In this project, we constructed and expressed three enzymes with chitinase activity: chitin deacetylase (AsCDA), chitin exonuclease (Cts2p), and chitin endoenuclease (FjChiB). We also established an E. coli expression platform to provide purified enzymes for activity characterization and downstream application development. To support DBTL cycles and provide quantitative, reproducible readouts, we designed three core measurement modules to evaluate catalytic performance and immobilized enzymes:
1.Chitinase Activity Assay by N-acetylglucosamine (GlcNAc)/Reducing Sugar
1) Purpose: To quantify the hydrolytic activity of Cts2p and FjChiB on colloidal chitin by measuring the release of reducing sugars.
2) Method: Hydrolytic activity was determined using a DNS-based colorimetric assay, with reducing sugar concentration calculated from a GlcNAc standard curve. Activity was expressed in U/mL, where one unit corresponds to the release of 1 µmol of reducing sugar per minute.
2.Determination of Chitin Deacetylase Activity by p-Nitroaniline (pNA) Release
1) Purpose: To measure AsCDA activity using a chromogenic substrate that liberates p-nitroaniline upon enzymatic cleavage.
2) Method: Enzyme activity was determined spectrophotometrically by monitoring pNA release at 405 nm, quantified against a pNA standard curve. One unit of activity was defined as the release of 1 µmol of pNA per minute, and results were reported as U/mL.
3.Enzyme Immobilization: Loading Efficiency, Activity Retention, and Reusability
3) Purpose: To evaluate immobilization yield and operational stability for practical applications.
4) Methods: Protein immobilization efficiency was assessed by comparing protein content before and after coupling to epoxy resin supports, while retained activity and reusability were measured using the DNS/pNA activity assays over multiple reaction cycles.
Chitinases are enzymes that hydrolyze chitin, a polymer of N-acetyl-D-glucosamine (GlcNAc), into soluble chitooligosaccharides or monomeric GlcNAc. To evaluate chitinase activity, it is necessary to measure the amount of reducing sugars released during enzymatic hydrolysis. The dinitrosalicylic acid (DNS) method, first described by Miller in 1959[1], is one of the most widely used colorimetric assays for quantifying reducing sugars. Under alkaline conditions, DNS reacts with the aldehyde groups of reducing sugars and is reduced to 3-amino-5-nitrosalicylic acid, producing a reddish-brown color. The intensity of this color, measured spectrophotometrically at 540 nm, is directly proportional to the concentration of reducing sugars present. By generating a standard curve with known concentrations of GlcNAc, the DNS method provides a reliable way to quantify the catalytic activity of chitinases.
In the DNS assay for chitinase activity, enzyme samples are incubated with colloidal chitin substrate under controlled conditions. During hydrolysis, chitinases release GlcNAc and other reducing sugars into the reaction mixture. After incubation, DNS reagent is added, and the mixture is heated to induce the colorimetric reaction. The resulting absorbance at 540 nm reflects the concentration of reducing sugars released. By referencing a GlcNAc standard curve, the absorbance values are converted into absolute amounts of reducing sugars. Enzyme activity is then calculated and expressed as units (U), where one unit is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute under the assay conditions.
Materials:
1. 1% (w/v) colloidal chitin suspension
2. Enzyme samples (purified or crude extracts, concentration pre-determined)
3. DNS reagent (commercial or freshly prepared)
4. N-acetylglucosamine (GlcNAc) standard stock solution (1 mg/mL), diluted to prepare a standard curve.
5. Distilled or deionized water
6. 40% sodium potassium tartrate solution (optional, for color stabilization)
7. 1.5 mL or 2.0 mL microcentrifuge tubes
8. Water bath at 42 °C for enzyme reaction
9. Boiling water bath for DNS reaction
10. Ice bath for rapid cooling
11. Microcentrifuge (up to 12,000 rpm)
12. Vortex mixer
13. Spectrophotometer or microplate reader capable of 540 nm detection
14. Pipettes and sterile tips
15. Protective equipment: lab coat, gloves, safety goggles
Procedures:
