scholarly journals Kinetics Properties of Polyphenol Oxidase in Hawthorn (Crataegus spp)

2014 ◽  
Vol 25 ◽  
pp. 29-38 ◽  
Author(s):  
Shahriar Saeidian
Keyword(s):  
Enzyme Activity ◽  
Polyphenol Oxidase ◽  
Thermal Inactivation ◽  
Kojic Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Ph ◽  
Km Value ◽  
Ph Range ◽  
Menten Constant

Polyphenol oxidase (PPO) from hawthorn was extracted and partially purified through (NH4)2SO4 precipitation, dialysis and ion exchange chromatography. The activity of polyphenol oxidase was investigated in Crataegus spp. Spectrophotometric method was used to assay the enzyme activity and the kinetic constants - maximum enzyme velocity (Vmax) and Michealis - Menten constant (Km). Of the substrates tested, catechol was the best substrate for PPO with a Km value of 2.2 mM. The optimum pH for PPO activity was found to be 7. The enzyme showed high activity over a broad pH range of 4 - 8. The optimal pH and temperature for enzyme activity were found to be 7 and 40-45 °C, respectively. km value for hawthorn PPO is calculated 22 mM for catechol and 6.7 mM for pyrogallol and 9.7 mM for L-dopa. As can be seen, affinity of PPOs for various substrates varies widely. The enzyme showed a broad activity over a broad pH and temperature range. The thermal inactivation studies showed that the enzyme is heat resistant. The enzyme showed the highest activity toward pyrogallol and no activity toward tyrosine. Of the inhibitors tested, the most potent inhibitors were kojic acid, cysteine and glycine , respectively

scholarly journals Isolation and Partial Purification of Polyphenol Oxidase from Seed of Melon (Cucumeropsis edulis)

2020 ◽  
Vol 11 (2) ◽  
pp. 9085-9096
Keyword(s):  
Metal Ions ◽  
Polyphenol Oxidase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Inhibitory Effects ◽  
Optimum Ph ◽  
Km Value ◽  
Menten Constant ◽  
Diphenolase Activity

Polyphenol oxidase (PPO) from Cucumeropsis edulis was extracted and partially purified through (NH4)2SO4 precipitation, dialysis, and ion-exchange chromatography on DEAE-Sephadex-A50. The spectrophotometric method was used to assay the enzyme activity in C. edulis using L-DOPA as substrate, the physicochemical properties such as the effect of pH and temperature, substrate specificity, kinetic constants - maximum enzyme velocity (Vmax), and Michaelis - Menten constant (Km) for three substrates namely, L-Dopa catechol and tyrosine were determined. The effects of inhibitors and metal ions on PPO activity were also investigated. The optimum pH and temperature values were found to be pH 6.5 and 50 °C, and the inhibitory effects of inhibitors such as ascorbic acid, EDTA, SDS, and metal ions were enhanced positively with increased concentration except with divalent metals such as Cu2+, Fe2+, and Zn2+ reflecting an activating effect on C. edulis PPO. Moreover, the enzyme solution showed both monophenolase and diphenolase activity with L-DOPA having the highest Vmax/Km value. However, the data obtained in this research provided a theoretical basis for the prevention of enzymatic browning of C. edulis during processing.

scholarly journals Decolorization of textile dyes by partially purified Pleurotus ostreatus laccase

2014 ◽  
Vol 8 (3) ◽  
pp. 64-69
Author(s):  
Shamam saady Abdulredha ◽  
Abdulkareem jasim Hashim ◽  
Abduljabar Abas Ali ◽  
Batool Imran Dheeb
Keyword(s):  
Solid State ◽  
Pleurotus Ostreatus ◽  
Specific Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Textile Dyes ◽  
Optimum Temperature ◽  
Optimum Conditions ◽  
Optimum Ph ◽  
Ph Range

Pleurotus ostreatus produced 2.93 U/mg of laccase in solid state fermentation (SSF) using barley bran as substrate under optimum conditions. The optimum SSF conditions were: pH 6.5; temperature, 25Cº; inoculums size 3.5 mm and moisture content, 1:1.5 w/v. Laccase was partially purified 8.29 fold with specific activity 17.5 U/mg by ion exchange chromatography after curd enzyme concentrated by dialysis against the solid sucrose. Partially purified laccase had an optimum pH of 6.5 and was stable in the pH range from 6.5 to 7.5. The optimum temperature was 45 Cº and it displayed considerable stability within the range 15 to 45 Cº with 1h incubation as well as The ability of partial purified laccase to decolorize of textile dyes showed that the blue H3R dye was completely decolorized in all concentrations within first min while yellow FG and red 3B dyes were decolorized in different percentage.

