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

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.

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Partial purification and characterization of an endo-α-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242

Biochemical Journal ◽

10.1042/bj2880475 ◽

1992 ◽

Vol 288 (2) ◽

pp. 475-482 ◽

Cited By ~ 30

Author(s):

I Ishii-Karakasa ◽

H Iwase ◽

K Hotta ◽

Y Tanaka ◽

S Omura

Keyword(s):

Enzyme Activity ◽

Culture Medium ◽

Gel Filtration ◽

Partial Purification ◽

Gastric Mucus ◽

Purification And Characterization ◽

Streptomyces Sp ◽

Optimum Ph ◽

New Type

For the purification of a new type of endo-alpha-N-acetylgalactosaminidase from the culture medium of Streptomyces sp. OH-11242 (endo-GalNAc-ase-S) [Iwase, Ishii, Ishihara, Tanaka, Omura & Hotta (1988) Biochem. Biophys. Res. Commun. 151, 422-428], a method for assaying enzyme activity was established. Using purified pig gastric mucus glycoprotein (PGM) as the substrate, oligosaccharides liberated from PGM were pyridylaminated, and the reducing terminal sugars of oligosaccharides larger than Gal beta 1-3GalNAc were analysed by h.p.1.c. The crude enzyme of endo-GalNAc-ase-S was prepared as an 80% (w/v) ammonium sulphate precipitate from the concentrated culture medium. The enzyme was partially purified by gel chromatofocusing and subsequent DEAE-Toyopearl chromatography. Endo-enzyme activity eluted around pI 4.8 on a gel chromatofocusing column and eluted with 0.19-0.25 M-NaCl on a DEAE-Toyopearl column. In the enzyme fraction obtained, no exo-glycosidases or proteases could be detected. The molecular mass of the enzyme was estimated as 105 kDa by gel filtration, and the optimum pH was 5.5. Endo-GalNAc-ase-S hydrolysed the O-glycosidic linkage between GalNAc and Ser (Thr) in 3H-labelled and unlabelled asialofetuin, liberating both the disaccharide (Gal beta 1-3GalNAc) and the tetrasaccharide [Gal beta 1-3 (Gal beta 1-4GlcNAc beta 1-6)GalNAc]. When endo-alpha-N-acetylgalactosaminidase from Alcaligenes sp. (endo-GalNac-ase-A) was incubated with 3H-labelled and unlabelled asialofetuin, only the disaccharide (Gal beta 1-3GalNAc) was liberated.

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Optimization of Growth Conditions for Purification and Production of L-Asparaginase bySpirulina maxima

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/1785938 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 12

Author(s):

Hanaa H. Abd El Baky ◽

Gamal S. El Baroty

Keyword(s):

Enzyme Activity ◽

Gel Filtration ◽

Growth Conditions ◽

Partial Purification ◽

Specific Enzyme ◽

Purification And Characterization ◽

Spirulina Maxima ◽

Optimum Ph ◽

Biomass Yields

L-asparaginase (L-AsnA) is widely distributed among microorganisms and has important applications in medicine and in food technology sectors. Therefore, the ability of the production, purification, and characterization of AsnA fromSpirulina maxima(SM) were tested. SM cultures grown in Zarrouk medium containing different N2(in NaNO3form) concentrations (1.25, 2.50, and 5.0 g/L) for 18 days contained a significant various quantity of dry biomass yields and AsnA enzyme levels. MS L-AsnA activity was found to be directly proportional to the N2concentration. The cultures of SM at large scales (300 L medium, 5 g/L N2) showed a high AsnA enzyme activity (898 IU), total protein (405 mg/g), specific enzyme activity (2.21 IU/mg protein), and enzyme yield (51.28 IU/L) compared with those in low N2cultures. The partial purification of crude MS AsnA enzyme achieved by 80% ammonium sulfate AS precipitated and CM-Sephadex C-200 gel filtration led to increases in the purification of enzyme with 5.28 and 10.91 times as great as that in SM crude enzymes. Optimum pH and temperature of purified AsnA for the hydrolyzate were 8.5 and 37 ± 0.2°C, respectively. To the best of our knowledge, this is the first report on L-asparaginase production inS. maxima.

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

Biochemical Journal ◽

10.1042/bj3080983 ◽

1995 ◽

Vol 308 (3) ◽

pp. 983-989 ◽

Cited By ~ 31

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.

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

Biochemical Journal ◽

10.1042/bj3160841 ◽

1996 ◽

Vol 316 (3) ◽

pp. 841-846 ◽

Cited By ~ 29

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.

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

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.550-553.1496 ◽

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.

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

HortScience ◽

10.21273/hortsci.32.3.488f ◽

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.

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

Anais da Academia Brasileira de Ciências ◽

10.1590/0001-3765201620150051 ◽

2016 ◽

Vol 88 (suppl 1) ◽

pp. 479-487 ◽

Cited By ~ 5

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.

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A Boophilus microplus vitellin-degrading cysteine endopeptidase

Parasitology ◽

10.1017/s0031182002002731 ◽

2003 ◽

Vol 126 (2) ◽

pp. 155-163 ◽

Cited By ~ 37

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.

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

10.1055/s-0038-1643813 ◽

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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