1. Prepare GlcNAc standard solutions in the range of 0, 0.1-1.0 mg/mL(Figure 1).
Figure 1. Glucose concentration gradient
2. Label reaction tubes for enzyme samples, substrate blanks, enzyme blanks, and standards.
3. Add 250 µL of 1% colloidal chitin to each sample and substrate blank tube.
4. Add 250 µL of enzyme solution to each sample and enzyme blank tube.
5. Add 500 µL of buffer to blanks or standards as required to make the reaction volume 1000 µL.
6. Mix gently and incubate all tubes at 42 °C for 60 minutes.
7. Stop the reaction by adding 2.0 mL DNS reagent to each tube.
8. Immediately mix and place the tubes in a boiling water bath for 5 minutes.
9. Cool the tubes rapidly in an ice bath until they reach room temperature.
10. Centrifuge the tubes at 12,000 rpm for 5 minutes to pellet undigested chitin.
11. Transfer the supernatant carefully to cuvettes or a 96-well plate.
12. Measure absorbance at 540 nm using a spectrophotometer or plate reader (Figure 2).
Figure 2. Glucose concentration gradient
13. Subtract blank values, plot absorbance against GlcNAc concentration to generate a standard curve, and calculate sample reducing sugar concentration (Figure 3).
Figure 3. Glucose standard curve
14. Convert reducing sugar concentration into µmol using the molecular weight of GlcNAc (203.19 g/mol).
15. Calculate enzyme activity (U) as µmol reducing sugar released per minute.
Figure 4. Calculate the total amount of reducing sugars produced in the reaction system (µmol). Note: that the units need to be converted before use.
Figure 5. Required U/mL (per mL of enzyme solution)
16. Express activity as U/mL by dividing by the volume of enzyme solution used (Figure 6).
Figure 6. The enzyme activity of different types of enzymes is determined by synergy, where ACF represents three free enzymes, AC, AF, and FC represent two enzymes combined, and ACF represents three enzymes combined.
The results in Figure 6 show significant differences in the effects of different enzyme combinations on enzyme activity. In the single-enzyme treatment groups, A (chitin deacetylase) and F (chitin endonuclease) exhibited moderate activity, while C (chitin exonuclease) had the lowest activity. Dual-enzyme combinations exhibited varying degrees of synergistic effects: the AF combination (deacetylase + endonuclease) had the highest activity (nearly 30 U/mL), significantly outperforming the other groups; the AC combination (deacetylase + exonuclease) was second; and the FC combination (endonuclease + exonuclease) had no significant synergistic effect. The activity of the triple-enzyme combination ACF was lower than that of the AF combination, but higher than that of the other dual-enzyme combinations.
This phenomenon may be due to the synergistic and competitive nature of the enzyme mechanisms: the significant synergistic effect of the AF combination suggests positive cooperation between the deacetylase and the endonuclease, with deacetylation potentially exposing more endonuclease sites. The weaker synergistic effect of exonuclease C with other enzymes may be related to its mode of action or steric hindrance. The triple-enzyme combination's activity did not reach the expected peak, suggesting negative regulation through substrate competition or product inhibition. These findings provide an important basis for optimizing enzyme combination formulas. It is recommended that the AF dual-enzyme system be given priority in practical applications, or the ratio be adjusted in the triple-enzyme system to avoid competitive inhibition.
Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolytic removal of acetyl groups from N-acetylglucosamine residues in chitin or chitooligosaccharides, generating chitosan and acetic acid derivatives. Evaluating CDA activity is essential for understanding its catalytic efficiency and potential applications in biomass conversion and biopolymer modification. A widely used method to quantify CDA activity is the chromogenic p-nitroaniline (pNA) assay[2]. In this method, synthetic substrates such as p-nitroacetanilide are cleaved by CDA, releasing free p-nitroaniline. pNA exhibits a strong absorbance peak at 405 nm under neutral to slightly alkaline conditions, making it suitable for spectrophotometric detection. The amount of pNA released is directly proportional to CDA activity, and when compared with a standard curve prepared from known concentrations of pNA, the enzyme activity can be quantified.
