scholarly journals Arthrobacter d-xylose isomerase: chemical modification of carboxy groups and protein engineering of pH optimum

1993 ◽  
Vol 296 (3) ◽  
pp. 685-691 ◽  
Author(s):  
K S Siddiqui ◽  
T Loviny-Anderton ◽  
M Rangarajan ◽  
B S Hartley
Keyword(s):  
Enzyme Activity ◽  
Protein Engineering ◽  
Coupling Reaction ◽  
Xylose Isomerase ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Native Enzyme ◽  
Ph Optimum ◽  
Isomerase Activity

To try to lower the pH optimum, the carboxy groups of Arthrobacter D-xylose isomerase were coupled to glycinamide using a water-soluble carbodi-imide. In conditions that substituted all of the 59 carboxy groups in the denatured monomer, a maximum of 30 groups/monomer reacted in the native enzyme, whether in presence or absence of ligands, and the enzyme remained fully active and tetrameric throughout the coupling reaction. Purification by f.p.l.c. ion-exchange chromatography gave broad symmetrical peaks with increased pI, suggesting that the modified enzymes are essentially homogeneous. However, they are less stable than native enzyme in 8 M urea or on heating (‘melting points’ of 59 degrees versus 73 degrees C for the apoenzymes and 67 degrees versus 81.5 degrees C for the Mg(2+)-enzymes). Kinetic studies of the D-fructose isomerase activity at 30 degrees C showed that the glycinamidylated enzyme had unaltered activation constant for Mg2+, and Km was also similar to that of the native enzyme at pH 7.3, but increased rapidly at higher pH rather than remaining constant. Vmax. was constant from pH 6.2 to 8.0, suggesting a reduced pKa for His-219, which controls Vmax. in the native enzyme (normally 6.0). Three mutants were constructed by protein engineering with a view to reducing the pH optimum of enzyme activity. Two of these, Glu140→Lys and Asp189→Lys, could be detected in crude extracts of Escherichia coli by SDS/PAGE, but could not be purified, whereas mutant Trp136→Glu was produced as a tetramer in amounts similar to the wild-type enzyme. However, it did not show any enzyme activity and was less stable in 0-9 M urea gradient PAGE.

Soybean seed pectinesterase

1998 ◽  
Vol 8 (4) ◽  
pp. 455-461 ◽  
Author(s):  
Oskar Markovič ◽  
Ralph L. Obendorf
Keyword(s):  
Methyl Esters ◽  
Simultaneous Detection ◽  
Gel Filtration ◽  
Soybean Seed ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Soluble Form ◽  
Ph Optimum ◽  
Ultrathin Layers ◽  
Ultra Filtration

AbstractMethanol accumulates in axis tissues of maturing soybean seeds, correlating with preharvest seed deterioration. Accumulation of methanol appears to be associated with the enzymic demethylation of pectin methyl esters by pectinesterase (PE; EC 3.1.1.11). To characterize PE in developing and maturing soybean (Glycine max (L.) Merrill) seeds, enzyme activity was assayed in axis and cotyledon tissues. Activity per g fresh weight was 20–25 times higher in axes than in cotyledons with highest activities between 45 and 60 days after flowering (DAF). Twenty to 33% of the total PE activity was in the ‘soluble’ form (extracted with water, 0.5 M sucrose, 1 M sucrose and water). Soluble and cell-wall-bound PE (subsequently extracted with 1 M NaCI) were purified and characterized from axes of seeds at 45–60 DAF. Purification of PE was achieved through concentration of extracts by ultra-filtration, precipitation with ammonium sulfate (30–80% saturation), dialysis, gel filtration on Sephadex G-75 columns, and ion exchange chromatography on CM Sepharose CL-6B. Further purification of both soluble and bound PE was by isoelectric focusing (IEF) on ultrathin layers of polyacrylamide gel with simultaneous detection of protein and PE activity. It was possible to follow seven bands exhibiting PE activity with pl values between 6.0 and 9.5 in 1 M NaCI-extracts of total homogenates. Differences in the IEF patterns of bound and soluble PE were observed. Whereas the bound enzyme exhibited more basic PE bands (pl 8–9.5), the soluble enzyme had more active bands at pl 6.5, 7.0 and 7.5. The Mr was close to 33 000 and the pH optimum was 7.8 for both soluble and bound PE.

