Purification and Characterization of Alkaline Polygalacturonases Produced by Paenibacillus polymyxa 20185 in Submerged Cultures

2012 ◽  
Vol 550-553 ◽  
pp. 1496-1502
Author(s):  
Hong Ju Jiang ◽  
Wei Zhen Xu ◽  
Jia Mei Song ◽  
Dong Ping Sun
Keyword(s):  
Gel Filtration ◽  
Paenibacillus Polymyxa ◽  
Maximal Activity ◽  
Purification And Characterization ◽  
Submerged Cultures ◽  
Liquid Cultures ◽  
Similar Molecular Weight ◽  
Sds Page ◽  
Alkaline Conditions

Two extracellular alkaline polygalacturonases from extracts of liquid cultures of Paenibacillus polymyxa 20185 were purified by gel filtration chromatography to homogeneity as judged by SDS-PAGE. The purified alkaline polygalacturonases (PG1 and PG2) had a similar molecular weight of 65 kDa, exhibited maximal activity at 50°C with pH 10.0, and were stable in alkaline conditions. The purified alkaline polygalacturonases activities were enhanced in the presence of Mg2+, and were resistant to inhibition by Mn2+, Zn2+and Cu2+. Michaelis-menten constants of PG1 and PG2 were found as 3.6mg/mL and 3.5mg/mL, respectively.

scholarly journals Production, purification and characterization of an exo-polygalacturonase from Penicillium janthinellum sw09

2016 ◽  
Vol 88 (suppl 1) ◽  
pp. 479-487 ◽  
Author(s):  
YUPING MA ◽  
SIWEN SUN ◽  
HUI HAO ◽  
CHUNPING XU
Keyword(s):  
Gel Filtration ◽  
Maximal Activity ◽  
Growth Conditions ◽  
Purification And Characterization ◽  
Penicillium Janthinellum ◽  
Soil Isolate ◽  
Sds Page ◽  
The Stability ◽  
Ph Range

ABSTRACT A soil isolate, Penicillium janthinellum sw09 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as exo-polygalacturonase (exo-PG). By optimizing growth conditions, P. janthinellum sw09 produced high amount of exo-PG (16.54 units/mL). The crude enzyme was purified by gel filtration chromatography and two exo-PG activity peaks (designated as PGI and PGII) were revealed. On SDS-PAGE analysis, purified PGII using DEAE-Sepharose FF column, was found to be a single band with a molecular mass of 66.2 kDa. The purified PGII exhibited maximal activity at the temperature of 45 oC and pH 5.0. The stability profiles show that PGII is more stable in the pH range of 4.0-8.0 and below 60 oC. The Km and Vmax for the enzyme was 1.74 mg/mL and 18.08 μmol/ (mL•min), respectively. Due to this enzymatic characterization, this pectinase is an attractive candidate for applications in degradation of pectin.

scholarly journals Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

2005 ◽  
Vol 387 (1) ◽  
pp. 271-280 ◽  
Author(s):  
Seonghun KIM ◽  
Sun Bok LEE
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Maximal Activity ◽  
Genome Database ◽  
Sds Page ◽  
Key Enzyme ◽  
Bivalent Metal Ions ◽  
Ammonium Sulphate Fractionation ◽  
Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

scholarly journals Purification and characterization of N-ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP2) from rat liver

1995 ◽  
Vol 308 (3) ◽  
pp. 983-989 ◽  
Author(s):  
I N Fleming ◽  
S J Yeaman
Keyword(s):  
Phosphatidic Acid ◽  
Rat Liver ◽  
Lysophosphatidic Acid ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Sds Page ◽  
Chromatography Columns ◽  
Triton X ◽  
Second Peak

N-Ethylmaleimide-insensitive phosphatidic acid phosphohydrolase (PAP; EC 3.1.3.4) was purified 5900-fold from rat liver. The enzyme was solubilized from membranes with octylglucoside, fractionated with (NH4)2SO4, and purified in the presence of Triton X-100 by chromatography on Sephacryl S300, hydroxyapatite, heparin-Sepharose and Affi-Gel Blue. Silver-stained SDS/PAGE indicated that the enzyme was an 83 kDa polypeptide. Sephacryl S-300 gel filtration also produced a second peak of enzyme activity, which was eluted from all of the chromatography columns at a different position from the purified enzyme. SDS/PAGE indicated that it contained three polypeptides (83 kDa, 54 kDa and 34 kDa), and gel filtration suggested that it was not an aggregate of the purified enzyme. Both forms were sensitive to inhibition by amphiphilic amines, Mn2+ and Zn2+, but not by N-ethylmaleimide. Purified PAP required detergent for activity, but was not activated by Mg2+, fatty acids or phospholipids. The enzyme was able to dephosphorylate lysophosphatidic acid or phosphatidic acid, and was inhibited by diacylglycerol and monoacylglycerol. No evidence was obtained for regulation of PAP by reversible phosphorylation.

