Difference between revisions of "Part:BBa K2944003"

Latest revision as of 02:58, 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. In our system, hydrogen peroxide reduction is catalyzed by Prussian Blue screen-printed inks, generating current.

Usage and Biology

Glucose oxidase (GOx) has mainly been isolated from Aspergillus niger although other filamentous fungi, such as those within the Penicillium genus, are also known to produce the enzyme (Hatzinikolaou et al., 1996; Eryomin et al., 2004). GOx catalyzes the oxidation of D-glucose to D-glucono-delta-lactone (which spontaneously converts to gluconate) and produces hydrogen peroxide as a by-product of molecular oxygen reduction (Bankar et al., 2009).

GOx is widely used commercially, including in the food industry where it is often utilized to remove oxygen from consumables and thereby prolonging their shelf life (Bankar et al., 2009). GOx is also used in biosensors such as to detect concentration of glucose in glucose monitors for diabetic patients (Petruccioli et al., 1999). Today, GOx is often paired with Prussian blue in biosensors as the electrocatalytic activity of Prussian blue with the hydrogen peroxide product of GOx reactions synergizes the conversion of a biological signal to an electrical one (Harper et al., 2010; Kim et al., 2018). Structurally, GOx consists of two identical subunits linked by disulfide bonds (Bankar et al., 2009). It has been shown that Arg-516 is the most important amino acid for the interaction of D-glucose with GOx (Witt et al., 2000). GOx from A. niger exhibits best activity in acidic pH ranging from 4.0 to 5.5 (Bankar et al., 2009).

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 which could be applied to identify amilCP post purification amongst other applications. 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:

1. Bankar, S. B., Bule, M. V., Singhal, R. S., & Ananthanarayan, L. (2009). Glucose oxidase - An overview. Biotechnology Advances. https://doi.org/10.1016/j.biotechadv.2009.04.003
2. Eryomin AN, Droshdenyuk AP, Zhavnerko GK, SemashkoTV, Mikhailova RV. Quartz sand as an adsorbent for purification of extracellular glucose oxidase from Penicillium funiculosum 46.1. Appl Biochem Microbiol 2004;40(2):178–85.
3. Harper, A., & Anderson, M. R. (2010). Electrochemical glucose sensors-developments using electrostatic assembly and carbon nanotubes for biosensor construction. Sensors, 10(9), 8248–8274. https://doi.org/10.3390/s100908248
4. Hatzinikolaou DG, Hansen OC, Macris BJ, Tingey A, Kekos D, Goodenough P, et al. A new glucose oxidase from Aspergillus niger characterization and regulation studies of enzyme and gene. Appl Microbiol Biotechnol 1996;46:371–81
5. Kim, J., Sempionatto, J. R., Imani, S., Hartel, M. C., Barfidokht, A., Tang, G., … Wang, J. (2018). Simultaneous Monitoring of Sweat and Interstitial Fluid Using a Single Wearable Biosensor Platform. Advanced Science, 5(10). https://doi.org/10.1002/ADVS.201800880
6. PetruccioliM, Federici F, Bucke C, Keshavarz T. Enhancement ofglucose oxidase production by Penicillium variabile P16. Enzyme Microb Technol 1999;24:397–401.
7. 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
8. Witt S, Wohlfahrt G, Schomburg D, Hecht H, Kalisz H. Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of β-D-glucose. J Biochem 2000;347:553–9.
9. 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




Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 717
  Illegal BamHI site found at 1027
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1051
  Illegal BsaI.rc site found at 2269