scholarly journals Characterization of Two α-l-Arabinofuranosidases from Acetivibrio mesophilus and Their Synergistic Effect in Degradation of Arabinose-Containing Substrates

2021 ◽  
Vol 9 (7) ◽  
pp. 1467
Author(s):  
Yajing Liu ◽  
Sonja Vanderhaeghen ◽  
Werner Feiler ◽  
Angel Angelov ◽  
Melanie Baudrexl ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Maximum Activity ◽  
Cellulolytic Bacterium ◽  
Strong Activity ◽  
Interesting Candidate ◽  
Xylanolytic Enzymes ◽  
Genetic Context

Arabinofuranosidases are important accessory enzymes involved in the degradation of arabinose-containing poly- and oligosaccharides. Two arabinofuranosidases from the recently described novel anaerobic cellulolytic bacterium Acetivibrio mesophilus, designated AmAraf51 and AmAraf43, were heterologously expressed in Escherichia coli and biochemically characterized. AmAraf51 not only removed arabinose moieties at O-3, O-2 and terminal O-5 positions of arabinose-containing oligosaccharides, but also exhibited exo-β-xylosidase side activity. In comparison, AmAraf43 preferably cleaved 1,3-linkages from arabinosyl disubstitutions. AmAraf51 and AmAraf43 demonstrated maximum activity at 70 °C and 57 °C, respectively. Judging from the genetic context and substrate specificity, AmAraf51 may decompose internalized arabino/xylo-oligosaccharides. The embedding of the AmAraf43 gene between genes for several putative xylanolytic enzymes, along with its enzymatic properties suggests that AmAraf43 cleaves arabinose decorations from heteroxylans extracellularly. The enzymes revealed completely converse activity profiles towards arabinan/arabinoxylan: AmAraf51 displayed strong activity on arabinan, while AmAraf43 prefers arabinoxylan. AmAraf51 dramatically stimulated the saccharification level of wheat arabinoxylan (WAX-RS) and sugar beet arabinan when administered along with xylanase M_Xyn10 or arabinanase PpAbn43, respectively. For WAX-RS degradation, the yield of arabinose and xylose was boosted 13.77-fold and 4.96-fold, respectively. The bifunctional activity, thermostability and high catalytic efficiency make AmAraf51 an interesting candidate for industrial applications.

Functional characterization of X-prolyl aminopeptidase from Toxoplasma gondii

Parasitology ◽  
2016 ◽  
Vol 143 (11) ◽  
pp. 1443-1449 ◽  
Author(s):  
MINGFA YANG ◽  
JUN ZHENG ◽  
HONGLIN JIA ◽  
MINGXIN SONG
Keyword(s):  
Escherichia Coli ◽  
Toxoplasma Gondii ◽  
Divalent Cations ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Strong Activity ◽  
Inhibition Of Growth ◽  
Aminopeptidase P ◽  
Insight Into

SUMMARYIn the present study, a recombinant aminopeptidase P (rTgAPP) from Toxoplasma gondii was expressed in Escherichia coli to evaluate its enzyme parameters. The rTgAPP showed strong activity against a synthetic substrate for aminopeptidase P at pH 8·0 with a Km value of 0·255 µm and a kcat value of 35·6 s−1. The overall catalytic efficiency (kcat/Km) of the rTgAPP was 139·6 × 105 M−1 s−1. The activity of rTgAPP was enhanced by the addition of divalent cations and inhibited by bestatin. Deletion of TgAPP gene in the parasite through a CRISPR/Cas9 system resulted in inhibition of growth indicating the importance of TgAPP. Thus our findings reveal that TgAPP is an active enzyme in T. gondii and provide an insight into the function of TgAPP.

scholarly journals Biochemical and Structural Characterization of the Subclass B1 Metallo-β-Lactamase VIM-4

2010 ◽  
Vol 55 (3) ◽  
pp. 1248-1255 ◽  
Author(s):  
Patricia Lassaux ◽  
Daouda A. K. Traoré ◽  
Elodie Loisel ◽  
Adrien Favier ◽  
Jean-Denis Docquier ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Thermal Stability ◽  
Pseudomonas Aeruginosa ◽  
Structural Characterization ◽  
Catalytic Efficiency ◽  
X Ray ◽  
Stabilizing Effect ◽  
Loop 2

ABSTRACTThe metallo-β-lactamase VIM-4, mainly found inPseudomonas aeruginosaorAcinetobacter baumannii, was produced inEscherichia coliand characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn2+at concentrations ranging from 0.4 to 100 μM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 Å. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn2+ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.

