Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5′-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.

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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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Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

Biochemical Journal ◽

10.1042/bj2880475 ◽

1992 ◽

Vol 288 (2) ◽

pp. 475-482 ◽

Cited By ~ 30

Author(s):

I Ishii-Karakasa ◽

H Iwase ◽

K Hotta ◽

Y Tanaka ◽

S Omura

Keyword(s):

Enzyme Activity ◽

Culture Medium ◽

Gel Filtration ◽

Partial Purification ◽

Gastric Mucus ◽

Purification And Characterization ◽

Streptomyces Sp ◽

Optimum Ph ◽

New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

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Purification and Characterization of Endoglucanase from local isolate of Aspergillus flavus

Baghdad Science Journal ◽

10.21123/bsj.10.3.844-853 ◽

2013 ◽

Vol 10 (3) ◽

pp. 844-853

Author(s):

Baghdad Science Journal

Keyword(s):

Aspergillus Flavus ◽

Specific Activity ◽

Gel Filtration ◽

Temperature Stability ◽

Exchange Chromatography ◽

Purification And Characterization ◽

Original Activity ◽

A Value ◽

Optimum Ph

Endoglucanase produced from Aspergillus flavus was purified by several steps including precipitation with 25 % ammonium sulphate followed by Ion –exchange chromatography, the obtained specific activity was 377.35 U/ mg protein, with a yield of 51.32 % .This step was followed by gel filtration chromatography (Sepharose -6B), when a value of specific activity was 400 U/ mg protein, with a yield of 48 %. Certain properties of this purified enzyme were investigated, the optimum pH of activity was 7 and the pH of its stability was 4.5, while the temperature stability was 40 °C for 60 min. The enzyme retained 100% of its original activity after incubation at 40 °C for 60 min; the optimum temperature for enzyme activity was 40 °C.

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Optimization of Growth Conditions for Purification and Production of L-Asparaginase bySpirulina maxima

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/1785938 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 12

Author(s):

Hanaa H. Abd El Baky ◽

Gamal S. El Baroty

Keyword(s):

Enzyme Activity ◽

Gel Filtration ◽

Growth Conditions ◽

Partial Purification ◽

Specific Enzyme ◽

Purification And Characterization ◽

Spirulina Maxima ◽

Optimum Ph ◽

Biomass Yields

L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA fromSpirulina maxima(SM) were tested. SM cultures grown in Zarrouk medium containing different N2(in NaNO3form) concentrations (1.25, 2.50, and 5.0 g/L) for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2concentration. The cultures of SM at large scales (300 L medium, 5 g/L N2) showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production inS. maxima.

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Partial Purification and Characterization of Inulinase Produced from a local isolate of Aspergillus niger J3

Journal of Biotechnology Research Center ◽

10.24126/jobrc.2010.4.2.129 ◽

2010 ◽

Vol 4 (2) ◽

pp. 78-85

Author(s):

Jasim M. Awdaa

Keyword(s):

Aspergillus Niger ◽

Gel Filtration ◽

Partial Purification ◽

Mercury Chloride ◽

Purification And Characterization ◽

Original Activity ◽

Local Isolate ◽

Optimum Ph ◽

Final Purification

Inulinase was produced from local isolate of Aspergillus niger J3. The inulinase was purified by two steps included precipitation by amonium sulphate at (30-80) % sa-turation and gel filtration on sephadex G100. The final purification folds and the yield of the enzyme were 3.15 times and 28.24%, respectively. The purified enzyme has the following characteristics: The optimum pH of the enzyme activity was 5.5. The enzyme was most stable at pH (4.5 - 6). The optimum temperature for its activity was 45c. The enzyme retained its original activity when incubates at (30-55) c for 20 minutes. Mercury chloride inhibited the enzyme completely at concentration of 10mM, cupper sulphate and calisium chloride inhibited the enzyme at concentrations of 85% and 7% respectively. It was revealed that the enzyme had the efficiency to hydrolyze 87% of 5% inulin solution when treated at 45c for 120 min.

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Purification and characterization of versatile peroxidase from Lentinus squarrosulus and its application in biodegradation of lignocellulosics

Journal of Applied Biotechnology & Bioengineering ◽

10.15406/jabb.2019.06.00205 ◽

2019 ◽

Vol 6 (6) ◽

pp. 280-286

Author(s):

Aarthi Ravichandran ◽

Ramya G Rao ◽

Maheswarappa Gopinath ◽

Manpal Sridhar

Keyword(s):

Finger Millet ◽

Nutritive Value ◽

Crop Residues ◽

Specific Activity ◽

Gel Filtration ◽

Versatile Peroxidase ◽

Purification And Characterization ◽

Optimum Ph ◽

Km Value

Versatile Peroxidases are high redox potential peroxidases capable of degrading lignin of lignocellulosic crop residues. Hence Versatile Peroxidases are prominent biocatalysts in upgrading lignocellulosic biomass for biotechnological applications. In the interest of exploiting the potential of Versatile Peroxidase in improving the digestibility of crop residues through delignification, a novel Versatile Peroxidase was purified and characterized from the immobilized cultures of native isolate Lentinus squarrosulus. The enzyme was purified with a specific activity of 62 U/mg through ion exchange and gel filtration chromatographic procedures. The enzyme possessed high affinity towards RB5 and manganese with a Km value of 6.84 µM for RB5 and 0.15 mM for manganese. The optimum temperature for oxidation was identified to be 30°C and optimum pH for manganese and RB5 oxidation was 5 and 3 respectively. Reactivity of the enzyme towards diverse substrates was investigated besides studying the effect of metal ions and inhibitors on RB5 oxidation. The enhanced potential of this purified Versatile Peroxidase in biodegradation of crop residues was demonstrated through augmentation of digestibility of finger millet and paddy straws by 20%.The results demonstrated that Versatile Peroxidase from Lentinus squarrosulus is capable of enhancing the nutritive value of crop residues through delignification