scholarly journals 307 Characterization of Amylolytic Activities of Tulip Bulb Scales

HortScience ◽  
1999 ◽  
Vol 34 (3) ◽  
pp. 495D-495
Author(s):  
Anil P. Ranwala ◽  
William B. Miller
Keyword(s):  
Enzyme Activity ◽  
Amylase Activity ◽  
Gel Filtration ◽  
Soluble Starch ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Product Analysis ◽  
Ph Optimum ◽  
Tulip Bulb ◽  
Tulipa Gesneriana

Amylolytic activities extracted from scales of tulip (Tulipa gesneriana L. cv. Apeldoorn) bulbs stored at 4 °C for 6 weeks under moist conditions were characterized. Anion exchange chromatography of enzyme extract on DEAE-Sephacel revealed three peaks of amylolytic activity. Three enzymes showed different electrophoretic mobilties on nondenaturing polyacrylamide gels. The most abundant amylase activity was purified extensively with phenyl-agarose chromatography, gel filtration on Sephacryl S-200, and chromatofocusing on polybuffer exchanger PBE 94. The purified amylase was determined to be an endoamylase based on substrate specificity and end product analysis. The enzyme had a pH optimum of 6.0 and a temperature optimum of 55 °C when soluble starch was used as the substrate. The apparent Km value for soluble starch was 1.28 mg/ml. The inclusion of 2 mM CaCl2 in the reaction mixture resulted in a 1.4-fold increase in the enzyme activity. The presence of calcium ions also enhanced the thermo-stability of the enzyme at higher temperatures. The enzyme was able to hydrolyze soluble starch, amylose, amylopectin, and beta-limit dextrin, but it had no activity against pullulan, inulin, maltose, or p-nitrophenyl alpha-glucopyranoside. Only maltooligosaccharides, having a degree of polymerization of 7 or more, were hydrolyzed to a significant extent by the enzyme. Exhaustive hydrolysis of soluble starch with the enzyme yielded a mixture of maltose and matlooligosaccharides. This amylase activity was not inhibited by alpha- or beta-cyclodextrin upto a concentration of 10 mM. Maltose at a 50 mM concentration partially inhibited the enzyme activity, whereas glucose had no effect at that concentration.

Pyruvate carboxylase of Aspergillus niger: kinetic study of a biotin-containg carboxylase

1969 ◽  
Vol 47 (7) ◽  
pp. 697-710 ◽  
Author(s):  
Helen A. Feir ◽  
Isamu Suzuki
Keyword(s):  
Enzyme Activity ◽  
Aspergillus Niger ◽  
Pyruvate Carboxylase ◽  
Active Sites ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Ph Optimum ◽  
Metal Cofactor ◽  
Michaelis Constants

Pyruvate carboxylase was partially purified from Aspergillus niger and the properties were studied. The enzyme was found to be cold-labile and protected by 25% glycerol. The pH optimum was determined to be 7.9–8.0. The enzyme was shown to be a biotin-containing enzyme by its inactivation by avidin and protection against such inactivation by biotin. The enzyme activity was stimulated by K+ ions and inhibited by Na+ ions. Acetyl-CoA had no effect on enzyme activity, but L-aspartate was inhibitory. Apparent Michaelis constants were determined for the substrates and metal cofactor involved, i.e. pyruvate, ATP, bicarbonate, and Mg2+.Initial-velocity studies were carried out at varied concentrations of substrates in order to determine the true Michaelis constants and to elucidate the kinetic mechanism of reaction. Product-inhibition studies were carried out with each product (ADP, Pi, and oxalacetate) in combination with every substrate (ATP, bicarbonate, and pyruvate). From these kinetic studies and the existing knowledge on biotin-containing carboxylases, a mechanism was proposed for the action of pyruvate carboxylase which involves three independently active sites on the enzyme, one for each substrate. The interactions between the sites were visualized as being mediated by carboxybiotin formed on the enzyme. A steady-state rate equation was derived that satisfied kinetic results obtained.

