Various neurotransmitters in the brain are closely related to amino acid metabolism in food. The glutamate is the precursor of γ-aminobutyric acid, and γ-aminobutyrate expression level can affect ASD symptoms such as sleep disorders and affective disorders. Studies have found that the blood levels of glutamate rise significantly in ASD.

This project develops a glutamate dehydrogenase activity detection system to verify if it could be used to diagnose ASD. In this project, we constructed an inak-gldh expression plasmid and transformed it into BL21(DE3) competent cells. We detected the expression level of inak-gldh by SDS-PAGE, and measured the enzyme activity by adding L-glutamate to the reaction system. Our experimental data indicated its significant effect on glutamic acid dehydrogenation. Therefore, our product will provide an efficient ASD-detecting biosensor.

Because the T7 promoter and T7 RNA polymerase have strong ability in translation and usually be used as protein expression, we chose pET28a-vector and E. coli BL21(DE3), with T7 promoter and T7 RNA polymerase respectively, to express our target protein inak-gldh. To achieve this, we optimized the DNA sequences of inak-gldh and inserted them into the HindIII and NcoI sites of the pET28a vector (Figure 1.), and transformed the recombinant plasmid into E. coli BL21(DE3) for protein expression.

To build the plasmid, we let the synthetic company synthesize the DNA fragment of optimized inak-gldh and inserted it into the HindIII and NcoI sites of the pET28a vector (Figure 2A). Then, we send it to the company for Sanger sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 2B), and the plasmid pET28a- inak-gldh was successfully constructed. And the last step was extracting the recombinant plasmid and transforming it into E. coli BL21(DE3) competent cells, so that can be used to express the inak-gldh proteins.

In order to verify the inak-gldh protein expression level, we cultured pET28a- inak-gldh containing BL21(DE3) strain in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.6. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used the SDS-PAGE method to verify the expression level of the target protein (Figure 3). As a result, we could significantly find the band at the correct size.

Glutamate dehydrogenase could use L-glutamate as a substrate, with reversible oxidation and deamination under the action of coenzyme (NAD + or NADP +), and the NADH or NADPH generated by the reaction has an obvious absorption peak at 340 nm. To detect glutamate dehydrogenase activity, the amount of NADPH generated by the hydrogenase catalytic reaction was measured spectrophotometrically. Absorption values at 340 nm were determined using a UV-visible spectrophotometer (Figure 4). One unit of enzymatic activity was defined as the production of 1 μ mol of the reduced product, NADPH, per OD600 cells per minute.

NADPH was diluted to 10 μM, 50 μM, 100 μM, 400 μM, 500 μM, and 500 μM, and the absorption peaks at 340nm at each concentration were measured by spectrophotometer. With the light absorption value of NADPH at 340 nm as the vertical coordinate and the corresponding concentration of NADPH (μM) as the abscissa, the linear fitting was performed to obtain the linear regression equation corresponding to the standard curve and the standard curve.

Cells containing the expression vector pET28a-inaK-gldh were centrifuged and washed twice with 100mM Tris-HCl (pH 8.0) buffer after induction. The standard reaction system contained bacterial cells (OD600-1.0), 100mM Tris-HC1 buffer (pH 8.0), sodium L-glutamate (final concentration 2mM), and NADP + (final concentration 0.5mM). The reaction was performed at 60°C in a 1.5mL centrifuge tube for 2min and was terminated by centrifugation of the bacteria at 12,000 rpm for 1min. The absorptive values at 340 nm were measured using a UV-visible spectrophotometer. As a result, the enzyme activity of glutamine dehydrogenase is around 567.92975 U/mL. So that the biosensor we constructed worked well.

The enzyme activity calculation formula is as follows

* Vsample: 0.05 mL, V total: 1×10^-3 L, ε: NADH molar extinction coefficient, 6.22×10³ L/mol/cm, d: Cuvette light diameter, 1 cm, ∆T: Reaction time, 2 min.

We have already collected the figures from our experiments. Glutamate dehydrogenase plays an important role in L-glutamate metabolism. After transforming the sequence-optimized gene inaK-gldh into the host strain, we can easily detect the metabolites NADPH using our biosensor. What’s more, we measured the activity of inaK-gldh, and found that the inaK-gldh worked well in the E. coli system.

The task of developing new portable tests and effective treatments is imminent. In the future, when the biosensor is improved, it may be applied to ASD detection, which can provide a new tool for disease diagnosis, and the earlier the diagnosis is made, the better the outcome.