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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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Partial purification and characterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen

Biochemical Journal ◽

10.1042/bj2370439 ◽

1986 ◽

Vol 237 (2) ◽

pp. 439-445 ◽

Cited By ~ 16

Author(s):

J Gomez-Cambronero ◽

S Velasco ◽

M Sanchez-Crespo ◽

F Vivanco ◽

J M Mato

Keyword(s):

Specific Activity ◽

Partial Purification ◽

Purification And Characterization ◽

Acetyl Coa ◽

Ph Optimum

The enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) was purified from rat spleen approx. 1500-fold in 1.6% yield. The specific activity of the purified enzyme was 0.317 +/- 0.089 mumol/min per mg of protein (mean +/- S.D., n = 6). The Km for the substrate acetyl-CoA was 137 +/- 13 microM and the pH optimum was about 8. Incubation of the purified enzyme was 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine followed by electrophoresis resulted in the incorporation of radioactivity into a protein of Mr 29,000. The enzyme was most active towards 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine as substrate, 1-palmitoyl-2-lyso-glycero-3-phosphocholine being a poor substrate. In addition, the enzyme preferred acetyl-CoA to palmitoyl-CoA or oleoyl-CoA as substrate.

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Partial purification and characterization of polyphenol oxidase from sugarcane (Saccharum officinarum L.)

Sugar Industry ◽

10.36961/si11437 ◽

2011 ◽

pp. 296-301 ◽

Cited By ~ 4

Author(s):

Zhengang Zhao ◽

Licai Zhu ◽

Shujuan Yu ◽

Michael Saska

Keyword(s):

Ascorbic Acid ◽

Chlorogenic Acid ◽

Polyphenol Oxidase ◽

Ph Value ◽

Saccharum Officinarum ◽

Partial Purification ◽

Optimum Conditions ◽

Purification And Characterization ◽

Triton X

Polyphenol oxidase (PPO) of sugarcane was extracted by using 0.02 mol/L phosphate buffer at pH = 6.8 containing 1.5% polyvinylpolypyrrolidone and 0.5% Triton X-100, and then partially purified by 80% ammonium sulfate fractionation, dialysis, and column chromatography on DEAE-Toyopearl 650M, Sephadex G-100. PPO activity was purified 37.6-fold with a recovery of 18.4%. The PPO showed activity to catechol, chlorogenic acid, 4-methylcatechol, caffeic acid and ferulic acid, but not to l-tyrosine. Optimum conditions (pH value and temperature) for PPO were determined using the five substances. PPO activity is quite thermostable between 20 and 30 °C. After heating for 10 min at 80 °C 90% of the activity is lost. Km and Vmax values of PPO were calculated for each substrate and the best substrate of PPO was chlorogenic acid. PPO was markedly inhibited by metal ions (Cu2+, Al3+, and Mg2+) at 1 and 10 mmol/L, and strongly inhibited by NaHSO3 and ascorbic acid at 1 mmol/L.

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Purifikasi dan Karakterisasi α-amilase Termostabil dari Bacillus stearothermophilus TII-12

Jurnal AgroBiogen ◽

10.21082/jbio.v7n1.2011.p56-62 ◽

2011 ◽

Vol 7 (1) ◽

pp. 56 ◽

Cited By ~ 1

Author(s):

Puji Lestari ◽

Nur Richana ◽

Abdul Aziz Darwis ◽

Khaswar Syamsu ◽

Untung Murdiyatmo

Keyword(s):

Bacillus Stearothermophilus ◽

Specific Activity ◽

Gel Filtration ◽

Total Activity ◽

Purification And Characterization ◽

Food Industries ◽

High Temperature Process ◽

Hydrolysis Products ◽

Starch Liquefaction

Purification and Characterization of Thermostable α-amylase from Bacillus stearothermophilus TII-12. Puji Lestari, Nur Richana, Abdul A. Darwis, Khaswar Syamsu, and Untung Murdiyatmo. Thermostable α-amylase is a potential enzyme employed in the starch processing and widely used in food industries, but this enzyme is still imported. The local enzyme production would be more economist and useful for its broad applications. Here we report α-amylase from indigenous bacteria TII-12 which was purified and characterized, as well as analyzed its hydrolysis product on cassava starch. The enzyme of Bacillus stearothermophilus TII-12 partially purified by ultrafiltration, acetone precipitation and gel filtration (Sephadex G-100) showed the reduced total activity, total protein and yield, but increased the specific activity. The enzyme had a Km of 1,06 mg/ml and Vmax of 1,21 mol/min, with optimal activity at pH 7 and 90oC. An apparent molecular mass was of 192.932,8 Dalton, as estimated by Native-Polyacrylamide Agarose Gel electrophoresis. Its activity was inhibited by the divalent cation chelator such as EDTA and CuSO4 but activated by calcium ion. Hydrolysis products of this enzyme on cassava starch were glucose, dextrin, maltose and oligosaccharides. After 24 hours of hydrolysis, the concentration of glucose and maltose reached 51.970 and 10.090 ppm, respectively. The thermostable α-amylase of TII-12 is an endo-α-amylase and prospective to be applied on starch liquefaction with high temperature process.

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