First cloning and characterization of aspartate aminotransferase from river buffalo (Bubalus bubalis)

Biologia ◽  
2011 ◽  
Vol 66 (6) ◽  
Author(s):  
Muhammad Shahid Nadeem ◽  
Naeem Rashid ◽  
Muzaffar Iqbal ◽  
Qurra-tul-Ann Afza Gardner ◽  
Muhammad Akhtar
Keyword(s):  
Aspartate Aminotransferase ◽  
Pyridoxal Phosphate ◽  
Specific Activity ◽  
Active Form ◽  
Bubalus Bubalis ◽  
Recombinant Enzyme ◽  
Ionization Mass ◽  
River Buffalo ◽  
Apparent Homogeneity

AbstractAspartate aminotransferase catalyzes the transfer of an amino group from L-aspartate to α-oxoglutarate. We have purified cytosolic isozyme, to apparent homogeneity, from the heart of river buffalo. In order to clone the enzyme total RNA was isolated and the cDNA encoding complete polypeptide of 413 amino acids was amplified by reverse transcriptase polymerase chain reaction. The cDNA and the deduced amino acid sequence exhibited 98.2% and 99.5% identities, respectively, with that of cow. The cDNA was overexpressed in Escherichia coli and the gene product was obtained in enzymologically active form. The recombinant enzyme was about 40% of the total cell proteins. The purified recombinant enzyme displayed specific activity, Km, temperature and pH stability values similar to that of the native enzyme. Edman degradation and electrospray ionization mass spectra showed that the recombinant enzyme consisted of three species having N-terminal sequences of MAPPSIF, APPSIF and PPSIF, although the second one was the predominant species. Conditions are also described, which in the mass spectral analysis give either the three species corresponding to the apo- or the holo-enzyme; the latter retaining pyridoxal phosphate bound to the protein. To our knowledge this is the first report on sequence determination, cloning and characterization of aspartate aminotransferase from river buffalo.

scholarly journals Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Dominic W. S. Wong ◽  
Victor J. Chan ◽  
Amanda A. McCormack ◽  
Ján Hirsch ◽  
Peter Biely
Keyword(s):  
Glucuronic Acid ◽  
Active Form ◽  
Schizophyllum Commune ◽  
Constitutive Expression ◽  
Recombinant Enzyme ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Glucuronoyl Esterase ◽  
Esterase Gene

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were Km 0.25 mM, Vmax 16.3 μM⋅min−1, and kcat 9.27 s−1 with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.

Characterization of the luteinizing hormone beta (LH-β) subunit gene in the Indian river buffalo (Bubalus bubalis)

2008 ◽  
Vol 155 (1) ◽  
pp. 63-69 ◽  
Author(s):  
Mallikarjuna Shivapura Basavarajappa ◽  
Sachinandan De ◽  
Manish Thakur ◽  
Tirtha Kumar Datta ◽  
Gaurav Dogra ◽  
...  
Keyword(s):  
Luteinizing Hormone ◽  
Bubalus Bubalis ◽  
River Buffalo ◽  
Β Subunit ◽  
Subunit Gene ◽  
Indian River

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

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
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.

scholarly journals Resolution and characterization of the glycine-cleavage reaction in pea leaf mitochondria. Properties of the forward reaction catalysed by glycine decarboxylase and serine hydroxymethyltransferase

1988 ◽  
Vol 255 (1) ◽  
pp. 169-178 ◽  
Author(s):  
J Bourguignon ◽  
M Neuburger ◽  
R Douce
Keyword(s):  
Pyridoxal Phosphate ◽  
Gel Filtration ◽  
Serine Hydroxymethyltransferase ◽  
Molar Ratios ◽  
Apparent Homogeneity ◽  
The Matrix ◽  
Lipoamide Dehydrogenase ◽  
Glycine Cleavage ◽  
Low Ionic Strength

High-molecular-mass proteins from pea (Pisum sativum) mitochondrial matrix retained on an XM-300 Diaflo membrane (‘matrix extract’) exhibited high rates of glycine oxidation in the presence of NAD+ and tetrahydropteroyl-L-glutamic acid (H4 folate) as long as the medium exhibited a low ionic strength. Serine hydroxymethyltransferase (SHMT) (4 x 53 kDa) and the four proteins of the glycine-cleavage system, including a pyridoxal phosphate-containing enzyme (‘P-protein’ 2 x 97 kDa), a carrier protein containing covalently bound lipoic acid (‘H-protein’ 15.5 kDa), a protein exhibiting lipoamide dehydrogenase activity (‘L-protein’; 2 x 61 kDa) and an H4 folate-dependent enzyme (‘T-protein’ 45 kDa) have been purified to apparent homogeneity from the matrix extract by using gel filtration, ion-exchange and phenyl-Superose fast protein liquid chromatography. Gel filtration on Sephacryl S-300 in the presence of 50 mM-KCl proved to be the key step in disrupting this complex. During the course of glycine oxidation catalysed by the matrix extract a steady-state equilibrium in the production and utilization of 5,10-methylene-H4 folate was reached, suggesting that glycine cleavage and SHMT are linked together via a soluble pool of H4 folate. The rate of glycine oxidation catalysed by the matrix extract was sensitive to the NADH/NAD+ molar ratios, because NADH competitively inhibited the reaction catalysed by lipoamide dehydrogenase.