Effect of temperature and pH on the production, activity, and stability of α-amylase from Bacillus subtilis V37

2021 ◽  
Vol 66 (1) ◽  
pp. 72-79
Author(s):  
Thuoc Doan Van ◽  
Hung Nguyen Phuc
Keyword(s):  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Animal Feed ◽  
Culture Conditions ◽  
Effect Of Temperature ◽  
Physical Parameters ◽  
Original Activity ◽  
Optimum Ph ◽  
Production Activity ◽  
Ph Range

The effect of physical parameters such as temperature and pH on the production, activity, and stability of α-amylase from Bacillus subtilis V37 was investigated. The results indicated that the optimum culture conditions for enzyme activity were pH 7.0 and 35 oC. The optimum pH and temperature for enzyme activity were 6.0 and 70 oC. The crude enzyme was found to be stable in the pH range of 5.0 to 7.0. The enzyme was stable for 1 h at a temperature from 30 to 80 oC; nearly 100% of enzyme activity remained at temperatures of 30 - 40 oC, and about 34% of original activity remained at a temperature of 80 oC. These features demonstrated that α-amylase from B. subtilis V37 can be applied in many areas such as the food, fermentation, and animal feed industries.

scholarly journals Preparation of Chitosan/Magnetic Porous Biochar as Support for Cellulase Immobilization by Using Glutaraldehyde

Polymers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 2672
Author(s):  
Haodao Mo ◽  
Jianhui Qiu
Keyword(s):  
Alkali Activation ◽  
Covalent Bonds ◽  
Ph Values ◽  
Maximum Reaction ◽  
Carboxymethyl Cellulose Sodium ◽  
Optimum Ph ◽  
Potential Applications ◽  
Km Value ◽  
Immobilized Cellulase ◽  
Menten Constant

In this work, porous biochar was obtained from sugarcane bagasse by alkali activation and pyrolysis and then magnetized with γ-Fe2O3 by calcination. After functionalization with chitosan and activation with glutaraldehyde, the as-prepared chitosan/magnetic porous biochar served as a support to immobilize cellulase by covalent bonds. The immobilization amount of cellulase was 80.5 mg cellulase/g support at pH 5 and 25 °C for 12 h of immobilization. To determine the enzymatic properties, 1% carboxymethyl cellulose sodium (CMC) (dissolved in 0.1 M buffer) was considered as a substrate for hydrolysis at different pH values (3–7) and temperatures (30–70 °C) for 30 min. The results showed that the optimum pH and temperature of the free and immobilized cellulase did not change, which were pH 4 and 60 °C, respectively. The immobilized cellulase had a relatively high activity recovery of 73.0%. However, it also exhibited a higher Michaelis–Menten constant (Km) value and a slower maximum reaction velocity (Vmax) value compared to the free enzyme. In the reusability assay, the immobilized cellulase showed initial glucose productivity of 330.9 mg glucose/g CMC and remained at 86.0% after 10 uses. In conclusion, the chitosan/magnetic porous biochar has great potential applications as a support for enzyme immobilization.

