Part:BBa_K2944003
pTDH3-GOx-tPGK1
This coding sequences encodes a constitutive yeast promoter regulating expression of the glucose oxidase enzyme from Aspergillus niger. The gene sequence has been optimized for Saccharomyces cerevisiae. Glucose oxidase catalyzes the oxidation of glucose to D-glucono-1,5-lactone and hydrogen peroxide.
Characterization by Mass Spectrometry.
In HPLC-MS, samples are purified using HPLC and are then analyzed using mass spectroscopy to determine the compound. This system can not only identify samples with high accuracy, but also quantitate them based on the area of the peak producing the signal of interest.
Preparation of Standard Curve
https://2019.igem.org/wiki/images/8/8f/T--Concordia-Montreal--GoxCharacterization.pdf
Activity of Glucose Oxidase was measured reacting the peroxide produce by the catalysis of glucose into gluconate with ammonium molybdate. The product of this reaction produces a yellow color that absorbs light at 405nm. The change in absorbance over time can be measured to determine if glucose oxidase is indeed present in the system.
Figure 1 – Absorbance of Cell Supernatant Over Time in the Presence of 100mM Ammonium Molybdate.
The sample was subjected to a solution of 10mM D-glucose and 100mM ammonium molybdate. The absorbance was measured periodically over a time frame of three hours.
Figure 2 Calibration Curve of the Absorbance at 405nm of Ammonium Molybdate
Table 1- Limit of Detection and Quantification of the Calibration Curve
The change in absorbance indicates that glucose oxidase is present in the system and can be detected. Furthermore, the similarity in the rate of change in absorbance further suggests the presence of enzymatic activity. However, the level of peroxide detected is too low to be used to accurately quantified the concentration of peroxide in the system and the amount of glucose oxidase in the sample. This is likely due to the low cell concentration in the sample Which was approximately 1.74*107 cells/ml.
Experimental
Table 1- Experimental Conditions
Sample prep
Standards
Authentic gluconolactone and gluconate standards were purchased from Sigma-Aldrich
Samples
Samples diluted 1:5 in cold acetonitrile and centrifuged at 21,000 RCF for 2 minutes
5 μL was injected into HPLC
HPLC Conditions
Machine:
1290 Infinity II LC system (Agilent Technologies)
Column:
Zorbax Eclipse Plus C18 column (50 x 2.1 mm, 1.8 uM; Agilent Technologies)
Column Temperature:
30°C
Solvents:
A: 0.1% formic acid in water
B: 0.1% formic acid in acetonitrile
Gradient:
2%B for 1 min, 85%B for 2 min, followed by re-equilibration for 2 min
MS Conditions
Machine:
Agilent 6545 quadrupole time-of-flight MS (QTOF-MS; Agilent Technologies)
Gas Settings:
Sheath gas flow rate: 10 L / min
Sheath gas temperature: 350 C
Drying gas flow rate: 12 L / min
Drying gas temperature: 325 C
Nebulizing gas: 55 psig
Ionization
Negative mode
Sample Analysis
Programs:
Agilent MassHunter Qualitative Analysis software
Agilent MassHunter Quantitative Analysis software
Compound confirmation
Exact mass of gluconolactone, [M-H]- : 177.0399
Exact mass of gluconate, [M-H]- : 195.0505
Samples were confirmed by comparison of retention time and exact mass to authentic standard
Exact mass was accurate to < 10 ppm
In HPLC-MS, samples are purified using HPLC and are then analyzed using mass spectroscopy to determine the compound. This system can not only identify samples with high accuracy, but also quantitate them based on the area of the peak producing the signal of interest.
Table 1Calibration Data
Figure 1-Figure 1- Standard Curves of Gluconolactone and Gluconate
Table 3- Sample Signals and Determined Concentration
Both samples show the production of gluconate although these values are low, they are still able to be quantified within the range of the calibration curve. The most likely reason for low production is a population of cells in the sample.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 717
Illegal BamHI site found at 1027 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1051
Illegal BsaI.rc site found at 2269
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