title: Microbial surface displaying formate dehydrogenase and its application in optical detection of formate.
abstract: In the present work, NAD(+)-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD(+), and the production of NADH can be detected spectrometrically at 340nm. After induction for 24h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50°C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4°C for one month with the half-life of 17days at 40°C and 38h at 50°C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5-700μM and a low limit of detection of 2μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.
authors.affiliation: Institute for Biosensing, Key Laboratory for Biosensors of Shangdong Province, and College of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071, China. Electronic address: liuah@qdu.edu.cn.
authors.forename: Aihua
authors.initials: A
authors.lastname: Liu
authors.affiliation: Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors.forename: Ruirui
authors.initials: R
authors.lastname: Feng
authors.affiliation: Institute for Biosensing, Key Laboratory for Biosensors of Shangdong Province, and College of Chemistry and Chemical Engineering, Qingdao University, Qingdao 266071, China; Laboratory for Biosensing, Qingdao Institute of Bioenergy & Bioprocess Technology, and Key Laboratory of Bioenergy, Chinese Academy of Sciences, 189 Songling Road, Qingdao 266101, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Liang
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D002175:Candida / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005560:Formate Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D005561:Formates / Q000032:analysis*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D007700:Kinetics; D057230:Limit of Detection; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D005561:Formates; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; C063113:ice nucleation protein; C030544:formic acid; D005560:Formate Dehydrogenases
keywords: Formate; Formate assay; Formate dehydrogenase; Microbial surface display; Tolerance to solvents