Catabolism of (R)-Amygdalin and (R)-Vicianin by Partially Purified ß-Glycosidases from Prunus serotina Ehrh. and Davallia trichomanoides

1984 ◽  
Vol 39 (3-4) ◽  
pp. 232-239 ◽  
Author(s):  
Gary Kuroki ◽  
Pauline A. Lizotte ◽  
Jonathan E. Poulton
Keyword(s):  
Ion Exchange ◽  
Deae Cellulose ◽  
Hydrolytic Activity ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Prunus Serotina ◽  
Artificial Substrates ◽  
Black Cherry ◽  
Significant Activity ◽  
Optimum Ph

Mature black cherry (Prunus serotina Ehrh.) seeds accum ulate high levels of the cyanogenic disaccharide (R)-amygdalin. Extracts from these seeds contain two β-glycosidases which have been identified and completely resolved by DEAE-cellulose ion-exchange chromatography. Amygdalin hydrolase hydrolyzed (R)-am ygdalin at an optimum pH of 5.5, releasing (R)-prunasin and D-glucose. This enzyme showed highest activity towards (R)-am ygdalin and failed to hydrolyze (R)-prunasin. linamarin, β-gentiobiose and cellobiose. A distinct β-glycosidase, prunasin hydrolase, displayed a pronounced preference for (R)-prunasin, hydrolyzing this cyanogenic monosaccharide at an optimum pH of 6.5 to mandelonitrile and D-glucose. Prunasin hydrolase was inactive towards (R)-am ygdalin, linamarin, and β-gentiobiose. Both enzymes showed significant activity towards the artificial substrates β-ONPGlu and β-PNPGlu but did not hydrolyze α-PNPGlu. In view of the pronounced specificity of these enzymes towards endogenous cyanogens, it is concluded that upon disruption of black cherry seeds (R)- amygdalin is catabolized to mandelonitrile in a stepwise manner (the sequential mechanism) by amygdalin hydrolase and prunasin hydrolase with (R)-prunasin serving as intermediate. Young fronds of Davallia trichomanoides are rich sources of (R)-vicianin (the β-vicianoside of (R)-mandelonitrile). A β-glycosidase, vicianin hydrolase, has been partially purified from frond extracts by ion-exchange chromatography. At the optimum pH of 6.0, this enzyme showed highest hydrolytic activity with (R)-vicianin, although both (R)-am ygdalin and (R)-prunasin could be hydrolyzed at approximately 15% of the rate observed with (R)-vicianin. It failed to hydrolyze β-gentiobiose, cellobiose, linamarin and α-PNPGlu. Closer exam ination revealed that (R)-vicianin and (R)-amygdalin were hydrolyzed at the aglycone-disaccharide bond (the simultaneous mechanism) yielding mandelonitrile and the respective disaccharides vicianose and β-gentiobiose

Characterisation of a Neutral Protease Produced by Micrococcus luteus

1996 ◽  
Vol 51 (5-6) ◽  
pp. 429-431 ◽  
Author(s):  
M.O. Ilori ◽  
O.O. Amund ◽  
O. Omidiji
Keyword(s):  
Molecular Weight ◽  
Citric Acid ◽  
Proteolytic Enzyme ◽  
Micrococcus Luteus ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Optimum Ph

Abstract A proteolytic enzyme produced by a cassava-ferment­ing strain of Micrococcus luteus was extracted and puri­fied 50-fold by gel filtration and ion exchange chromatography. The optimum pH for the enzyme was 7.0, the optimum temperature 25 °C, the apparent molecular weight 42 kDa and the Km value, 0.45 mg ml-1 with casein as substrate. The enzyme was stimulated by Ca2+ and Mg2+ but inhibited by Zn2+ and Co2+ ions. Other inhibitors were EDTA, KCN, citric acid and L-cysteine indicating the enzyme to be a metalloprotease.

Purification and properties of an exocellular β-glucosidase of Candida molischiana (Zikes) Meyer and Yarrow capable of hydrolyzing soluble cellodextrins

1985 ◽  
Vol 63 (11) ◽  
pp. 1160-1166 ◽  
Author(s):  
Pierre Gondé ◽  
Robert Ratomahenina ◽  
Alain Arnaud ◽  
Pierre Galzy
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Purification And Properties ◽  
Optimum Ph

The exocellular enzyme β-glucosidase of Candida molischiana was studied. This strain is able to ferment soluble cellodextrins. The enzyme was partially purified by ion-exchange chromatography and gel filtration. The molecular weight of this enzyme was 120 000; its optimum pH was between 4 and 4.5 and its optimum temperature was 60 °C. This enzyme was active against different soluble glucosides and was inhibited by p-chloromercuribenzoate, gluconolactone, and glucose. A "glucosyltransferase" activity appeared in the presence of ethanol. The biosynthesis of the enzyme was constitutive but repressed by glucose.