Purification and characterization of tonin

1976 ◽  
Vol 54 (9) ◽  
pp. 788-795 ◽  
Author(s):  
S. Demassieux ◽  
R. Boucher ◽  
C. Grisé ◽  
J. Genest
Keyword(s):  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Potassium Phosphate ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Sedimentation Equilibrium ◽  
Angiotensin I ◽  
Ph Optimum ◽  
Ammonium Sulphate Precipitation

Tonin was purified from rat submaxillary glands by differential centrifugation, ammonium sulphate precipitation, gel filtration on Sephadex G150, and by ion-exchange chromatography on DEAE-cellulose, phospho-cellulose, SP-Sephadex C25, and SP-Sephadex C50. Purified tonin was shown to be homogeneous by analytical electrophoresis and by analytical ultracentrifugation analysis. Purified tonin was very stable when stored in buffers of low pH values or when incubated at high temperatures in neutral solutions. The molecular weight estimated by sedimentation equilibrium was 28 700. The pH optimum was near 6.8 in a 0.1 M potassium phosphate buffer. The Michaelis–Menten constant for tonin using angiotensin I as substrate was about 4 × 10−5 M. Tonin activity was strongly inhibited by plasma. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by plasma was of the non-competitive type.

Simultaneous Spectrophotometric Determination of Salbutamol by Coupling with Diazotized 4-Aminobenzoic acid

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Hind Hadi ◽  
Gufran Salim
Keyword(s):  
Pharmaceutical Preparations ◽  
Coupling Reaction ◽  
Dosage Forms ◽  
Water Soluble ◽  
Trace Determination ◽  
Aminobenzoic Acid ◽  
Optimum Conditions ◽  
Colored Product ◽  
Versus Concentration

A simple, rapid and sensitive spectrophotmetric method for trace determination of salbutamol (SAL) in aqueous solution and in pharmaceutical preparations is described. The method is based on the diazotization coupling reaction of the intended compound with 4-amino benzoic acid (ABA) in alkaline medium to form an intense orange, water soluble dye that is stable and shows maximum absorption at 410 nm. A graph of absorbance versus concentration indicates that Beer’s law is obeyed over the concentration range of 0.5-30 ppm, with a molar absorbtivity 3.76×104 L.mol-1 .cm-1 depending on the concentration of SAL. The optimum conditions and stability of the colored product have been investigated and the method was applied successfully to the determination of SAL in dosage forms.

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

1971 ◽  
Vol 17 (3) ◽  
pp. 183-191 ◽  
Author(s):  
Philip J Garry
Keyword(s):  
Enzyme Activity ◽  
Sodium Fluoride ◽  
Phosphate Buffer ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Serum Cholinesterase ◽  
Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

scholarly journals Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 325 ◽  
Author(s):  
He Chen ◽  
Jie Huang ◽  
Binyun Cao ◽  
Li Chen ◽  
Na Song ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Plantarum ◽  
Industrial Development ◽  
Extraction Efficiency ◽  
Specific Activity ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Kinetic Studies ◽  
Predicted Values ◽  
Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

scholarly journals Adsorption of heavy metals by road deposited solids

2013 ◽  
Vol 67 (11) ◽  
pp. 2622-2629 ◽  
Author(s):  
Chandima Gunawardana ◽  
Ashantha Goonetilleke ◽  
Prasanna Egodawatta
Keyword(s):  
Heavy Metals ◽  
Kinetic Studies ◽  
Field Studies ◽  
Water Soluble ◽  
Trivalent Cation ◽  
Urban Road ◽  
High Charge Density ◽  
Road Surfaces ◽  
Adsorption Desorption ◽  
Exchangeable Form

