iGEM 2025 — Team subcat-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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Notebook

### Main Timeline

The project schedule is organized in monthly units, where each month is standardized to a duration of 30 days for consistency. Within this framework, the timeline is divided into smaller increments, with each individual grid cell representing a span of three consecutive days. This breakdown allows the timeline to be viewed at a fine enough scale for tracking progress.

#### February:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |
| Theoretical knowledge learning |  |  |  |  |  |  |  |  |  |  |
| Preliminary experiment |  |  |  |  |  |  |  |  |  |  |

#### March:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |
| Theoretical knowledge learning |  |  |  |  |  |  |  |  |  |  |
| Preliminary experiment |  |  |  |  |  |  |  |  |  |  |

#### April:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |
| Theoretical knowledge learning |  |  |  |  |  |  |  |  |  |  |
| Project brain storm |  |  |  |  |  |  |  |  |  |  |

#### May:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |
| Project introduction |  |  |  |  |  |  |  |  |  |  |
| Theoretical knowledge training |  |  |  |  |  |  |  |  |  |  |

#### June:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |
| Project brain storm |  |  |  |  |  |  |  |  |  |  |
| Experimental design |  |  |  |  |  |  |  |  |  |  |

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Laboratory safety training |  |  |  |  |  |  |  |  |  |  |
| Implement experimental plan |  |  |  |  |  |  |  |  |  |  |

#### July:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |
| Project problems discussion |  |  |  |  |  |  |  |  |  |  |
| Record experiment report |  |  |  |  |  |  |  |  |  |  |

#### August:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Implement experimental plan |  |  |  |  |  |  |  |  |  |  |
| Project problems discussion |  |  |  |  |  |  |  |  |  |  |
| Analysis of experimental results |  |  |  |  |  |  |  |  |  |  |

#### September:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Project problems discussion |  |  |  |  |  |  |  |  |  |  |
| Wiki writing |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Preparation of defense materials |  |  |  |  |  |  |  |  |  |  |

#### October:

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Preparation of defense materials |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Project review |  |  |  |  |  |  |  |  |  |  |

#### Record of Main Experimental Content

##### Construction of recombination plasmids

###### pET-28a(+)-AsCDA/Cts2p/FjChiB

###### Ⅰ. Obtain target gene fragments by PCR

**Goal:** PCR technology is used to amplify the target gene fragment to provide DNA fragments for subsequent infusion recombination.

**Materials:**

1) 2x PrimeStarMix
2) Gene template
3) Double distilled water (ddH2O)
4) Primer-F
5) Primer-R
6) PCR tube
7) PCR thermal cycler

PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.

![Figure 1 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/1.webp)

Figure 1 Experimental record

**Procedures:**

1) Aseptically prepare the PCR reaction system. The recommended total reaction volume is 50 µL: add 25 µL of 2×PrimeStar Mix, 1 µL of template DNA, 1 µL of Primer-F, and 1 µL of Primer-R. Add ddH2O to the remaining volume to 50 µL. After preparing the reaction solution, briefly centrifuge to remove any bubbles.

2) Place the reaction tube in a PCR instrument and set the reaction program:

3) Initial denaturation: 95℃ for 3 minutes to fully unwind the double-stranded structure of the template DNA;

4) Cycling: Perform 35 cycles, each cycle consisting of:

5) Denaturation at 95℃ for 30 seconds

6) Annealing at 55℃ for 30 seconds (the specific temperature can be adjusted based on primer design);

7) Extension at 72℃ for 2 minutes (the PrimeStar Mix has an extension rate of approximately 1000 bp/10 seconds; the time can be adjusted based on the target fragment size);

8) End extension: 72℃ for 1 minute to ensure complete extension of all fragments.

9) After the reaction, if not used immediately, the product can be stored at 4℃ for a short period of time.

10) To obtain purified DNA fragments, separate the PCR products by electrophoresis on a 1%–1.2% agarose gel. Cut out the target band and use a DNA recovery kit to extract the fragment for subsequent infusion recombination.

![Figure 2 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/2.webp)

Figure 2 Experimental record

##### Protein expression and purification

###### 3.1 Induction of protein expression for 6xHis-AsCDA/Cts2p/FjChiB

###### Ⅰ. Amplification culture for correct colony

**Goal:** The pET-28a(+)-AsCDA/Cts2p/FjChiB recombinant protein was efficiently expressed in Escherichia coli BL21, and bacterial samples were collected at different times and temperatures for SDS-PAGE analysis of protein expression.

**Materials:**

• LB liquid medium
• E. coli BL21 transformed with pET-28a(+)-AsCDA/Cts2p/FjChiB
• Kanamycin solution
• Isopropyl β-D-thiogalactopyranoside (IPTG) inducer
• Iso-thermic shaker
• Spectrophotometer (for OD600 measurement)
• 1.5 mL centrifuge tubes and sterile pipette tips

![Figure 3 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/3.webp)

Figure 3 Experimental record

**Procedures:**

1) **Inoculation and Preculture**

2) Add 100 µL of the recombinant bacterial culture to 100 mL of LB liquid medium. Also add an appropriate amount of kanamycin (e.g., 100 µg/mL) to ensure selective pressure.

3) Incubate the culture flask at 37℃ on a constant-temperature shaker, shaking vigorously to ensure aeration.

4) **Growth Monitoring**

5) Measure the OD600 value of the culture every 30 minutes until the culture reaches the logarithmic growth phase (OD600 ≈ 0.4–0.6), which usually takes approximately 2–3 hours.

6) At this stage, the bacteria are actively growing and suitable for IPTG induction.

7) **Protein Induction**

8) Add IPTG to the culture to a final concentration of 1 mM and gently mix to initiate recombinant protein expression.

9) Parallel cultures at different temperatures (e.g., 25℃ and 37℃) can be set up to compare expression efficiency and solubility.

4. **Sampling and Monitoring**

10) At 0.5 h, 1 h, 3 h, and 6 h after induction, collect equal volumes of bacterial culture samples (e.g., 1–2 mL) and place them in centrifuge tubes.

11) Samples can be frozen immediately or added to lysis buffer for subsequent SDS-PAGE analysis of protein expression.

**Notes:**

• IPTG solution should be prepared fresh or stored frozen in aliquots to avoid repeated freeze-thaw cycles.
• The induction temperature affects protein solubility and folding. Low temperatures (25℃) generally favor soluble protein expression.
• When sampling, perform the procedure as quickly as possible to prevent continued bacterial growth or protein degradation.

![Figure 4 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/4.webp)

Figure 4 Experimental record

###### Ⅱ: Protein crude extraction and SDS-PAGE

**Goal:** Crude protein was extracted from the induced expression E. coli BL21 culture medium, and the expression and size of the target protein were analyzed by

![Figure 5 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/5.webp)

Figure 5 Experimental record

**SDS-PAGE.**

**Materials for protein crude extraction:**

• Induced bacterial suspension (approximately 500 µL per induction condition)
• Centrifuge
• Double-distilled water (ddH2O)
• