• Microbiology and Biotechnology Letters
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• v.50 no.2
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• pp.235-239
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• 2022

Psychrophiles have evolved to produce cold-adapted enzymes to enable survival in low-temperature environments. In this study, the cold adaptation of 5-enolpyruvylshikimate-3-phosphate synthase (CpsEPSPS) from Colwellia psychrerythraea, a model psychrophile, was analyzed. The optimum temperature for the activity of CpsEPSPS was found to be 25℃, with 35% activity remaining at 5℃. However, the unfolding temperature of CpsEPSPS was 54℃. This phenomenon is frequently observed in cold-active enzymes. As is the cases for most cold-active enzymes, the K_m values of CpsEPSPS for its substrates were higher than those of Escherichia coli EPSPS. These results indicate that CpsEPSPS is cold-adapted, but not perfectly.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.25-31
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• 2006

Changes of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in the glyphosate-tolerant Roundup Ready soybean were examined using purified CP4 EPSPS produced in cloned Escherichia coli as a control. CP4 EPSPS in genetically modified soybean was detected by twodimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) with databases. CP4 EPSPS in soybean products was resolved on 2-DE by first isoelectric focusing (IEF) based on its characteristic pI of 5.1, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) based on its molecular mass of 47.5 kDa. We quantified various percentages of soybean CP4 EPSPS. The quantitative analysis was performed using a 2D software program on artificial gels with spots varying in Gaussian volumes. These results suggested that 2-DE image analysis could be used for quantitative detection of GM soybean, unlike Western blotting.

• Journal of Microbiology and Biotechnology
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• v.28 no.8
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• pp.1384-1390
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• 2018

Glyphosate inhibits the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway. A mutant of EPSPS from Pantoea sp. was identified using site-directed mutagenesis. The mutant showed significantly improved glyphosate resistance. The mutant had mutations in three amino acids: Gly97 to Ala, Thr 98 to Ile, and Pro 102 to Ser. These mutation sites in Escherichia coli have been studied as significant active sites of glyphosate resistance. However, in our research, they were found to jointly contribute to the improvement of glyphosate tolerance. In addition, the level of glyphosate tolerance in transgenic Arabidopsis confirmed the potentiality of the mutant in breeding glyphosate-resistant plants.

• Plant Biotechnology Reports
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• v.4 no.4
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• pp.329-334
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• 2010

Multiple sequence alignments showed that the prolines at the 25th, 129th, 153rd, 242nd, 322nd, and 434th amino acids in 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium sp. strain CP4 are strongly conserved in various prokaryotic EPSPS proteins. Single point mutations of the conserved prolines to alanine (P25A, P153A, P242A, P322A, and P434A) were introduced in the CP4 EPSPS in order to investigate the importance of the conserved prolines for the enzyme properties. The point mutations caused decreases in substrate binding affinity and catalytic efficiency as well as the glyphosate resistance, in general. Especially, the 25th and 242nd prolines located in the polypeptide hinges connecting top and bottom domains of CP4 EPSPS as well as the 434th proline at the C-terminus of the enzyme turned out to be crucial for the enzyme activity.

• Food Science and Biotechnology
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• v.17 no.5
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• pp.1092-1096
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• 2008

Commercial food products from major cities of Coahuila, Mexico were screened to identify residues of transgenic deoxyribonucleic acid (DNA) and/or proteins. After performed, an inventory on all products that contained a soybean-based ingredient in a commercial grocery store in the city of Saltillo, Coahuila, Mexico, 245 food products were identified and grouped in 15 classes according to the soybean ingredient as well as the manufacturing process used for their elaboration. Similar sampling was made for the different food classes in the cities of Monclova, Piedras Negras, and Torreon. A total of 88 samples were analyzed and DNA was extracted by the hexadecyltrimethyl-ammonium bromide (CTAB) technique with slight modification to obtain better DNA quality (1). In addition, segments of the transgenic genes one that codifies for 5-enolpyruvylshikimate-3-phosphate synthase (epsps), cry 1A, and the cauliflower mosaic virus (CaMV) promoter were amplified using polymerase chain reaction (PCR). The transgenic proteins 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) and insecticidal crystal protein (Cry 1Ab/Ac) were identified using double antibody sandwich-enzymatic linked immunoassay analysis (DAS-ELISA). Presence of transgenic genes and/or proteins was identified in 35.3% of the commercial products samples.