Gene cloning and characterization of thiourocanate hydratase from Burkholderia sp. HME13

2019 ◽  
Vol 167 (3) ◽  
pp. 333-341
Author(s):  
Hisashi Muramatsu ◽  
Haruna Miyaoku ◽  
Syuya Kurita ◽  
Hidenori Matsuo ◽  
Takehiro Kashiwagi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Crude Extract ◽  
Maximum Activity ◽  
Luria Bertani ◽  
Sole Nitrogen Source ◽  
Gene Encoding ◽  
Gene Cloning And Characterization

Abstract A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The Km and Vmax values of thiourocanate hydratase towards thiourocanic acid were 30 μM and 7.1 μmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida, but thiourocanate hydratase had no urocanase activity.

Characterization of L-fucose isomerase from Paenibacillus rhizosphaerae to produce L-fuculose from hydrolyzed fucoidan and commercial fucose

2019 ◽  
Author(s):  
Muhammad Waheed Iqbal ◽  
Tahreem Riaz ◽  
Shahid Mahmood ◽  
Yingying Zhu ◽  
Dawei Ni ◽  
...  
Keyword(s):  
Industrial Applications ◽  
Maximum Activity ◽  
Lysosomal Disease ◽  
Simple Method ◽  
Enzymatic Production ◽  
Native Molecular Mass ◽  
Anti Cancer ◽  
Viral Hepatitis B ◽  
High Production

Abstract Background L-fuculose is an expensive and rare sugar used against different kinds of diseases such as HIV, anti-cancer, anti-viral, Hepatitis-B, human lysosomal disease (fucosidosis), and cardio-protective drugs. The enzymatic way of converting L-fucose into L-fuculose would be an effective method with great industrial applications. The purpose of this research is to introduce a high production of L-fuculose from cheap and natural sources (fucoidan) and commercially source (Sigma-Aldrich) by a recombinant enzyme L-fucose isomerase from Paenibacillus rhizosphaerae (Pa-LFI).Results Fucose containing polysaccharide (FPs) called fucoidan was extracted, hydrolyzed and characterized by U. pinnatifida for enzymatic production of L-fuculose. The FPs provide 35.9% of fucose along with few other monosaccharides. Pa-LFI was characterized and purified with a single band at 65 kDa. It showed an activity of 104.5 U mg -1 and exhibited as a hexamer with native molecular mass 396 kDa. The maximum activity for recombinant Pa-LFI was detected at pH 6.5 and 50 °C in 1 mM of Mn 2+ . The melting temperature observed 75 °C and half-life at 50 °C was 12.6 h. The isomerizing activity of Pa-LFI with aldose substrate (L-fucose) was higher exposing K m , k cat and k cat / K m 86.2 mM, 32831 min -1 and 335 min -1 mM -1 respectively. The conversion ratio of L-fuculose from 100 g L -1 of FPs and commercial fucose after the equilibrium state was about 6% (5.6 g L -1 ) and 30% (30.2 g L -1 ) respectively.Conclusion Pa-LFI catalyzed the reaction to convert L-fucose into L-fuculose. The enzyme will be helpful in the production of L-fuculose with an efficient and simple method without producing any by-product.

scholarly journals First Characterization of CTX-M-15-Producing Escherichia coli Strains Belonging to Sequence Type (ST) 410, ST224, and ST1284 from Commercial Swine in South America

2016 ◽  
Vol 60 (4) ◽  
pp. 2505-2508 ◽  
Author(s):  
Ketrin C. Silva ◽  
Marina Moreno ◽  
Carlos Cabrera ◽  
Beny Spira ◽  
Louise Cerdeira ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
South America ◽  
Insertion Sequence ◽  
Sequence Type ◽  
Content Type ◽  
First Time ◽  
Genetic Context ◽  
New Reservoir

ABSTRACTWe report for the first time the isolation of CTX-M-15-producingEscherichia colistrains belonging to sequence type (ST) 410, ST224, and ST1284 in commercial swine in Brazil. TheblaCTX-M-15gene was located on F-::A9::B1 and C1::A9::B1 IncF-type plasmids, surrounded by a new genetic context comprising the IS26insertion sequence truncated with the ISEcp1element upstream ofblaCTX-M-15. These results reveal that commercial swine have become a new reservoir of CTX-M-15-producing bacteria in South America.

scholarly journals Molecular and Biochemical Characterization of a β-Fructofuranosidase from Xanthophyllomyces dendrorhous

2008 ◽  
Vol 75 (4) ◽  
pp. 1065-1073 ◽  
Author(s):  
Dolores Linde ◽  
Isabel Macias ◽  
Luc�a Fern�ndez-Arrojo ◽  
Francisco J. Plou ◽  
Antonio Jim�nez ◽  
...  
Keyword(s):  
Total Mass ◽  
Structural Model ◽  
Biochemical Characterization ◽  
Sucrose Solution ◽  
Catalytic Efficiency ◽  
Maximum Activity ◽  
Xanthophyllomyces Dendrorhous ◽  
Glycosyl Hydrolases ◽  
Mature Polypeptide

ABSTRACT An extracellular β-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous was characterized biochemically, molecularly, and phylogenetically. This enzyme is a glycoprotein with an estimated molecular mass of 160 kDa, of which the N-linked carbohydrate accounts for 60% of the total mass. It displays optimum activity at pH 5.0 to 6.5, and its thermophilicity (with maximum activity at 65 to 70�C) and thermostability (with a T 50 in the range 66 to 71�C) is higher than that exhibited by most yeast invertases. The enzyme was able to hydrolyze fructosyl-β-(2→1)-linked carbohydrates such as sucrose, 1-kestose, or nystose, although its catalytic efficiency, defined by the k cat/Km ratio, indicates that it hydrolyzes sucrose approximately 4.2 times more efficiently than 1-kestose. Unlike other microbial β-fructofuranosidases, the enzyme from X. dendrorhous produces neokestose as the main transglycosylation product, a potentially novel bifidogenic trisaccharide. Using a 41% (wt/vol) sucrose solution, the maximum fructooligosaccharide concentration reached was 65.9 g liter−1. In addition, we isolated and sequenced the X. dendrorhous β-fructofuranosidase gene (Xd-INV), showing that it encodes a putative mature polypeptide of 595 amino acids and that it shares significant identity with other fungal, yeast, and plant β-fructofuranosidases, all members of family 32 of the glycosyl-hydrolases. We demonstrate that the Xd-INV could functionally complement the suc2 mutation of Saccharomyces cerevisiae and, finally, a structural model of the new enzyme based on the homologous invertase from Arabidopsis thaliana has also been obtained.

scholarly journals Characterization of an Agrobacterium tumefaciensd-Psicose 3-Epimerase That Converts d-Fructose to d-Psicose

2006 ◽  
Vol 72 (2) ◽  
pp. 981-985 ◽  
Author(s):  
Hye-Jung Kim ◽  
Eun-Kyung Hyun ◽  
Yeong-Su Kim ◽  
Yong-Joo Lee ◽  
Deok-Kun Oh
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Molecular Mass ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Maximal Activity ◽  
Turnover Number ◽  
Conversion Yield ◽  
Equilibrium Ratio

ABSTRACT The noncharacterized gene previously proposed as the d-tagatose 3-epimerase gene from Agrobacterium tumefaciens was cloned and expressed in Escherichia coli. The expressed enzyme was purified by three-step chromatography with a final specific activity of 8.89 U/mg. The molecular mass of the purified protein was estimated to be 132 kDa of four identical subunits. Mn2+ significantly increased the epimerization rate from d-fructose to d-psicose. The enzyme exhibited maximal activity at 50°C and pH 8.0 with Mn2+. The turnover number (k cat) and catalytic efficiency (k cat/Km ) of the enzyme for d-psicose were markedly higher than those for d-tagatose, suggesting that the enzyme is not d-tagatose 3-epimerase but d-psicose 3-epimerase. The equilibrium ratio between d-psicose and d-fructose was 32:68 at 30°C. d-Psicose was produced at 230 g/liter from 700-g/liter d-fructose at 50°C after 100 min, corresponding to a conversion yield of 32.9%.

