Purification and characterization of rat intestinal peroxidase. Its activity towards 2-t-butyl-4-methoxyphenol (BHA)

Impure preparations of rat intestinal peroxidase were shown to aggregate at low ionic strengths and to disaggregate at higher values. This aggregation was accompanied by a decrease in specific activity, which could lead to hysteretic behaviour of reaction progress curves. Advantage was taken of this reversible aggregation to obtain a relatively pure extract, which was subsequently purified to apparent homogeneity by affinity chromatography on concanavalin A-Sepharose followed by hydrophobic chromatography. The purified enzyme did not show the ionic-strength-dependent aggregation behaviour, behaving as a monomer of Mr 50,000. The purified enzyme was shown to catalyse the peroxidatic conversion of the commonly used antioxidant 2-t-butyl-4-methoxyphenol (butylated hydroxyanisole, BHA) to form 3,3′-di-t-butyl-2,2′-dihydroxy-5,5′-dimethoxybiphenyl, with a Km value of 176 microM and a maximum velocity of 8 mumol/min per mg. The specificity constant, kcat./Km, for this substrate was similar to that shown towards the substrate guaiacol.

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The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

Biochemical Journal ◽

10.1042/bj2370415 ◽

1986 ◽

Vol 237 (2) ◽

pp. 415-420 ◽

Cited By ~ 40

Author(s):

C R Goward ◽

R Hartwell ◽

T Atkinson ◽

M D Scawen

Keyword(s):

Large Scale ◽

Bacillus Stearothermophilus ◽

Specific Activity ◽

Gel Filtration ◽

Exchange Chromatography ◽

Purification And Characterization ◽

Km Value ◽

Extraneous Material ◽

Sepharose 4B

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.

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Purification and Characterization of a Polyextremophilicα-Amylase from an Obligate HalophilicAspergillus penicillioidesIsolate and Its Potential for Souse with Detergents

BioMed Research International ◽

10.1155/2015/245649 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 13

Author(s):

Imran Ali ◽

Ali Akbar ◽

Mohammad Anwar ◽

Sehanat Prasongsuk ◽

Pongtharin Lotrakul ◽

...

Keyword(s):

Specific Activity ◽

Amylase Activity ◽

Gel Filtration ◽

Soluble Starch ◽

Ammonium Sulfate Precipitation ◽

Purification And Characterization ◽

Apparent Homogeneity ◽

Sds Page ◽

Good Potential

An extracellularα-amylase from the obligate halophilicAspergillus penicillioidesTISTR3639 strain was produced and enriched to apparent homogeneity by ammonium sulfate precipitation and Sephadex G100 gel filtration column chromatography. The mass of the purified amylase was estimated to be 42 kDa by SDS-PAGE. With soluble starch as the substrate it had a specific activity of 118.42 U·mg−1andVmax⁡andKmvalues of 1.05 µmol·min−1·mg−1and 5.41 mg·mL−1, respectively. The enzyme was found to have certain polyextremophilic characteristics, with an optimum activity at pH 9, 80°C, and 300 g·L−1NaCl. The addition of CaCl2at 2 mM was found to slightly enhance the amylase activity, while ZnCl2, FeCl2, or EDTA at 2 mM was strongly or moderately inhibitory, respectively, suggesting the requirement for a (non-Fe2+or Zn2+) divalent cation. The enzyme retained more than 80% of its activity when incubated with three different laundry detergents and had a better performance compared to a commercial amylase and three detergents in the presence of increasing NaCl concentrations up to 300 g·L−1. Accordingly, it has a good potential for use as anα-amylase in a low water activity (high salt concentration) and at high pH and temperatures.

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Partial Purification and Characterization of Catalase from Banana Peels

Baghdad Science Journal ◽

10.21123/bsj.13.2.392-398 ◽

2016 ◽

Vol 13 (2) ◽

pp. 392-398

Author(s):

Baghdad Science Journal

Keyword(s):