• Journal of Plant Biotechnology
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• v.38 no.4
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• pp.272-277
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• 2011

Plants expressing Agrobacterium sp. strain CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) are known to be resistant to glyphosate, a potent herbicide that inhibits the activity of the endogenous plant EPSPS. In order to develop herbicide-resistant rice, we prepared transgenic rice plants with CP4 EPSPS gene under the control of CaMV 35S promoter for over-expression. A recombinant plasmid was transformed into rice via Agrobacterium-mediated transformation. A large number of transgenic rice plants were obtained with glyphosate and most of the transformants showed fertile. The integration and expression of CP4 EPSPS gene from regenerated plants was analyzed by Southern and northern blot analysis. The transgenic rice plants had CP4 EPSPS enzyme activity levels more than 15-fold higher than the wild-type plants. EPSPS enzyme activity of transgenic rice plants was also identified by strip-test method. Field trial of transgenic rice plants further confirmed that they can be selectively survived at 100% by spay of glyphosate (Roundup$^{(R)}$) at a regular dose used for conventional rice weed control.

• Journal of Applied Biological Chemistry
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• v.49 no.2
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• pp.65-68
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• 2006

• Korean Journal of Environmental Biology
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• v.39 no.4
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• pp.445-451
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• 2021

In this study, we described the production of an antibody to living modified organisms (LMOs) containing the gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens strain CP4 EPSPS provides resistance to the herbicide glyphosate (N- (phosphonomethyl) glycine). These LMOs were approved and have recently been used in the feed, food production, and processing industries in South Korea. Highly efficient monoclonal antibody (mAb) production is crucial for developing assays that enable the proper detection and quantification of the CP4 EPSPS protein in LMOs. This study describes the purification and characterization of recombinant CP4 EPSPS protein in E. coli BL21 (DE3) based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The production of mAbs was undertaken based on the standard operating procedure of Abclon, Inc.(South Korea), and the purity of the mAbs was assessed using SDS-PAGE. The following five mAb clones were produced: 2F2, 4B9, 6C11, 10A9, and 10G9. To verify the efficiency and specificity of the five developed mAbs, we performed Western blotting analysis using the LM (living modified) cotton crude extracts. All mAbs could detect the CP4 EPSPS protein in the LM cotton traits MON1445 and MON88913 with high specificity, but not in any other LM cottons or non-LM cottons. These data indicate that these five mAbs to CP4 EPSPS could be successfully used for the further development of antibody-based detection methods to target CP4 EPSPS protein in LMOs.

• Food Science and Biotechnology
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• v.15 no.6
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• pp.954-958
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• 2006

A comprehensive safety evaluation was conducted to assess the potential allergenicity of newly introduced proteins in genetically modified (GM) crops. We assessed the allergenicity of CP4 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in GM soybeans. This assessment was performed by IgE immunoblotting with soy-allergic children's sera, amino acid sequence homology with known allergens, and the digestibility of CP4 EPSPS. No differences in IgE-antigen binding by immunoblotting were found between GM soy samples and the corresponding non-GM samples. Based on the comparison of EPSPS amino acid sequence homology with current allergen databases, no known allergen was found. In addition, CP4 EPSPS protein was rapidly digested by simulated gastric fluid (SGF). Taken together, these results indicate that GM soybeans have no allergenicity in children and are as safe as conventional soybeans.

• Korean Journal of Food Science and Technology
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• v.36 no.3
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• pp.369-374
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• 2004

Genetically modified (GM) soybean Roundup Ready carries Agrobacterium sp. CP4 gene, which expresses 5-enolpyruvylshikimate-3-phosphate synthase (CP4EPSPS). CP4EPSPS in GM soybeans and soybean curds was screened using CP4EPSPS-specific polyclonal and monoclonal antibodies (pab and mab, respectively) by immunoblotting. Isolated recombinant CP4EPSPS was detected at detection limits of $0.006\;and\;0.0006{\mu}g$, whereas those of CP4EPSPS expressed in GM soybean were $0.001\;and\;0.0001{\mu}g$g, using mab and pab, respectively. From nine screened soybean curds, two had positive results with pab Immunoblotting method with pab and mab developed in this study could be applied to screen glyphosate-tolerant GM soybeans in soybean products.