scholarly journals A study of production, purification and characterization of amylases from Escherichia coli for medical uses

2018 ◽  
Vol 9 (SPL1) ◽  
Author(s):  
Baydaa Abood Hassan
Keyword(s):  
Escherichia Coli ◽  
Incubation Temperature ◽  
High Titer ◽  
Synthetic Medium ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Ammonium Sulphate Precipitation ◽  
Enhanced Production ◽  
Sds Page

This study was conducted in the laboratories of Biology Department,facultyofScience,which deal with isolation,purification and characterization ofof amylase by Escherichia coli which carried out for enhanced production of amylase using starch (1%) asthe substrate of enzyme, the production was carried out by submerged fermentation, the best conditions were the isolated ofamylase in synthetic medium, it gave high titer of amylase activity, the ammonium sulfate as nitrogen source, incubation period 48 h, the starch as carbon source,, incubation temperature 30 °C and pH = 7, The enzyme was purified using ammonium sulphate precipitation(60%) anddialysis, the purified amylase had a maximum activity at pH =7,the amylase was stable with pH values ranging between (7 - 8) and in temperature 30 °C also amylase was stable in (30- 40 ) °C analyses of the amylase for molecular weight was carried out by SDS-PAGE electrophoresiswhich revealed 52 KDa.

scholarly journals Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine

1999 ◽  
Vol 340 (3) ◽  
pp. 639-647 ◽  
Author(s):  
Gyles E. COZIER ◽  
Raff A. SALLEH ◽  
Christopher ANTHONY
Keyword(s):  
Escherichia Coli ◽  
Electron Transfer ◽  
Active Site ◽  
Marked Decrease ◽  
Glucose Dehydrogenase ◽  
Catalytic Efficiency ◽  
Prosthetic Group ◽  
Competitive Inhibitors ◽  
A Site

The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH 7 and pH 8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process.

scholarly journals Characterization of a Novel IncHI2 Plasmid Carrying Tandem Copies ofblaCTX-M-2in afosA6-Harboring Escherichia coli Sequence Type 410 Strain

2016 ◽  
Vol 60 (11) ◽  
pp. 6742-6747 ◽  
Author(s):  
Qinglan Guo ◽  
Baixing Ding ◽  
Thomas Jové ◽  
Nicole Stoesser ◽  
Vaughn S. Cooper ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
The United States ◽  
Sequence Type ◽  
Clinical Strain ◽  
Content Type ◽  
Lactamase Gene ◽  
Genetic Context

ABSTRACTThe extended-spectrum β-lactamase geneblaCTX-M-2is mainly associated with ISCR1embedded in complexsul1-type integrons, but information on the genetic context of plasmids harboring the ISCR1-blaCTX-M-2module remains limited. In this study, ablaCTX-M-2-harboring plasmid (pYD786-1) belonging to the sequence type 2 (ST2)-IncHI2 plasmid type and isolated from anEscherichia coliST410 clinical strain was sequenced and analyzed. pYD786-1 belongs to the APEC-O1-R-type IncHI2 plasmids, which are widely distributed in human, poultry, and livestock strains. It contains a multidrug resistance mosaic region (MRR) consisting of a Tn21::In2 transposon backbone augmented by acquisition of duplicate ISCR1-blaCTX-M-2modules. Tn2411, a Tn21::In2 precursor, likely played a role in the generation of the MRR in pN13-01290_23, the putative progenitor plasmid of pYD786-1, found in a foodborneSalmonellastrain. Tn21/Tn2411::In::ISCR1-blaCTX-M-2derivatives, including pYD786-1, have been identified in strains from Europe, South America, and the United States, suggesting potential global dissemination of theblaCTX-M-2modules mediated by this vehicle.

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