In the pNA-based CDA activity assay, enzyme samples are incubated with a chromogenic substrate (e.g., p-nitroacetanilide) under controlled conditions. CDA catalyzes deacetylation, leading to the release of p-nitroaniline into the reaction mixture. The liberated pNA is detected spectrophotometrically by measuring absorbance at 405 nm. The absorbance value is converted into concentration using a pNA standard curve (µg/mL), and subsequently into µmol amounts using its molecular weight (138.12 g/mol). One unit of CDA activity (U) is defined as the amount of enzyme required to release 1 µmol of pNA per minute under the assay conditions.
Materials:
1. p-nitroacetanilide substrate solution (1 mg/mL, freshly prepared in buffer or suitable solvent)
2. Phosphate buffer (250 mM, pH 7.4)
3. Enzyme samples (purified)
4. p-nitroaniline (pNA) standard stock solution (1 mg/mL), diluted to prepare standard curve.
5. Distilled or deionized water
6. Ice bath for rapid cooling
7. 1.5 mL or 2.0 mL microcentrifuge tubes
8. 37 °C water bath or incubator
9. Vortex mixer
10. Spectrophotometer or microplate reader capable of 405 nm detection
11. Pipettes and sterile tips
12. Protective equipment: lab coat, gloves, safety goggles
Procedures:
1. Prepare a series of pNA standards in the range of 0–200 µg/mL (Figure 7).
Figure 7. p-Nitroaniline standard curve sample reaction
2. Label tubes for enzyme samples, substrate blanks, enzyme blanks, and standards.
3. For each reaction, add 150 µL p-nitroacetanilide solution, 40 µL phosphate buffer, and 10 µL enzyme solution to a tube, making a total reaction volume of 200 µL.
4. For blanks, replace enzyme solution or substrate solution with buffer as appropriate.
5. Mix gently and incubate all tubes at 37 °C for 30 minutes.
6. Stop the reaction immediately by placing tubes in an ice bath.
7. Vortex briefly to ensure mixing after termination.
8. Transfer the reaction mixture to cuvettes or a 96-well plate.
9. Measure absorbance at 405 nm using a spectrophotometer or plate reader.
10. Subtract blank values, plot absorbance against pNA concentration to generate a standard curve, and calculate the amount of pNA released in each sample.
11. Convert pNA concentration (µg/mL) into µmol using the molecular weight of pNA (138.12 g/mol).
12. Calculate enzyme activity (U) as µmol pNA released per minute.
13. Express activity as U/mL by dividing by the volume of enzyme solution used (0.01 mL).
Figure 8. p-Nitroacetanilide standard curve
The enzyme activity assay is performed using the same method described above, with a MW of pNA = 138.12 μg/μmol. This method's advantage lies in the rapid generation of a strong absorbance signal using the chromogenic substrate, making it suitable for routine screening of CDA activity. The linear relationship between absorbance and pNA concentration ensures accurate quantification within the assay range, and the short incubation time allows for efficient parallel assays.
However, this method also has limitations. The synthetic p-nitroacetanilide substrate does not fully mimic natural chitin or chitosan oligosaccharide substrates, so the absolute activity values obtained may not directly represent the enzyme's activity against natural polysaccharides. Furthermore, incomplete substrate solubility or spontaneous hydrolysis can lead to elevated background absorbance, necessitating careful blank subtraction to ensure data accuracy.