scholarly journals Purification and characterization of an extracellular (1 → 6)-β-glucanase from the filamentous fungus Acremonium persicinum

1996 ◽  
Vol 316 (3) ◽  
pp. 841-846 ◽  
Author(s):  
Stuart M. PITSON ◽  
Robert J. SEVIOUR ◽  
Barbara M. McDOUGALL ◽  
Bruce A. STONE ◽  
Maruse SADEK
Keyword(s):  
Molecular Mass ◽  
Filamentous Fungus ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Eisenia Bicyclis ◽  
Ph Optimum ◽  
Hydrolysis Products ◽  
Sds Page ◽  
Hydrolysis Of

An endo-(1 → 6)-β-glucanase has been isolated from the culture filtrates of the filamentous fungus Acremonium persicinum and purified by (NH4)2SO4 precipitation followed by anion-exchange and gel-filtration chromatography. SDS/PAGE of the purified enzyme gave a single band with an apparent molecular mass of 42.7 kDa. The enzyme is a non-glycosylated, monomeric protein with a pI of 4.9 and pH optimum of 5.0. It hydrolysed (1 → 6)-β-glucans (pustulan and lutean), initially yielding a series of (1 → 6)-β-linked oligoglucosides, consistent with endo-hydrolytic action. Final hydrolysis products from these substrates were gentiobiose and gentiotriose, with all products released as β-anomers, indicating that the enzyme acts with retention of configuration. The purified enzyme also hydrolysed Eisenia bicyclis laminarin, liberating glucose, gentiobiose, and a range of larger oligoglucosides, through the apparent hydrolysis of (1 → 6)-β- and some (1 → 3)-β-linkages in this substrate. Km values for pustulan, lutean and laminarin were 1.28, 1.38, and 1.67 mg/ml respectively. The enzyme was inhibited by N-acetylimidazole, N-bromosuccinimide, dicyclohexylcarbodi-imide, Woodward's Regent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO4 and some metal ions, whereas D-glucono-1,5-lactone and EDTA had no effect.

scholarly journals Isolation, Purification, and Characterization of Fungal Laccase from Pleurotus sp.

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Sunil S. More ◽  
Renuka P. S. ◽  
Pruthvi K. ◽  
Swetha M. ◽  
S. Malini ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Laccase Activity ◽  
Gel Filtration ◽  
Industrial Enzymes ◽  
Purification And Characterization ◽  
Sds Page ◽  
Concomitant Reduction ◽  
The One ◽  
Inhibited Enzyme

Laccases are blue copper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. They are currently seen as highly interesting industrial enzymes because of their broad substrate specificity. A positive strain was isolated and characterized as nonspore forming Basidiomycetes Pleurotus sp. Laccase activity was determined using ABTS as substrate. Laccase was purified by ionexchange and gel filtration chromatography. The purified laccase was a monomer showed a molecular mass of 40±1 kDa as estimated by SDS-PAGE and a 72-fold purification with a 22% yield. The optimal pH and temperature were 4.5 and 65°C, respectively. The Km and Vmax⁡ values are 250 (mM) and 0.33 (μmol/min), respectively, for ABTS as substrate. Metal ions like CuSO4, BaCl2, MgCl2, FeCl2, ZnCl2 have no effect on purified laccase whereas HgCl2 and MnCl2 moderately decrease enzyme activity. SDS and sodium azide inhibited enzyme activity, whereas Urea, PCMB, DTT, and mercaptoethanol have no effect on enzyme activity. The isolated laccase can be used in development of biosensor for detecting the phenolic compounds from the effluents of paper industries.

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 Partial Purification and Characterization of Catalase from Banana Peels

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.

scholarly journals Purification and Characterization of a Prominent Polygalacturonase Isozyme Produced by Phomopsis cucurbitae in Decayed Muskmelon Fruit

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 488F-489
Author(s):  
J.X. Zhang ◽  
B.D. Bruton ◽  
C.L. Biles
Keyword(s):  
Latent Infection ◽  
Gel Filtration ◽  
Central American ◽  
Purification And Characterization ◽  
Fruit Storage ◽  
Sds Page ◽  
Fruit Decay ◽  
Isozyme Activity

Phomopsis cucurbitae is a latent infecting pathogen that infects unripe muskmelon fruit, but causes decay after harvest. This fungus causes severe losses during muskmelon fruit storage and marketing in the U.S., Japan, and some Central American countries. Previous studies showed that the fungus produced the cell wall-degrading enzyme polygalacturonase (PG) in both culture and muskmelon fruit tissue. The role of P. cucurbitae PG in the fruit decay process and its relation to latent infection is not well-understood. A prominent PG isozyme produced by the fungus in decayed fruit was purified to homogeneity by a sequence of extraction, ultrafiltration, preparative isoelectric focusing, anion exchange, and gel filtration chromatography. This isozyme exhibited endo-activity, a molecular weight of 54 kDa according to SDS-PAGE, and a pI of 4.2 based on IEF-PAGE. Isozyme activity was optimal at 40–45°C and pH 5.0. It had a Km of 44.7 g/ml and a Vmax of 0.313. The purified isozyme also effectively macerated mature muskmelon fruit tissues. This isozyme was the most prominent of the PG isozymes produced by P. cucurbitae in decaying fruit, and may play an important role in postharvest decay.

