Partial purification, characterization and wheat bran degradation studies of a new phytase from the Bacillus megaterium EBD 9-1 strain

2017 ◽  
Vol 42 (3) ◽  
Author(s):  
Elif Demirkan ◽  
Tuba Sevgi ◽  
Dilara Akcakoca ◽  
Figen Ersoy
Keyword(s):  
Gel Filtration ◽  
Partial Purification ◽  
The Novel ◽  
Bacterial Identification ◽  
Ammonium Sulfate Precipitation ◽  
16S Rrna Analysis ◽  
Sequence Identity ◽  
And Storage ◽  
Identification And Characterization

AbstractObjective:The present study was designed to report the bacterial identification and characterization of a new phytase enzyme from aMethods:sp. strain was identified based on 16S rRNA analysis. The phytase was partially purified through ammonium sulfate precipitation and Sephadex G100 gel filtration steps, and characterized for its activity and stability.Results:The new isolate EBD 9-1 showed 100% sequence identity withConclusion:Consequently, due to the characteristics such as significant stability at higher temperatures, alkaline pH and storage of the novel phytase enzyme produced by

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

1992 ◽  
Vol 288 (2) ◽  
pp. 475-482 ◽  
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.

scholarly journals Identification and Characterization of the Phage Gene sav, Involved in Sensitivity to the Lactococcal Abortive Infection Mechanism AbiV

2009 ◽  
Vol 75 (8) ◽  
pp. 2484-2494 ◽  
Author(s):  
Jakob Haaber ◽  
Geneviève M. Rousseau ◽  
Karin Hammer ◽  
Sylvain Moineau
Keyword(s):  
Gel Filtration ◽  
Point Mutations ◽  
Whole Genome ◽  
Wild Type ◽  
Abortive Infection ◽  
Native Form ◽  
Phage Gene ◽  
Infection Mechanism ◽  
Identification And Characterization

ABSTRACT Lactococcus lactis phage mutants that are insensitive to the recently characterized abortive infection mechanism AbiV were isolated and analyzed in an effort to elucidate factors involved in the sensitivity to AbiV. Whole-genome sequencing of the phage mutants p2.1 and p2.2 revealed mutations in an orf that is transcribed early, indicating that this orf was responsible for AbiV sensitivity. Sequencing of the homologous regions in the genomes of other AbiV-insensitive mutants derived from p2 and six other lactococcal wild-type phages revealed point mutations in the homologous orf sequences. The orf was named sav (for sensitivity to AbiV), and the encoded polypeptide was named SaV. The purification of a His-tagged SaV polypeptide by gel filtration suggested that the polypeptide formed a dimer in its native form. The overexpression of SaV in L. lactis and Escherichia coli led to a rapid toxic effect. Conserved, evolutionarily related regions in SaV polypeptides of different phage groups are likely to be responsible for the AbiV-sensitive phenotype and the toxicity.

scholarly journals Tannin Degradation by a Novel Tannase Enzyme Present in Some Lactobacillus plantarum Strains

2014 ◽  
Vol 80 (10) ◽  
pp. 2991-2997 ◽  
Author(s):  
Natalia Jiménez ◽  
María Esteban-Torres ◽  
José Miguel Mancheño ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Tannic Acid ◽  
Plant Material ◽  
Aliphatic Alcohol ◽  
Methyl Gallate ◽  
Content Type ◽  
Sequence Identity ◽  
Tannin Degradation ◽  
Identification And Characterization

ABSTRACTLactobacillus plantarumis frequently isolated from the fermentation of plant material where tannins are abundant.L. plantarumstrains possess tannase activity to degrade plant tannins. AnL. plantarumtannase (TanBLp, formerly called TanLp1) was previously identified and biochemically characterized. In this study, we report the identification and characterization of a novel tannase (TanALp). While all 29L. plantarumstrains analyzed in the study possess thetanBLpgene, the genetanALpwas present in only four strains. Upon methyl gallate exposure, the expression oftanBLpwas induced, whereastanALpexpression was not affected. TanALpshowed only 27% sequence identity to TanBLp, but the residues involved in tannase activity are conserved. Optimum activity for TanALpwas observed at 30°C and pH 6 in the presence of Ca2+ions. TanALpwas able to hydrolyze gallate and protocatechuate esters with a short aliphatic alcohol substituent. Moreover, TanALpwas able to fully hydrolyze complex gallotannins, such as tannic acid. The presence of the extracellular TanALptannase in someL. plantarumstrains provides them an advantage for the initial degradation of complex tannins present in plant environments.

scholarly journals Identification and Characterization of a Porcine Torovirus

1998 ◽  
Vol 72 (5) ◽  
pp. 3507-3511 ◽  
Author(s):  
A. Kroneman ◽  
L. A. H. M. Cornelissen ◽  
M. C. Horzinek ◽  
R. J. de Groot ◽  
H. F. Egberink
Keyword(s):  
Longitudinal Study ◽  
Neutralizing Antibodies ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Nucleocapsid Gene ◽  
Sequence Identity ◽  
Young Pigs ◽  
Porcine Torovirus ◽  
Identification And Characterization

ABSTRACT A porcine torovirus (PoTV) was identified and characterized; it is a novel member of the genus Torovirus (familyCoronaviridae, order Nidovirales), closely related to but clearly distinct from the already recognized equine torovirus (ETV) and bovine torovirus (BoTV) representatives. Immunoelectron microscopy of feces from piglets revealed elongated, 120- by 55-nm particles which were recognized by a torovirus-specific antiserum. Amplification by reverse transcriptase (RT) PCR with primers designed to detect conserved regions (on the basis of the genomes of BoTV strain Breda and ETV strain Berne) resulted in the identification of the 489-bp nucleocapsid gene, encoding a 18.7-kDa protein. The sequence identity in this region between PoTV and both ETV and BoTV was only about 68%, whereas the latter two show 81% identity. Neutralizing antibodies directed against ETV were found in sera of adult and young pigs. In all 10 herds sampled, seropositive animals were present, and 81% of randomly selected adult sows possessed antibodies. A longitudinal study with RT PCR showed that piglets shed virus in the feces for 1 or more days, starting 4 to 14 days after weaning.

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.

Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis

1986 ◽  
Vol 32 (10) ◽  
pp. 765-771 ◽  
Author(s):  
A. Gálvez ◽  
M. Maqueda ◽  
E. Valdivia ◽  
A. Quesada ◽  
E. Montoya
Keyword(s):  
Broad Spectrum ◽  
Gel Filtration ◽  
Basal Medium ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Gram Negative Bacteria ◽  
Broad Spectrum Antibiotic ◽  
Streptococcus Faecalis ◽  
Group D

Streptococcus faecalis S-48 produces a broad spectrum antibiotic, active against Gram-positive and Gram-negative bacteria. This substance is produced in solid and liquid media and also in a defined basal medium. It is sensitive to protease, pronase, or trypsin, heating at 70 °C, and alkaline pH, but resistant to treatment with lipase, lysozyme, alkaline phosphatase, DNAase, RNAase, acidic or neutral pHs, and also lower temperatures (60 °C). Several organic solvents cause precipitation, but not inactivation. This antibiotic has been partially purified by gel filtration and further ion-exchange chromatography. Its molecular weight has been estimated close to 2000. The biological activity of this antagonistic substance against the selected indicator strains, Streptococcus faecalis S-47 and Escherichia coli U-9, is bactericidal. The characterization of this substance, initially classified as a bacteriocin, indicates that it is an antibiotic of peptidic nature. The significance of antibiotic occurrence in group D of the genus Streptococcus is also discussed.

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