scholarly journals Purification and Characterization of a Prolyl Aminopeptidase from Debaryomyces hansenii

2003 ◽  
Vol 69 (1) ◽  
pp. 227-232 ◽  
Author(s):  
Tomás Bolumar ◽  
Yolanda Sanz ◽  
M.-Concepción Aristoy ◽  
Fidel Toldrá
Keyword(s):  
Enzyme Activity ◽  
Gel Filtration ◽  
Debaryomyces Hansenii ◽  
Cysteine Proteases ◽  
Anion Exchange Chromatography ◽  
Cell Extract ◽  
Exchange Chromatography ◽  
Prolyl Aminopeptidase ◽  
Ph Range ◽  
Meat Fermentation

ABSTRACT A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45°C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg2+, which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The Km for proline-7-amido-4-methylcoumarin was 40 μM. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.

scholarly journals ISOLASI DAN KARAKTERISASI ENZIM b-1,3-ENDOGLUKANASE DARI TANAMAN KUBIS (Brassica oleracea cv. Capitata L.)

2010 ◽  
Vol 15 (2) ◽  
pp. 99-105
Author(s):  
Y. Sri Manuhara
Keyword(s):  
Ion Exchange ◽  
Brassica Oleracea ◽  
Hydrophobic Interaction Chromatography ◽  
Inhibition Zone ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Isolation And Characterization ◽  
Sds Page ◽  
Optimum Ph

Isolation and characterization of β-1,3-endoglucanase from cabbage (Brassica oleracea var. capitata L.) have been done. It showed 40° C of optimum temperature, and optimum pH is 7. After the purification with hydrophobic interaction chromatography and ion exchange chromatography, it’s activity was increased. Based on SDS-PAGE analysis, β-1,3-endoglucanase have molecular weight around 48 kD. Antifungal activity of β-1,3-endoglukanase show that it has best inhibition zone on Fusarium solanii at extract from ion exchange chromatography.

scholarly journals Purification of a 75 kDa protein from the organelle matrix of human neutrophils and identification as N-acetylglucosamine-6-sulphatase

2005 ◽  
Vol 387 (3) ◽  
pp. 841-847 ◽  
Author(s):  
Shengyuan XU ◽  
Linshu ZHAO ◽  
Anders LARSSON ◽  
Emanuel SMEDS ◽  
Marion KUSCHE-GULLBERG ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Matrix Protein ◽  
Heparan Sulphate ◽  
Subcellular Fractionation ◽  
Exchange Chromatography ◽  
Human Neutrophils ◽  
Km Value ◽  
Protein Nature ◽  
Primary Granules ◽  
Ph Range

A 75 kDa protein was purified to homogeneity from granule extracts of normal human granulocytes using Sephadex G-75 chromatography, Mono-S cation exchange chromatography and chromatofocusing. The protein consisted of one chain with a molecular mass of 75 kDa, as determined by SDS/PAGE. Tryptic peptide analysis by MALDI-TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS and sequence analysis by MS/MS identified the protein to be N-acetylglucosamine-6-sulphatase (EC 3.1.6.14). The identity of the protein was confirmed by demostrating enzymatic activity towards the substrate N-acetylglucosamine 6-sulphate. The enzyme was active over a broad pH range with an optimum of pH 7.0, and showed a Km value of 13.0 mM and a Vmax value of ∼1.8 μM/min per mg. The enzyme also showed O-desulphation activity towards heparan sulphate-derived saccharides. Subcellular fractionation of neutrophil organelles showed the presence of enzymatic activity mainly in the same fractions as primary granules. Furthermore, PMA treatment of the neutrophils induced release of the enzyme, indicating its matrix protein nature. The presence of N-acetylglucosamine-6-sulphatase in human neutrophils implies that neutrophils may play a role in the modulation of cell surface molecules and extracellular matrix by O-desulphation.

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