Introduction

In our project, we aimed to develop an enzyme cocktail primarily composed of chitinase and glucanase. In order to select the best enzyme candidates for ArMOLDgeddon, wild-type enzymes and modified variants using two main assays: a protein concentration assay and an enzyme activity assay. With the goal of maximizing accessibility and reproducibility—especially for high school and educational labs with limited equipment—we prioritized straightforward and economical methods. Both assays rely on a single low-cost reagent: Coomassie Brilliant Blue G-250 dye for protein quantification and 3,5-dinitrosalicylic acid (DNS) for enzyme activity. This design ensures that our protocols are not only easy to execute but also readily repeatable by other iGEM teams.

  1. Protein Concentration Assay (Bradford method): quantifies protein expression levels.
  2. Enzyme Activity Assay (DNS method): measures substrate breakdown to determine enzymatic hydrolysis rates.

Part 1: Protein Concentration Assay
(Bradford method)

Background
The Bradford protein assay is a widely used colorimetric method to measure protein concentration. In our project, it was used to assess protein expression and calculate total protein mass—essential for deriving specific activity (total enzyme activity per mg protein).
Principle
The Bradford Assay relies on the binding of Coomassie Brilliant Blue G-250 dye to proteins: the dye will transform from its original brownish-red form in acidic solution to a blue form when binding to proteins via hydrophobic and ionic interactions, resulting in an increase in absorbance at 595 nm, which can be measured using a colorimeter. [1] Since the intensity of the blue color is proportional to the concentration of protein, protein concentration of our samples can be determined by comparison to a standard curve prepared with known Bovine Serum Albumin (BSA) concentrations.
Protocol
1. Preparation of protein standard curve
Apparatus and materials
  • Apparatus: pipette, vortex mixer, 96-well plate, 1.5 mL centrifuge tubes, microplate reader
  • Materials: coomassie brilliant blue G-250, BSA solution (1 mg/mL), ddH₂O
Procedures
  • Prepare 1 mg/mL BSA solution (a protein standard) as the stock solution from any higher concentration solution.
  • Generate BSA dilutions as summarized in Table 1-1. Vortex after each dilution step.
  • Combine 40 µL of each standard with 200 µL of G-250 dye, vortex, and transfer 200 µL to a 96-well plate. Include three technical replicates per concentration.
  • Measure absorbance at 595 nm using a microplate reader.
  • Plot absorbance versus concentration. A standard curve with R² > 0.995 is considered valid. The resulting linear equation is used to determine protein concentrations in unknown samples.
Table 1-1 | Preparation of BSA standard concentrations.
2. G-250 assay for protein samples
Apparatus and materials
  • Apparatus: pipette, vortex mixer, 96-well plate, 1.5 mL centrifuge tubes, microplate reader
  • Materials: coomassie brilliant blue G-250, protein eluate, ddH₂O
Procedures
  • Mix 40 µL of protein sample with 200 µL G-250, vortex, and load 200 µL into the plate. Include three technical replicates per sample.
  • Measure absorbance at 595 nm using a microplate reader.
  • Calculate protein concentration using the valid standard curve.
  • Dilute and reassay if the value exceeds the curve’s range.
3. Controls and Validation
  • A standard curve with BSA serves as a positive control and a unit calibrator.
  • A reagent blank (ddH₂O mixed with G-250 dye) was included to establish the baseline absorbance and confirm that the color change was specifically due to protein-dye binding.
  • Triplicate measurements ensure reproducibility.
  • Samples exceeding the linear range are systematically diluted and remeasured to maintain accuracy.
Result
Fig. 1-1 | Preparation of protein standard curve. (a) Serial BSA dilutions in a 96-well plate; (b) BSA standard calibration curve.

Table 1-2 | Target protein concentrations.
*Note 1: SDS-PAGE band intensities analyzed by ImageJ were used to estimate target protein (enzyme) concentrations.

*Note 2: Inclusion body formation prevented concentration measurement for this protein.

Fig. 1-2 | Concentrations of wild-type chitinases (a), ProteinMPNN-modified PrChiA (b), and wild-type glucanases (c).
*Note: Inclusion body formation prevented concentration measurement for this protein.
Discussion
We successfully quantified target protein mass in our samples, with most enzyme candidates showing sufficient expression. However, noticeable discrepancies exist between proteins from different batches, highlighting the importance of repeating experiments.

Though ProteinMPNN modification of GlxChiB unexpectedly decreased its solubility, PrChiA showed a successful enhancement, demonstrating that AI-guided protein design can improve protein properties with a higher success rate and less experimental effort compared to traditional directed evolution. This contrast highlights that computational predictions are not always accurate and must be experimentally validated, guiding careful candidate selection in the next Design phase.