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

HortScience ◽  
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.

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

1988 ◽  
Vol 250 (2) ◽  
pp. 501-507 ◽  
Author(s):  
M Valoti ◽  
L Della Corte ◽  
K F Tipton ◽  
G Sgaragli
Keyword(s):  
Specific Activity ◽  
Maximum Velocity ◽  
Purification And Characterization ◽  
Reaction Progress ◽  
Reversible Aggregation ◽  
Aggregation Behaviour ◽  
Apparent Homogeneity ◽  
Specificity Constant ◽  
Km Value

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.

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

1996 ◽  
Vol 314 (1) ◽  
pp. 321-326 ◽  
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.

scholarly journals Partial purification of collagenase and gelatinase from human polymorphonuclear leucocytes. Analysis of their actions on soluble and insoluble collagens

1982 ◽  
Vol 203 (1) ◽  
pp. 209-221 ◽  
Author(s):  
G Murphy ◽  
J J Reynolds ◽  
U Bretz ◽  
M Baggiolini
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Polymorphonuclear Leucocyte ◽  
Partial Purification ◽  
Type I ◽  
Polymorphonuclear Leucocytes ◽  
Gelatinase Activity ◽  
Apparent Homogeneity

The separation and further purification of human polymorphonuclear-leucocyte collagenase and gelatinase, using modifications of the method of Cawston & Tyler [(1979) Biochem J. 183, 647-656], are described. The final preparations yielded collagenase of specific activity 260 units/mg and gelatinase of specific activity 13 000 units/mg. Gelatinase was purified to apparent homogeneity in a latent form, and analysis of the activation of 125I-labelled latent enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel-filtration techniques suggested that no peptide material was lost on conversion into the active form. The purified natural inhibitors alpha 2-macroglobulin, tissue inhibitor of metalloproteinases (‘TIMP’) and amniotic-fluid inhibitor of metalloproteinases all inhibited the two polymorphonuclear-leucocyte metalloproteinases, but the last two inhibitors were slow to act and complete inhibition was difficult to attain. Collagenase degraded soluble types I and III collagen equally efficiently, but soluble type II collagen less well. Gelatinase alone had little activity on these substrates, although it enhanced the action of collagenase. Gelatinase was capable of degrading soluble types IV and V collagen at 25 degrees C, whereas collagenase was only active at higher temperatures when the collagens were susceptible to trypsin activity. By using tissue preparations of insoluble collagens (type I, II or IV) the activity of leucocyte collagenase was low and gelatinase activity was negligible, as measured by the solubilization of hydroxyproline-containing material. The two enzymes together were two or three times more effective in the degradation of these insoluble collagens.

Expression of Gene for β-glucanase from Paenibacillus jamilae Bg1 in Pichia pastoris and Characteristics of Recombinant Enzyme

2019 ◽  
Vol 35 (4) ◽  
pp. 15-23 ◽  
Author(s):  
T.L. Gordeeva ◽  
A.N. Kalinina ◽  
A.V. Serkina ◽  
A.S. Fedorov ◽  
S.P. Sineoky
Keyword(s):  
Pichia Pastoris ◽  
Specific Activity ◽  
Recombinant Enzyme ◽  
Gene Encoding ◽  
Resource Center ◽  
Putative Signal Peptide ◽  
The Russian Federation ◽  
Paenibacillus Macerans ◽  
Positive Effect

The isolation, heterologous expression and characterization of a new thermostable β-glucanase from Paenibacillus jamilae is described. The bgl26 gene from the P. jamilae Bg1 VKPM B-13093 strain consisting of 714 nucleotides encodes endo-1,3-1,4-β-glucanase (EC 3.2.1.73) containing 213 amino acids and 24 residues of the putative signal peptide in N-end area. The nucleotide sequence of the bgl26 gene and the amino acid sequence of the mature Bgl26 protein have the greatest homology with the sequence of the Paenibacillus macerans endo-l,3-l,4-β-glucanase (82 and 88%, respectively). A fragment of the gene encoding the mature protein was expressed in Pichia pastoris. Purified recombinant enzyme Bgl26 was active towards barley β-glucan. The optimal pH for the enzyme to work was 7,0, and the optimum temperature range was 40-45 °C. The specific activity of β-glucanase was at the level of 6650 U/mg of protein, Km and Vmax were equal to 6.4 ± 0.3 mg/mL and 9450.1 ± 471.2 umol/(min-mg), respectively. The recombinant protein Bgl26 was characterized by high pH and thermal stability, as well as resistance to digestive enzymes. It is also shown that Co2+ ions have a positive effect on the activity of the enzyme. β-glucanase, β-glucan, Paenibacillus jamilae, Pichia pastoris The work was financially supported by the Ministry of Science and Higher Education of the Russian Federation (Unique Project Identifier RFMEFI60717X0179) and was carried out using the Multipurpose Scientific Installation of National Bio-Resource Center «All-Russian Collection of Industrial Microorganisms», NRC «Kurchatov Institute» - GOSNIIGENETIKA.

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