Gel Filtration ◽

Maximum Velocity ◽

Kinetic Studies ◽

Maximal Activity ◽

Partial Purification ◽

Purification And Characterization ◽

Km Value ◽

Almost All ◽

Kinetics Of

Catalase (EC 1.11.1.6) is a well known enzyme which exists in almost all living creatures exposing to oxygen (such as plants, bacteria, and animals). It is a very necessary enzyme to protect the cell from oxidative detriment by reactive oxygen species (ROS). The aim of this study is the partial purification and characterization of Catalase enzyme from Banana peels. In this study, fresh banana peels are treated with 70 % ethanol ,further separated with chloroform ,water and ethyl acetate respectively .The supernatant of the enzymatic sample which is treated with chloroform is loaded into gel filtration column with Sephadex G-100 (1.0 x 90 cm) equilibrated with pH7 buffer media (phosphate buffer 0.1 M). Kinetic studies of the purified enzyme activity are measured and characterized .The maximal activity (26.04 units/mg) of catalase is observed with chloroform buffer extraction. The kinetics of catalase; Michalis constant Km and maximum velocity Vmax is determined using Linweaver- Burk plot, The Km value for catalase (434.7mM), Vmax (100 m mole min -1). Characterization results demonstrate that the optimal pH for activity is (7.6). And the optimal temperature for activity is 30?C .The present study indicates that Banana peels is a good source of catalase enzyme.

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(335) Purification and Characterization of ADP-glucose Pyrophosphorylase from Apple Leaves

HortScience ◽

10.21273/hortsci.40.4.1083c ◽

2005 ◽

Vol 40 (4) ◽

pp. 1083C-1083

Author(s):

Rui Zhou ◽

Lailiang Cheng

Keyword(s):

Enzyme Activity ◽

Thermal Inactivation ◽

Specific Activity ◽

Purification And Characterization ◽

Apple Leaves ◽

Apparent Homogeneity ◽

Adp Glucose Pyrophosphorylase ◽

High Concentrations ◽

Dual Functions

Apple leaf ADP-glucose pyrophosphorylase was purified over 1400-fold to apparent homogeneity with a specific activity of 58.9 units per mg of protein. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolysis 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 of 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 monomerization of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerization of the small subunits of the enzyme induced by DTT. These findings indicate that the binding of PGA may have dual functions in regulating apple leaf AGPase activity—activating the enzyme and rendering the enzyme with a conformation more stable to thermal inactivation.

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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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Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides

Biochemical Journal ◽

10.1042/bj3140321 ◽

1996 ◽

Vol 314 (1) ◽

pp. 321-326 ◽

Cited By ~ 9

Author(s):

Kai-Fai LEE ◽

Yen-Chywan LIAW ◽

Pang-Chui SHAW

Keyword(s):

Initial Rate ◽

Specific Activity ◽

Overlapping Genes ◽

Purification And Characterization ◽

Native Molecular Mass ◽

Apparent Homogeneity ◽

Molecular Masses ◽

Chromatographic Media ◽

Lambda Dna

The two overlapping genes coding for EcoHK31I methyltransferase have previously been cloned, sequenced and expressed [Lee, Kam and Shaw (1995) Nucleic Acids Res. 23, 103–108]. Here we describe protocols developed to purify polypeptides α and β together or separately, to apparent homogeneity by various chromatographic media. M.EcoHK31I is a heterodimer with a native molecular mass of 61 kDa. Its specific activity towards non-methylated lambda DNA was 3.0×105 units per mg of protein. The respective denatured molecular masses of polypeptides α and β were 38 and 23 kDa, and their pI values were 8.7 and 6.8. Initial rate kinetic parameters of the native enzyme were 2.0 nM, 0.58 μM and 3 min-1 for KmDNA, KmAdoMet and kcat. respectively, where AdoMet stands for S-adenosyl-L-methionine. Fully active enzyme was reconstituted by co-purifying the two separately synthesized polypeptides, and activity assays confirmed our previous finding that two polypeptides were needed to methylate substrate DNA.

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Purification and Characterization of a Carbonyl Reductase from Human Liver, which is Competent in the Reduction of 6-Pyruvoyl-Tetrahydropterin

Pteridines ◽

10.1515/pteridines.1989.1.4.189 ◽

1989 ◽

Vol 1 (4) ◽

pp. 189-198 ◽

Cited By ~ 1

Author(s):

Petra Steinerstauch ◽

Yoshitomo Sawada ◽

Walter Leimbacher ◽

Sandro Ghisla ◽

Hans-Christoph Curtius

Keyword(s):

