Characterization of Affinity-Purified Isoforms of<i>Acinetobacter calcoaceticus</i>Y1 Glutathione Transferases

Chee, C. P.; Tan, İlhan; Alias, Zazali

doi:10.1155/2014/750317

Cited by 5 publications

(4 citation statements)

References 37 publications

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“…In the present work, GPx and GST were purified using column chromatography resulting in 3-and 2-fold purified yield of 42.4 and 39.8%, respectively. Similar findings were reported by Yamashita et al (2012), Chee et al (2014), andIbrahim et al (2016). Furthermore, our purified GST and GPx showed a single protein band weighing 25 and 18 kDa, respectively, following SDS-PAGE and pIs of 7 and 5, respectively.…”

Section: Discussionsupporting

confidence: 90%

“…Multiple isoforms of glutathione s-transferases were scarcely reported in bacterial cells in comparison to eukaryotic cells (Chee et al, 2014). In the present work, GPx and GST were purified using column chromatography resulting in 3-and 2-fold purified yield of 42.4 and 39.8%, respectively.…”

Section: Discussionmentioning

confidence: 88%

“…Furthermore, our purified GST and GPx showed a single protein band weighing 25 and 18 kDa, respectively, following SDS-PAGE and pIs of 7 and 5, respectively. However, Chee et al (2014) mentioned that they purified and characterized two isoforms of glutathione stransferase from Acinetobacter calcoaceticus Y1; GST1 and GST2. The first isoform showed a single protein band at 23 kDa following SDS-PAGE while GST2 formed some aggregates due to posttranslational modification presenting a molecular weight of 115 kDa.…”

Section: Discussionmentioning

confidence: 99%

“…The concentrated purified GST and GPx solutions were subjected to quantification of the protein content as well as determination of the specific activity as previously determined by Zhang et al (2007) and the enzymatic assay kit. The purified GST and GPx samples were subjected to Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (12% SDS-PAGE) and isoelectric focusing (IEF) that were carried out according to Laemmli (1970) and Chee et al (2014). For characterization of the purified enzymes, GST and GPx solutions were divided into a number of aliquots to investigate the effect of temperature and pH on the enzymatic properties.…”

Section: Purification and Characterization Of Gst And Gpx Enzymesmentioning

confidence: 99%

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Stress-Based Production, and Characterization of Glutathione Peroxidase and Glutathione S-Transferase Enzymes From Lactobacillus plantarum

Al-Madboly

Ali

Fakharany

et al. 2020

Front. Bioeng. Biotechnol.

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More attention has been recently directed toward glutathione peroxidase and s-transferase enzymes because of the great importance they hold with respect to their applications in the pharmaceutical field. This work was conducted to optimize the production and characterize glutathione peroxidase and glutathione s-transferase produced by Lactobacillus plantarum KU720558 using Plackett-Burman and Box-Behnken statistical designs. To assess the impact of the culture conditions on the microbial production of the enzymes, colorimetric methods were used. Following data analysis, the optimum conditions that enhanced the s-transferase yield were the De Man-Rogosa-Sharp (MRS) broth as a basal medium supplemented with 0.1% urea, 0.075% H 2 O 2 , 0.5% 1-butanol, 0.0125% amino acids, and 0.05% SDS at pH 6.0 and anaerobically incubated for 24 h at 40 • C. The optimum s-transferase specific activity was 1789.5 U/mg of protein, which was ∼12 times the activity of the basal medium. For peroxidase, the best medium composition was 0.17% urea, 0.025% bile salt, 7.5% Na Cl, 0.05% H 2 O 2 , 0.05% SDS, and 2% ethanol added to the MRS broth at pH 6.0 and anaerobically incubated for 24 h at 40 • C. Furthermore, the optimum peroxidase specific activity was 612.5 U/mg of protein, indicating that its activity was 22 times higher than the activity recorded in the basal medium. After SDS-PAGE analysis, GST and GPx showed a single protein band of 25 and 18 kDa, respectively. They were able to retain their activities at an optimal temperature of 40 • C for an hour and pH range 4-7. The 3D model of both enzymes was constructed showing helical structures, sheet and loops. Protein cavities were also detected to define druggable sites. GST model had two large pockets; 185Å3 and 71 Å3 with druggability score 0.5-0.8. For GPx, the pockets were relatively Al-Madboly et al.Enzymes Production From Lactobacillus plantarum smaller, 71 Å3 and 32 Å3 with druggability score (0.65-0.66). Therefore, the present study showed that the consortium components as well as the stress-based conditions used could express both enzymes with enhanced productivity, recommending their application based on the obtained results.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%