and in adults. CAF 1 is present only in the caterpillar. The larval heart responds to both factors; the adult heart responds only to CAF 2. 6. Partial purification of CAF 1 and CAF 2 by reverse-phase HPLC gives a single peak of bioactivity in each case. 7. The biological activity of CAF 1 is destroyed by α-chymotrypsin, but not by trypsin. CAF 2 is not attacked by trypsin or by α-chymotrypsin. Treatment with performic acid or cyanogen bromide destroys the activity of both CAF 1 and CAF 2.

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