Difference between revisions of "Part:BBa K2944003"

Revision as of 01:56, 22 October 2019

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.

Experimental

1. HPLC-MS overview
2. Preparation of Standard Curve
3. HPLC-MS for activity of glucose oxidase
4. Results

We chose to characterize glucose oxidase with HPLC-MS which is a highly accurate, quantitative method. In HPLC-MS, samples are first purified using HPLC. Analysis is then done with Mass Spectrometry (MS). In mass spectrometry, the sample is ionized. The bonds in the molecules break part in different fragments as per their bonding strengths, forming fragments with quantitative mass to charge ratios, plotted on the x-axis. The abundance of these fragments is recorded along the y-axis. The HPLC-MS is therefore a characterizing fingerprint for glucose oxidase activity. 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.

Figure 1 Results: HPLC-MS (High Performance Liquid Chromatography- Mass Spectrometry) of glucose oxidase in solution to quantify the products of catalysis. Image built using Prism 6.

Preparation of Standard Curve

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 2 – 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 3 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 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 1 Calibration Data

Figure 4- Standard Curves of Gluconolactone and Gluconate

Table 2- 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.

Results

Figure 1 Mass Spectrometry Results. Image built using Prism 6.

There are two superimposed traces: gluconolactone standard and yeast supernatant mixed 50/50 with 40 g/L glucose. The traces are of extracted ion 195.0505 (exact [M-H]^- of gluconate. The gluconolactone standard is predominately gluconate upon resuspension in water.

The mass spectrum in the inset is the total ion count of the standard at 0.5 mins between the indicated m/z range. The two masses which are not the exact mass of gluconate are also part of the standard. Their identities are confirmed using the online database mzCloud. Annotated in the figure is we believe these masses correspond to based on the papers referenced below.

• • • Characterization done by Matthew Tiranardi.

Thankyou Lauren Narcross for her technical assistance!

References:

Taylor, V. F., March, R. E., Longerich, H. P., & Stadey, C. J. (2005). A mass spectrometric study of glucose, sucrose, and fructose using an inductively coupled plasma and electrospray ionization. International Journal of Mass Spectrometry, 243(1), 71–84. doi: 10.1016/j.ijms.2005.01.001

Zhang, Z., Gibson, P., Clark, S. B., Tian, G., Zanonato, P. L., & Rao, L. (2007). Lactonization and Protonation of Gluconic Acid: A Thermodynamic and Kinetic Study by Potentiometry, NMR and ESI-MS. Journal of Solution Chemistry, 36(10), 1187–1200. doi: 10.1007/s10953-007-9182-x

Sequence and Features