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.

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 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 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

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

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
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.

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 three extracellular (1→3)-β-d-glucan glucohydrolases from the filamentous fungus Acremonium persicinum

1995 ◽  
Vol 308 (3) ◽  
pp. 733-741 ◽  
Author(s):  
S M Pitson ◽  
R J Seviour ◽  
B M McDougall ◽  
J R Woodward ◽  
B A Stone
Keyword(s):  
Filamentous Fungus ◽  
Gel Filtration ◽  
High Specificity ◽  
Major Product ◽  
Ph Optimum ◽  
Sds Page ◽  
Molecular Masses ◽  
Monomeric Proteins ◽  
Ph Range

Three (1-->3)-beta-D-glucanases (GNs) were 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. Homogeneity of the purified proteins was confirmed by SDS/PAGE, isoelectric focusing and N-terminal amino acid sequencing. All three GNs (GN I, II and III) are non-glycosylated, monomeric proteins with apparent molecular masses, estimated by SDS/PAGE, of 81, 85 and 89 kDa respectively. pI values for the three enzymes are 5.3, 5.1, and 4.4 respectively. The pH optimum for GN I is 6.5, and 5.0 for GN II and III. All three purified enzymes displayed stability over the pH range 4.5-10.0. Optimum activities for GN I, II and III were recorded at 65, 55 and 60 degrees C respectively, with both GN II and III having short-term stability up to 50 degrees C and GN I up to 55 degrees C. The purified GNs have high specificity for (1-->3)-beta-linkages and hydrolysed a range of (1-->3)-beta- and (1-->3)(1-->6)-beta-D-glucans, with laminarin from Laminaria digitata being the most rapidly hydrolysed substrate of those tested. K(m) values for GN I, II, and III against L. digitata laminarin were 0.1, 0.23 and 0.22 mg/ml respectively. D-Glucono-1,5-lactone does not inhibit any of the three GNs, some metals ions are mild inhibitors, and N-bromosuccinimide and KMnO4 are strong inhibitors. All three GNs acted in an exo-hydrolytic manner, determined by the release of alpha-glucose as the initial and major product of hydrolysis of (1-->3)-beta-D-glucans, and confirmed by viscometric analysis and the inability to cleave periodate-oxidized laminarin, and may be classified as (1-->3)-beta-D-glucan glucohydrolases (EC 3.2.1.58).

scholarly journals Purification and characterization of a novel NADPH(NADH)-dependent glyoxylate reductase from spinach leaves. Comparison of immunological properties of leaf glyoxylate reductase and hydroxypyruvate reductase

1986 ◽  
Vol 239 (3) ◽  
pp. 653-659 ◽  
Author(s):  
L A Kleczkowski ◽  
D D Randall ◽  
D G Blevins
Keyword(s):  
High Specificity ◽  
Maximal Activity ◽  
Purification And Characterization ◽  
Leaf Extracts ◽  
Hydroxypyruvate Reductase ◽  
Ph Range ◽  
Glyoxylate Reductase ◽  
Spinach Leaves

A novel reductase displaying high specificity for glyoxylate and NADPH was purified 3343-fold from spinach leaves. The enzyme was found to be an oligomer of about 125 kDa, composed of four equal subunits of 33 kDa each. A Km for glyoxylate was about 14-fold lower with NADPH than with NADH (0.085 and 1.10 mM respectively), but the maximal activity, 210 mumol/min per mg of protein, was similar with either cofactor. Km values for NADPH and NADH were 3 and 150 microM respectively. Optimal rates with either NADPH or NADH were found in the pH range 6.5-7.4. The enzyme also showed some reactivity towards hydroxypyruvate with rates less than 2% of those observed for glyoxylate. Results of immunological studies, using antibodies prepared against either glyoxylate reductase or spinach peroxisomal hydroxypyruvate reductase, suggested substantial differences in molecular structure of the two proteins. The high rates of NADPH(NADH)-glyoxylate reductase in crude leaf extracts of spinach, wheat and soya bean (30-45 mumol/h per mg of chlorophyll) and its strong affinity for glyoxylate suggest that the enzyme may be an important side component of photorespiration in vivo. In leaves of nitrogen-fixing legumes, this reductase may also be involved in ureide breakdown, utilizing the glyoxylate produced during allantoate metabolism.

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.

Purification and Characterization of an Extracellular Cold-Active Protease Produced by the Psychrotrophic Bacterium serratia Sp. WJ39

2014 ◽  
Vol 618 ◽  
pp. 330-334 ◽  
Author(s):  
Xiu Ling Ji ◽  
Muhammad Kamran Taj ◽  
Xiao Bo Lu ◽  
Lian Bing Lin ◽  
Qi Zhang ◽  
...  
Keyword(s):  
Residual Activity ◽  
Leather Industry ◽  
Purification And Characterization ◽  
Suitable Candidate ◽  
Optimal Activity ◽  
Sds Page ◽  
Cold Active ◽  
Active Protease ◽  
Ph Range

Proteases have diverse applications in a wide variety of industries, such as in detergent, leather, food, pharmaceutical and silk. The extracellular cold-active protease was purified from the psychrotrophic bacteriumSerratiasp. WJ39 from a meat factory. The protease was cold-active with a molecular mass of 47.6 kDa estimated on SDS-PAGE. It showed an optimal activity at pH of 8 and was stable at pH 6 to 10, while its optimal temperature was 37°C and it was stable at 0-25°C, even remained 35% residual activity at 0°C. The protease was totally inhibited by PMSF which was telling that the purified enzyme was a serine protease. The properties like moderate thermostability, activity in a broad pH range and resistance to metal ions make this enzyme a suitable candidate for the possible use in food and leather industry.

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