The research study discussed in the paper investigated the adsorption/desorption behaviour of heavy metals commonly deposited on urban road surfaces, namely, Zn, Cu, Cr and Pb, for different particle size ranges of solids. The study outcomes, based on field studies and batch experiments, confirmed that road deposited solids particles contain a significantly high amount of vacant charge sites with the potential to adsorb additional heavy metals. Kinetic studies and adsorption experiments indicated that Cr is the most preferred metal element to associate with solids due to the relatively high electronegativity and high charge density of trivalent cation (Cr3+). However, the relatively low availability of Cr in the urban road environment could influence this behaviour. Comparing total adsorbed metals present in solids particles, it was found that Zn has the highest capacity for adsorption to solids. Desorption experiments confirmed that a low concentration of Cu, Cr and Pb in solids was present in water-soluble and exchangeable form, whilst a significant fraction of adsorbed Zn has a high likelihood of being released back into solution. Among heavy metals, Zn is considered to be the most commonly available metal among road surface pollutants.

Synthesis and Characterization of Novel PEPPSI type Pd–BICAAC Complexes

2022 ◽  
Author(s):  
Márton Nagyházi ◽  
Balázs Almási ◽  
Ádám Lukács ◽  
Attila Bényei ◽  
Tibor Nagy ◽  
...  
Keyword(s):  
High Activity ◽  
Coupling Reaction ◽  
Kinetic Studies ◽  
Elemental Mercury ◽  
Palladium Complexes ◽  
Synthesis And Characterization ◽  
Heck Coupling ◽  
Moisture Resistant

A series of bicyclic alkylamino carbenes (BICAAC) (where N-aryl = dipp, mes, 2,6-dimethyl-4-(dimethylamino)phenyl, 5a-d) and their novel air- and moisture-resistant pyridine (pyridine, 4 dimethylaminopyridine) containing palladium PEPPSI-type Pd(II) complexes (6a-e) were synthetized and characterized. The new palladium complexes have shown high activity in Mizoroki–Heck coupling reaction even at as low as 100 ppm loading (TON up to 10000). Kinetic studies revealed that reactions carried out in the presence of elemental mercury resulted in decrease in activity. It indicates that the coupling reaction may have both molecular and Pd(0)-mediated catalytic paths.

Purification and Characterization of Laccase fromChaetomium thermophilium and Its Role in Humification

1998 ◽  
Vol 64 (9) ◽  
pp. 3175-3179 ◽  
Author(s):  
Benny Chefetz ◽  
Yona Chen ◽  
Yitzhak Hadar
Keyword(s):  
Municipal Solid Waste ◽  
Solid Waste ◽  
Laccase Activity ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Water Soluble ◽  
Type I ◽  
Light Spectrum ◽  
Solid Media ◽  
Wide Range

ABSTRACT Chaetomium thermophilium was isolated from composting municipal solid waste during the thermophilic stage of the process.C. thermophilium, a cellulolytic fungus, exhibited laccase activity when it was grown at 45°C both in solid media and in liquid media. Laccase activity reached a peak after 24 h in liquid shake culture. Laccase was purified by ultrafiltration, anion-exchange chromatography, and affinity chromatography. The purified enzyme was identified as a glycoprotein with a molecular mass of 77 kDa and an isoelectric point of 5.1. The laccase was stable for 1 h at 70°C and had half-lives of 24 and 12 h at 40 and 50°C, respectively. The enzyme was stable at pH 5 to 10, and the optimum pH for enzyme activity was 6. The purified laccase efficiently catalyzed a wide range of phenolic substrates but not tyrosine. The highest levels of affinity were the levels of affinity to syringaldazine and hydroxyquinone. The UV-visible light spectrum of the purified laccase had a peak at 604 nm (i.e., Cu type I), and the activity was strongly inhibited by Cu-chelating agents. When the hydrophobic acid fraction (the humic fraction of the water-soluble organic matter obtained from municipal solid waste compost) was added to a reaction assay mixture containing laccase and guaiacol, polymerization took place and a soluble polymer was formed. C. thermophilium laccase, which is produced during the thermophilic stage of composting, can remain active for a long period of time at high temperatures and alkaline pH values, and we suggest that this enzyme is involved in the humification process during composting.

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