Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies1

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya(wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3´,4´-trihydroxychalcone and 4,3´,4´-trihydroxy-3-methoxychalcone to the corresponding 3,3´-biflavanones, as mixtures of racemic and mesoforms.

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

Biochemical Journal ◽

10.1042/bj3080733 ◽

1995 ◽

Vol 308 (3) ◽

pp. 733-741 ◽

Cited By ~ 25

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).

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

Biochemical Journal ◽

10.1042/bj20041053 ◽

2005 ◽

Vol 387 (1) ◽

pp. 271-280 ◽

Cited By ~ 58

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.

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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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Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

Applied and Environmental Microbiology ◽

10.1128/aem.66.1.252-256.2000 ◽

2000 ◽

Vol 66 (1) ◽

pp. 252-256 ◽

Cited By ~ 19

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 ).

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Screening and Characterization of Fibrinolytic Protease Producing Bacillus circulans from Mangrove Sediments Pitchavaram, South East Coast of India

International Letters of Natural Sciences ◽

10.18052/www.scipress.com/ilns.28.10 ◽

2014 ◽

Vol 28 ◽

pp. 10-16 ◽

Cited By ~ 1

Author(s):

R. Sadeesh Kumar ◽

R. Rajesh ◽

S. Gokulakrishnan ◽

J. Subramanian

Keyword(s):

Gel Filtration ◽

Fibrinolytic Enzyme ◽

Bacillus Circulans ◽

Health Issues ◽

Medical Sciences ◽

Fibrinolytic Enzymes ◽

Leading Role ◽

Sds Page ◽

Bacterial Sources

Regulation and production of Fibrinolytic enzymes from bacterial sources especially from Bacillus strains has taken a leading role in the medical sciences for the treatment of cardiovascular disorders as it removes thrombus or clots adding to its significant role in curing human health issues saving millions. Significant progress has been made during the last few years on the studies of fibrinolytic enzymes in identifying, cloning, purification, characterization and overproduction of these for commercialization in medical sciences and in fields like detergents development. Production of fibrinolytic enzyme from Bacillus circulans was done using Nutrient broth medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous with other species of same genus, as examined by SDS−PAGE and sephadex G-75 column chromatography. The enzyme had an optimal pH of 7-12, an optimal temperature of 50 °C, for fibrin hydrolysis. The molecular mass estimated by gel filtration was 24 to36 KDa. Further studies for characterization and structural elucidation are necessary for their medicinal applications and molecular biological characteristics.

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PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

IIUM Engineering Journal ◽

10.31436/iiumej.v18i2.654 ◽

2017 ◽

Vol 18 (2) ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Dzun Noraini Jimat ◽

Intan Baizura Firda Mohamed ◽

Azlin Suhaida Azmi ◽

Parveen Jamal

Keyword(s):

Specific Activity ◽

Hot Spring ◽

Gel Filtration ◽

Maximum Activity ◽

Exchange Chromatography ◽

Bacillus Sp ◽

Sds Page ◽

Fast Flow ◽

Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60Â°C.

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Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

Journal of Bacteriology ◽

10.1128/jb.181.1.91-99.1999 ◽

1999 ◽

Vol 181 (1) ◽

pp. 91-99 ◽

Cited By ~ 111

Author(s):

Hisayo Ono ◽

Kazuhisa Sawada ◽

Nonpanga Khunajakr ◽

Tao Tao ◽

Mihoko Yamamoto ◽

...

Keyword(s):

Molecular Mass ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Apparent Molecular Mass ◽

Optimum Temperature ◽

Sds Page ◽

Halomonas Elongata ◽

Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

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Purification and characterization of cytosolic pyruvate kinase from banana fruit

Biochemical Journal ◽

10.1042/bj3520875 ◽

2000 ◽

Vol 352 (3) ◽

pp. 875-882 ◽

Cited By ~ 11

Author(s):

William L. TURNER ◽

William C. PLAXTON

Keyword(s):

Pyruvate Kinase ◽

Specific Activity ◽

Gel Filtration ◽

Banana Fruit ◽

Ph Optimum ◽

Cytosolic Ph ◽

Pep Carboxylase ◽

Sds Page ◽

Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

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Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24

Biochemical Journal ◽

10.1042/bj3190977 ◽

1996 ◽

Vol 319 (3) ◽

pp. 977-983 ◽

Cited By ~ 11

Author(s):

Jeong Heon KO ◽

Cheorl Ho KIM ◽

Dae-Sil LEE ◽

Yu Sam KIM

Keyword(s):

Molecular Mass ◽

Specific Activity ◽

Gel Filtration ◽

Higher Plant ◽

Sds Page ◽

Thermus Caldophilus ◽

Internal Peptide ◽

Adp Glucose Pyrophosphorylase ◽

Fructose 1,6 Bisphosphate

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73–78 °C and its half-life was 30 min at 95 °C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.

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Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00053.x ◽

2005 ◽

Vol 37 (6) ◽

pp. 363-370 ◽

Cited By ~ 16

Author(s):

Ye-Yun Li ◽

Chang-Jun Jiang ◽

Xiao-Chun Wan ◽

Zheng-Zhu Zhang ◽

Da-Xiang Li

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Maximum Activity ◽

Exchange Chromatography ◽

Optimum Activity ◽

Development Of Resistance ◽

Sds Page ◽

C 50 ◽

Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

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