Phosphoglucomutase (PGM) is a ubiquitous enzyme that is expressed in all organisms from bacteria to plants to animals and controls a key branch point for carbohydrate metabolism. PGM catalyzes the interconversion of glucose-1-phosphate and glucose-6-phosphate. In this process, the enzyme links various catabolic pathways to yield energy ATP or reducing power NAD(P)H, and several anabolic pathways, such as to lead to the synthesis of polysaccharides [1][2][3][4].Due to its importance, measurements for the enzymatic activity of PGM have been widely performed, by the use of optical methods with coupled enzyme system [3][4][5][6][7][8][9][10][11], or the use of a combination of ion/molecule reactions and FT-ICR mass spectrometry [12]. However, these measurements either result in a long detection time, a large amount of enzyme consumption, or complex procedures which need to be performed by skilled personnel. The importance of amperometric enzyme-based biosensors has increased considerably in recent years thanks to the advantages of being highly sensitive, rapid, accurate, economical and easy-to-handle for specific measurement of target analyte in complex matrices such as blood, food product and This is the Pre-Published Version 2 environmental sample. To the best of our knowledge, there is no report for the determination of PGM activity using an amperometric biosensor.In this work, we present an amperometric determination of PGM activity with a bienzyme screen-printed biosensor. As shown in Fig.1, the principle is as follows: Phosphoglucomutase (PGM, EC 5.4.2.2, from rabbit muscle, Sigma) converts glucose-1-phosphate to glucose-6-phosphate. Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49, from Leuconostoc mesenteroides, Sigma) catalyzes the specific dehydrogenation of glucose-6-phosphate by consuming NAD + . The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by salicylate hydroxylase (SHL, EC 1.14.13.1, from Pseudomonas sp., GDS Technology Inc., USA) in the presence of oxygen to produce catechol, which results in a detectable signal due to its oxidation at the working electrode.The bienzyme electrode for the measurement of PGM activity is based on the detection of glucose-6-phosphate, the product of the PGM-catalyzed reaction. From literature, glucose-6-phosphate biosensors have been developed by several methods, including the combinations of G6PDH and various mediators, or the combination of phosphatase and glucose oxidase [13][14][15].In this work, the successful coupling of SHL and G6PDH on a screen-printed electrode can also serve as a glucose-6-phosphate biosensor.A potentiostat EP30 (Biometria, Germany) and a computer installed with the software FIABOLO were used. The screen-printed electrode (BioSensorTrend, Germany) with a 2-electrode configuration was composed of a platinum (Pt) working electrode (diameter: 1 mm) and an Ag/AgCl reference/counter electrode. A measuring cell (volume: 1 ml), connected to a syringe and installed with two connectors (cathode an...