title: Bacterial cell-surface displaying of thermo-tolerant glutamate dehydrogenase and its application in L-glutamate assay.
abstract: In this paper, glutamate dehydrogenase (Gldh) is reported to efficiently display on Escherichia coli cell surface by using N-terminal region of ice the nucleation protein as an anchoring motif. The presence of Gldh was confirmed by SDS-PAGE and enzyme activity assay. Gldh was detected mainly in the outer membrane fraction, suggesting that the Gldh was displayed on the bacterial cell surface. The optimal temperature and pH for the bacteria cell-surface displayed Gldh (bacteria-Gldh) were 70°C and 9.0, respectively. Additionally, the fusion protein retained almost 100% of its initial enzymatic activity after 1 month incubation at 4°C. Transition metal ions could inhibit the enzyme activity to different extents, while common anions had little adverse effect on enzyme activity. Importantly, the displayed Gldh is most specific to l-glutamate reported so far. The bacterial Gldh was enabled to catalyze oxidization of l-glutamate with NADP(+) as cofactor, and the resultant NADPH can be detected spectrometrically at 340nm. The bacterial-Gldh based l-glutamate assay was established, where the absorbance at 340nm increased linearly with the increasing l-glutamate concentration within the range of 10-400μM. Further, the proposed approach was successfully applied to measure l-glutamate in real samples.
authors: Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, and College of Chemistry and Chemical Engineering, Ocean University of China, 238 Songling Road, Qingdao 266100, China; Laboratory for Biosensing, and Key Laboratory of Biofuels, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors: Jianxia
authors: J
authors: Song
authors: Laboratory for Biosensing, and Key Laboratory of Biofuels, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors: Bo
authors: B
authors: Liang
authors: Laboratory for Biosensing, and Key Laboratory of Biofuels, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors: Dongfei
authors: D
authors: Han
authors: Laboratory for Biosensing, and Key Laboratory of Biofuels, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors: Xiangjiang
authors: X
authors: Tang
authors: Laboratory for Biosensing, and Key Laboratory of Biofuels, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors: Qiaolin
authors: Q
authors: Lang
authors: Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, and College of Chemistry and Chemical Engineering, Ocean University of China, 238 Songling Road, Qingdao 266100, China.
authors: Ruirui
authors: R
authors: Feng
authors: Key Laboratory of Marine Chemistry Theory and Technology of Ministry of Education, and College of Chemistry and Chemical Engineering, Ocean University of China, 238 Songling Road, Qingdao 266100, China. Electronic address: lhhan@ouc.edu.cn.
authors: Lihui
authors: L
authors: Han
authors: Laboratory for Biosensing, and Key Laboratory of Biofuels, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China. Electronic address: liuah@qibebt.ac.cn.
authors: Aihua
authors: A
authors: Liu
mesh_terms: D000838:Anions / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001709:Biotechnology; D002462:Cell Membrane / Q000201:enzymology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000838:Anions; D001425:Bacterial Outer Membrane Proteins; D004800:Enzymes, Immobilized; D029968:Escherichia coli Proteins; D008670:Metals; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein; D018698:Glutamic Acid; D005969:Glutamate Dehydrogenase
keywords: Bacterial surface display; Enzyme inhibition; Thermo-tolerant glutamate dehydrogenase; l-Glutamate; l-Glutamate detection