Human Liver ◽

Specific Activity ◽

Polyclonal Antibodies ◽

Potassium Phosphate ◽

Side Chain ◽

Purification And Characterization ◽

Single Polypeptide Chain ◽

Apparent Homogeneity ◽

Single Polypeptide

Summary An enzyme which reduces 6-pyruvoyl-tetrahydropterin has been purified to apparent homogeneity from human liver. It consists of a single polypeptide chain with a molecular weight of 35 kDa, has an isoelectric point of 5.9 ± 0.1 and contains no glycosyl residues. The pure enzyme has a specific activity of 450 mU/mg protein at pH 7.0 in 10 mM potassium phosphate buffer. It converts 6-pyruvoyl-tetrahydropterin to 6-lactoyltetrahydropterin by transfer of the pro 4R-hydrogen of NADPH to form the side chain -OH at position C(2') of the substrate. Km values are 1.8 J..lM for 6-pyruvoyl-tetrahydropterin and 5.5 J..lM for NADPH. Polyclonal antibodies raised against the purified enzyme recognize 6-pyruvoyl-tetrahydropterin reductase in Western blot and ELISA but do not cross-react with human sepiapterin reductase. The enzyme appears to be identical with aldose reductase.

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Occurrence of fatty acid epoxide hydrolases in soybean (Glycine max). Purification and characterization of the soluble form

Biochemical Journal ◽

10.1042/bj2820711 ◽

1992 ◽

Vol 282 (3) ◽

pp. 711-714 ◽

Cited By ~ 49

Author(s):

E Blée ◽

F Schuber

Keyword(s):

Glycine Max ◽

Gel Filtration ◽

Soluble Form ◽

Soluble Enzyme ◽

Purification And Characterization ◽

Epoxide Hydrolases ◽

Major Activity ◽

Apparent Homogeneity ◽

A Minor

Epoxide hydrolases catalysing the hydration of cis-9,10-epoxystearate into threo-9,10-dihydroxystearate have been detected in soybean (Glycine max) seedlings. The major activity was found in the cytosol, a minor fraction being strongly associated with microsomes. The soluble enzyme, which was purified to apparent homogeneity by (NH4)2SO4 fractionation, hydrophobic, DEAE- and gel-filtration chromatographies, has a molecular mass of 64 kDa and a pI of 5.4.

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Purification and Characterization of Asparaginase fromPhaseolus vulgarisSeeds

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2015/309214 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 2

Author(s):

Saleh A. Mohamed ◽

Mohamed F. Elshal ◽

Taha A. Kumosani ◽

Alia M. Aldahlawi

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Lymphoblastic Leukemia ◽

Potential Candidate ◽

Purification And Characterization ◽

Apparent Homogeneity ◽

Wide Range ◽

Optimum Ph ◽

Glutaminase Activity ◽

Low Activity

L-asparaginase from bacteria has been used in treatment of acute lymphoblastic leukemia. The aim of this study was to purify and characterize L-asparaginase fromPhaseolus vulgarisseeds instead of microbial sources. L-asparaginase was purified to apparent homogeneity. The enzyme has molecular mass of 79 kDa. The purified asparaginase had very low activity toward a number of asparagine and glutamine analogues. L-asparaginase was free from glutaminase activity. Kinetic parameters, Km andVmax of purified enzyme, were found to be 6.72 mM and 0.16 μM, respectively. The enzyme had optimum pH at 8.0. The enzyme showed high stability at alkaline pH (pH 7.5–9.0) when incubated for up to 24 h. L-asparaginase had the same temperature optimum and thermal stability at 37°C. K+was able to greatly enhance the activity of asparaginase by 150% compared with other metals tested. In conclusion, L-asparaginase showed no glutaminase activity and good stability over a wide range of physiological conditions, and thus it could be used as a potential candidate for treatment of acute lymphoblastic leukemia.

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Purification and characterization of two isoforms of native α amylase from Ok-Rong mango (Mangifera indica Linn. cv. Ok-Rong)

Turkish Journal of Biochemistry ◽

10.1515/tjb-2016-0218 ◽

2017 ◽

Vol 42 (6) ◽

Author(s):

Raksmont Ubonbal ◽

Saijai Porsoongnoen ◽

Jureerut Daduang ◽

Sompong Klaynongsruang ◽

Sakda Daduang

Keyword(s):

High Temperatures ◽

Fruit Ripening ◽

Specific Activity ◽

Mangifera Indica ◽

Ripening Stage ◽

Purification And Characterization ◽

Tropical Plant ◽

Short Period ◽

Structure Properties

AbstractIntroduction:The tropical plant amylases involved in the fruit ripening stage is outstanding for their high activities in converting starch to sugars within a short period at high temperatures over 40°C.Methods:The α amylase iso-enzymes from Ok-Rong mango (Results:The enzyme was purified 105-fold with a final specific activity of 59.27 U mgConclusion:Two α amylase iso-enzymes were classified as members of the low-pI group of amylases with identical structure, properties and functions. They are mesophilic with high possibilities for application for many purposes.

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