Enzyme immobilization is a widely used strategy to enhance enzyme stability, reusability, and catalytic efficiency for industrial and environmental applications. Epoxy resins such as LX-1000EP provide a solid support with reactive epoxy functional groups that can form covalent bonds with amino, hydroxyl, or sulfhydryl groups on enzyme molecules[3]. By immobilizing enzymes onto a resin matrix, the biocatalyst can be separated from the reaction mixture, allowing repeated use without significant activity loss. For chitin-active enzymes, immobilization offers an effective way to improve operational stability in biomass degradation processes and to lower the cost of enzyme applications by extending enzyme lifespan. To evaluate immobilization efficiency, both protein binding and retained enzymatic activity need to be measured.
Enzyme immobilization on epoxy resin LX-1000EP is based on covalent coupling between the epoxy groups of the resin and the functional groups of amino acid residues on the enzyme surface. During the immobilization process, the enzyme is incubated with the resin under appropriate pH and temperature conditions to allow for covalent binding. Protein immobilization efficiency is determined by measuring the grayscale value of the protein gel in the supernatant before and after immobilization using SDS-PAGE.
Materials:
1. Epoxy resin carrier LX-1000EP
2. Purified enzyme solution
3. PBS buffer (0.1 M, pH 7.0)
4. 15 mL centrifuge columns or tubes
5. Centrifuge capable of 4000 rpm
6. Shaking or mixing instrument, horizontal mode, 25 °C
7. SDS-PAGE kit and reagents
8. ImageJ software for densitometry analysis
9. Pipettes and sterile tips
10. Protective equipment
Procedures:
1. Weigh 0.5 g of epoxy resin LX-1000EP into a 15 mL centrifuge column.
2. Add 3 mL PBS buffer, wash three times at 4000 rpm for 1 min each, discard the supernatant after each wash.
3. Add enzyme solution at a ratio of 10 mg enzyme per g carrier; for 0.5 g resin add 2 mL enzyme solution at 5 mg/mL. Record this as pre-immobilization supernatant.
4. Place the tube horizontally on a shaker at 25 °C, allow reaction for 12 hours to ensure resin suspension and sufficient contact.
5. After reaction, centrifuge at 4000 rpm for 1 min, collect the supernatant as post-immobilization supernatant.
Figure 9. Purified proteins of AsCDA and FjChiB and SDS-page images of proteins before and after immobilization
6. Wash the resin by adding 2 mL PBS buffer, gently mixing, centrifuging at 4000 rpm for 1 min, and discarding the wash. Repeat this washing step twice.
7. Take aliquots of pre-immobilization and post-immobilization supernatants, dilute fivefold if necessary, and analyze by SDS-PAGE.
8. Use ImageJ software to calculate the gray value of protein bands.
Figure 10. Image J software was used for calculation and analysis
9. Calculate immobilization efficiency (%) using the formula:
Figure 11. Calculate immobilization efficiency formula
Figure 12. Calculation of grayscale values of the two proteins before and after immobilization
The results in Figure 12 show significant differences between the two proteins before and after immobilization. Grayscale analysis shows that the integrated density of AsCDA was approximately 800,000 before immobilization, decreasing to approximately 650,000 after immobilization, resulting in an immobilization efficiency of 17.84%. The integrated density of FjChiB was approximately 850,000 before immobilization, decreasing to approximately 640,000 after immobilization, resulting in an immobilization efficiency of 24.59%. This indicates that both proteins experienced some loss during the immobilization process, with FjChiB having a relatively higher immobilization efficiency.
Discussion suggested that the difference in immobilization efficiency may be related to protein characteristics: FjChiB, as an endonuclease, may possess more surface-active groups (such as amino groups), making it easier to bind to the support; whereas the steric structure of AsCDA may limit its effective binding to the support. The immobilization efficiency of both proteins did not exceed 25%, suggesting that the current immobilization method (possibly epoxy or amino coupling) has room for optimization, such as adjusting the support pore size, activation conditions, or reaction time. Furthermore, the retained protein activity after immobilization requires further verification, as the grayscale value only reflects protein quantity, not activity.
