Difference between revisions of "Part:BBa K2934000"

Revision as of 07:26, 21 October 2019

Glucose Oxidase-Histag A. niger optimized for B. subtilis

This part is coding for the Glucose Oxidase (GOx) enzyme, which catalyzes the oxidation of glucose to D-glucono-lacton and releases hydrogen peroxide. The sequence includes His-tag (6xHis), connected with seven amino acids linker protein at the N-terminus of the protein. The enzyme derived from A. niger and is codon-optimized for B. subtilis.

Usage and Biology

figure 1: the enzymatic reaction of glucose oxidase

Glucose oxidase (GOx) catalyzes the oxidation of glucose by using molecular oxygen to create hydrogen peroxide and D-glucono lactone. GOx requires the cofactor flavin adenine dinucleotide (FAD) that acts as an electron acceptor in a redox reaction. During the reaction, FAD is reduced to FADH2, followed by oxidation using molecular oxygen, which is then reduced to hydrogen peroxide [1].

References

[1] Frederick KR, Tung J, Richard S. Emerick F, Chamberlain SH, Vasavada A, Steven R, Chakraborty S, Schopter LM, Schopter LM, Massey V. 1990. Glucose Oxidase from Aspergillus niger. J Biol Chem 265:3793–3802.

Sequence and Features

Glucose Oxidase Activity Assay

Glucose Oxidase (GOx) catalyzes the oxidation of glucose to D-gluconic-lactone and hydrogen peroxide. Therefore, a precise method to determine the amount of hydrogen peroxide found in the solution could indicate the activity of the glucose oxidase. In this experiment, we used the assistance of another commercial enzyme- horseradish peroxidase (HRP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). Using hydrogen peroxide, the HRP catalyzes the oxidation of reduced ABTS, which is colorless, to oxidized ABTS, which is turquoise. While using excess ABTS and HRP, the absorbance of 416 nm light could indicate to us the amount of hydrogen peroxide created by the glucose oxidase, and therefore - the glucose oxidase activity.

To verify that the absorbance-activity relation remains linear under the experiment's conditions, we performed the experiment using commercial GOx with increasing activity, measured the 416 nm light absorbance and plotted a graph to demonstrate that relation. Fortunately, we have proved the absorbance-activity relation linearity under the experiment's conditions (Figure 2).

figure 2: Calibration curve for glucose oxidase activity – change in absorbance at 416 nm in different activity values

After the cloning process had been completed, we performed the activity test on both the supernatant and the lysate of genetically engineered bacteria as well as WT bacteria as control. The first colony tested contained a gene for glucose oxidase and the AmyE signal peptide. The absorbance detected in the recombinant Bacteria was significantly higher, in both supernatant and lysate (Figure 3). The evaluated activity of glucose oxidase is 0.46U/ml in the bacterial supernatant and 0.45U/ml in the lysate solution.

figure 3: The activity of glucose oxidase and AmyE in different samples, indicated by the absorbance at 416 nm.

Glucose Oxidase pH Influence

The assay of both glucose oxidase and catalase relied on another enzyme present in the solution, horseradish peroxidase (HRP). Although the HRP was added to the solution in excess, we wanted to verify that the only variant is the glucose oxidase activity, i.e. that the other parameters of the experiment– HRP, ABTS, and hydrogen peroxide - are not affected by the pH level. Therefore, we had conducted a test using constant amounts of hydrogen peroxide, HRP and ABTS, and pH level as the sole variable. After we measured the absorbance, no significant difference observed between the different PH levels, implies that the only parameter afflicted by the pH is the enzyme teste (Figure 4).

figure 4: verification of the method reliability by measuring HRP absorbance at 416nm for different pH levels.

After we had verified the reliability of the method, we have performed the activity test. The results (Figure 5) indicate that glucose oxidase is nearly unaffected by the pH decrease.

figure 5: Glucose oxidase activity in various pH levels by measuring the absorbance at 416nm.