Contribution for iGEM Community
We selected the Bradford method for its simplicity, low cost, and speed. It requires only one reagent and takes minutes to perform, making it ideal for high-school laboratories. This enhances the accessibility and repeatability of our protocol, offering practical benefits to future iGEM teams.

Part 2: Enzyme Activity Assay
(DNS method)

Background
The DNS assay quantifies reducing sugars released by enzymatic hydrolysis of substrates such as chitin and glucan. It reflects total enzyme activity and, combined with protein concentration, enables specific activity calculation—key for comparing enzyme efficiency.
Principle
DNS is an aromatic compound that reacts with the free aldehyde or ketone groups of reducing sugars under alkaline and high-temperature conditions, forming a reddish-brown 3-amino-5-nitrosalicylic acid complex. The formation of the complex increases absorbance at 540 nm, which can be quantified using colorimeter [2]. Because the intensity of the resulting color is proportional to the concentration of reducing sugars,the amount of substrate broken down can be determined by comparison to a standard curve prepared with known glucose concentrations.
Protocol
1. Preparation of reducing sugar standard curve
Apparatus and materials
  • Apparatus: pipette, vortex mixer, 96-well plate, 1.5 mL centrifuge tubes, microplate reader, digital dry bath
  • Materials: DNS reagent, reducing sugar solution (1 mg/mL), ddH₂O
Procedures
  • Prepare 1 mg/mL reducing sugar (glucose for glucanases or N-acetyl-glucosamine for chitinases) solution as the stock solution.
  • Generate sugar dilutions as summarized in Table 2-1. Vortex after each dilution step.
  • Mix 200 μL of reducing sugar solution of each concentration with 200 μL of DNS reagent (triplicate).
  • Incubate at 100°C in a dry bath for 10 min.
  • Transfer 200 µL to a 96-well plate and measure absorbance at 540 nm using a microplate reader.
  • Plot absorbance versus concentration. A standard curve with R² > 0.995 is considered valid. The resulting linear equation is used to determine reducing sugar concentrations in unknown samples.
Table 2-1 | Preparation of reducing sugar standard concentrations.
2. DNS assay for enzyme activity
Apparatus and materials
  • Apparatus: pipette, vortex mixer, 96-well plate, 1.5 mL centrifuge tubes, microplate reader, digital dry bath
  • Materials: DNS reagent, enzyme supernatant (samples), substrate solution, ddH₂O
Procedures
  • Prepare substrate solutions at a concentration of 2 mg/mL (colloidal chitin, laminaran, lichenan, and pustulan).
  • Mix 200 μL of substrate solutions with 1 μL of enzyme sample and incubate at 37℃ for 10 min (triplicate).
  • Add 200 µL DNS reagent and incubate at 100°C in a dry bath for 10 min.
  • Transfer 200 µL to a 96-well plate and measure absorbance at 540 nm using a microplate reader.
  • One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from corresponding substrate under 37℃. Calculate enzyme activity (U/mL) as:
X = (n · c · 1000)/(t · M)

where X is the enzyme activity of the sample (U/mL), n is the dilution factor, c is the glucose concentration (mg/mL) calculated from the glucose standard curve regression equation, t is the reaction time (min), and M is the molecular weight of reducing sugar (glucose, 180.16 g/mol; N-acetyl-glucosamine, 221.21 g/mol).
  • Specific activity is defined as the number of enzyme activity units per mg of protein (U/mg), representing the catalytic efficiency of the enzyme per unit protein mass. It can be calculated as:

    Specific Activity (U/mg) = Enzyme Activity (U) / Enzyme Mass (mg)
3. Controls and Validation
  • Standard curves with glucose or N-acetyl-glucosamine serve as positive controls and unit calibration.
  • A critical negative control was implemented: the supernatant from cells transformed with an empty plasmid (lacking the target enzyme gene) was put through the identical expression and induction process. This control was essential to confirm that the observed reducing sugar signal was generated by our target enzymes and not by endogenous background activity from the host cells.
  • Substrate-only controls (substrate mixed with DNS reagent but no enzyme) were also included to rule out any non-enzymatic breakdown of the substrate.
  • All measurements were performed in triplicate to ensure reliability.
Result
Fig. 2-1 | Preparation of reducing sugar standard curve. Serial dilutions of glucose (a) and N-acetyl-glucosamine (b) in a 96-well plate; standard calibration curves of glucose (c) and N -acetyl-glucosamine (d).

Table 2-1 | Chitinase activities on 1% colloidal chitin.