scholarly journals A Boophilus microplus vitellin-degrading cysteine endopeptidase

Parasitology ◽  
2003 ◽  
Vol 126 (2) ◽  
pp. 155-163 ◽  
Author(s):  
A. SEIXAS ◽  
P. C. DOS SANTOS ◽  
F. F. VELLOSO ◽  
I. DA SILVA VAZ ◽  
A. MASUDA ◽  
...  
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Boophilus Microplus ◽  
Hard Tick ◽  
Purification And Characterization ◽  
Basic Residue ◽  
Cysteine Endopeptidase ◽  
Sds Page

Here we describe the purification and characterization of a vitellin (VT) degrading cysteine endopeptidase (VTDCE) from eggs of the hard tick Boophilus microplus. A homogeneous enzyme preparation was obtained by chromatographic fractionation on ion-exchange and gel filtration columns and an autolysis step. This step consisted of incubation of a semipurified enzyme (after the first ion-exchange chromatography) at pH 4·0 that dissociated the enzyme from VT, to which VTDCE is naturally tightly associated. The enzyme purity was confirmed by capillary and native gel electrophoresis, and SDS–PAGE suggested the enzyme is a dimer of 17 and 22 kDa. VTDCE was active upon several synthetic substrates, with a preference for a hydrophobic or a basic residue in P1, and a hydrophobic residue in P2. VTDCE also hydrolysed haemoglobin, albumin, gelatin and vitellin. VTDCE is inactive in the absence of DTT and was totally inhibited by E-64, indicating it is a cysteine endopeptidase. Our results suggest that VTDCE is a major enzyme involved in yolk processing during B. microplus embryogenesis.

PURIFICATION AND CHARACTERIZATION OF A PROTEIN C ACTIVATOR FROM THE VENOM OF AGKISTRODON CONTORTRIX CONTORTRIX

1987 ◽  
Author(s):  
Carolyn L Orthner ◽  
Prabir Bhattacharya ◽  
Dudley K Strikland
Keyword(s):  
Protein C ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Agkistrodon Contortrix ◽  
Purification And Properties ◽  
Sds Page ◽  
Single Protein ◽  
Agkistrodon Contortrix Contortrix

There are two recent reports on the purification and properties of a protein C activator (PCA) from the venom of the Southern copperhead snalce. The purification of a 37,000 Mr nonenzymatic PCA (Martinoli and Stocker, Thrcmb. Res. 43, 253, 1976) as well as of a 20,000 Mr thrombin-like enzyme (Klein and Walker, Biochem. ,25, 4175, 1986) have been described. The purpose of this investigation was to purify and further characterize the PCA(s) from this vencm. A PCA has been isolated by sulphopropyl-Sephadex followed by gel filtration chromatography resulting in approximately a 100-fold purification with a 50% yield. PCA appeared as a single band on SDS-PAGE with an estimated Mr of 32,000 or 37,000 in the absence or presence of β-mercaptoethanol, respectively. High pressure gel permeation cinematography of PCA in Tris-buffered saline, pH 7.5 resulted in a single protein peak with a Mr of 39,000 which was coincident with activity. PCA was a potent activator of human protein C (PC) with a Km for PC of 0.6uM and a Vm of 0.02 sec-1. In addition, PCA catalyzed the arnidolysis of Tosyl-gly-pro-arg-p-nitroanilide (TGPRpNA) with a Km of 1.1 irM and a Vim of 66 sec-1. The rate of arnidolysis of five other pept idyl-arginyl-pNA substrates each tested at 1.0 mM was < 10% that of TGPRpNA. PCA was inhibited by nitrophenylguanidi-nobenzoate (NPGB), phenylmethylsulphonylflouride, D-phe-pro-arg-chloromethyi_ketone (PPACK) and soybean trypsin inhibitor indicating that PCA is a serine protease. The active site concentration of PCA as measured by NPGB titration was 90% that of the protein concentration. Measurement of the rate of PCA inhibition at varying levels of PPACK indicated that it had a Ki of 34uM .and an aUcylation rate constant of 0.09 min-1. PCA activation of PC was completely inhibited by CaC12 with an apparent Ki of 99uM. Since neither PCA arnidolysis of TGPRpNA nor inhibition by PPACK was affected by Ca2+, the effect of this metal was likely on the substrate PC. In summary, a PCA has been purified to homogeneity and has properties which are distinct from those reported. PCA premises to be a useful enzyme in studies of PC and its activation.

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