Bacterial cell-surface displaying of thermo-tolerant glutamate dehydrogenase and its application in l-glutamate assay |
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| Affiliation: | 1. 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;2. Laboratory for Biosensing, and Key Laboratory of Biofuels, Qingdao Institute of Bioenergy & Bioprocess Technology, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China;1. A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, 119071, Moscow, Russian Federation;2. Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, bld. 2, 119071 Moscow, Russian Federation;3. NBICS Center, National Research Centre “Kurchatov Institute”, Akad. Kurchatova sqr 1, 123182 Moscow, Russian Federation;1. Department of Medicine and Surgery, University of Parma, Italy;2. Department of Chemistry, Life Science and Environmental Sustainability, University of Parma, Italy;1. College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, People’s Republic of China;2. College of Forest Resources Environment, Nanjing Forestry University, Nanjing 210037, People’s Republic of China;1. Department of Bioprocess and Polymer Engineering, School of Chemical and Energy Engineering, Faculty of Engineering, Universiti Teknologi Malaysia, 81310, Skudai, Johor, Malaysia;2. School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia;3. Synthetic Biology and Cell Factory Unit, Malaysia Genome Institute, Jalan Bangi, 43000, Kajang, Selangor, Malaysia;1. Department of Microbiology, Biochemistry and Molecular Genetics, New Jersey Medical School, Rutgers University, Newark, NJ, 07103, USA;2. State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China |
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| 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 340 nm. The bacterial-Gldh based l-glutamate assay was established, where the absorbance at 340 nm increased linearly with the increasing l-glutamate concentration within the range of 10 − 400 μM. Further, the proposed approach was successfully applied to measure l-glutamate in real samples. |
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| Keywords: | Bacterial surface display Thermo-tolerant glutamate dehydrogenase Enzyme inhibition |
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