Fig. 2-2 | Comparative analysis of chitinase activities. (a) Specific activities of wild-type chitinases; (b) ProteinMPNN-modified PrChiA; (c) relative activities of top four chitinase candidates.
*Note: no data for some constructs due to inclusion bodies (PrChiA and PrChiA-4) or low activity (GlxChiB, PrChiA-1, PrChiA-2 and PrChiA-6).
Table 2-2 | Glucanase activities across different substrates.
*Note: Enzyme activities were measured using their primary substrates (Bglu1 on lichenan, BglS27 on laminaran and Fl Glu30 on pustulan).

Fig. 2-3 | Comparative analysis of glucanase activities. (a) Specific activities of glucanases on their primary substrates; (b) specific activities of all glucanases tested on lichenan; (c) relative activities on lichenan, with Bglu1 set as 100%.
Fig. 2- 4 | Time-course activities of Bc Chi (a), rMvEChi (b), and Bglu1 (c).
Discussion
Most wild-type enzyme candidates did not exhibit satisfactory activity, differing from previous reports for reasons that remain unclear. After discovering that Bglu1, a β-1,3-1,4-glucanase, exhibited significantly better performance in breaking down lichenan—composed of glucose units linked by both β-1,3 and β-1,4 glycosidic bonds—than BglS27, a β-1,3-glucanase tested here on laminaran (a β-1,3-glucan), we decided to assess the ability of all three glucanases (BglS27, Bglu1, and FlGlu30) on lichenan instead, as BglS27 showed insufficient activity on laminaran for meaningful comparison.

Among all glucanase and chitinase samples, Bglu1 and BcChiA1 displayed the highest hydrolytic activity. Chitinase activity curves presents that BcChiA1 shows rapid early hydrolysis but plateaus quickly, while rMvEChi hydrolyzes more gradually yet maintains activity over time, indicating complementary kinetic profiles. We also observed that BglS27 and FlGlu30 showed relatively higher activity toward lichenan, which may be attributed to the high purity of the substrate (≥90%) or to limited substrate specificity in their hydrolytic activity.

Contribution for iGEM Community
The DNS method is highly cost-effective and versatile. It can be applied to any experiment involving reducing sugars (e.g., glucose, fructose, xylose), such as polysaccharide hydrolysis, fermentation monitoring, food science, or metabolic studies. With minimal equipment—pipettes, tubes, a heat source, and a colorimeter—school labs can perform robust enzyme comparisons, enhancing cross-project utility.

In addition, a critical aspect of our analysis was the determination of specific activity (enzyme activity per mg of protein). This parameter is essential because it provides a normalized measure of catalytic efficiency, allowing for a fair comparison between different enzymes and engineered variants, irrespective of their expression levels or purification yields. By using specific activity, we could objectively identify not just the enzyme with the highest total activity, but the most efficient catalyst, which delivers the most catalytic power per unit of protein. This is crucial for downstream applications and economic enzyme usage, and it establishes a standardized metric that other iGEM teams can adopt to benchmark their own enzyme performance against ours.

Future Improvement

  1. Protein Concentration Assay (Bradford method)
  • A key limitation of the Bradford assay is its variable dye-binding response to different proteins. Future work should incorporate an alternative method, such as the Bicinchoninic Acid (BCA) assay or absorbance at 280 nm, to cross-validate protein concentrations. This is crucial for ensuring that specific activity calculations, which directly influence candidate selection, are accurate and comparable across diverse enzyme variants.
  • We noticed that some protein samples themselves had a slight inherent color. For instance, residual components from the LB medium (which is light yellow) could contribute to the absorbance reading. A simple improvement would be to include a "sample blank" for each colored sample. This means mixing the sample with just water (instead of the dye) and subtracting its absorbance from the final result, giving a more accurate reading.
  1. Enzyme Activity Assay (DNS method)
  • Our current assay used a fixed amount of substrate. A more advanced experiment would be to test how the enzyme activity changes when we give it different amounts of substrate. This could help us find the optimal substrate concentration for each enzyme, revealing which one is truly the "hungriest" and most efficient.
  • We used a standard curve, but it would be even better to include a known, commercially available chitinase or glucanase in every experiment as a "positive control". This would act as a benchmark, helping us confirm that our experimental process was working correctly every single time and making our comparison to other studies easier.

Reference

  1. Bradford, Marion M. "A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-dye Binding." Analytical Biochemistry, vol. 72, no. 1–2, May 1976, pp. 248–54, doi:10.1016/0003-2697(76)90527-3.
  2. Miller, G. L. "Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar." Analytical Chemistry, vol. 31, no. 3, Mar. 1959, pp. 426–28, doi:10.1021/ac60147a030.
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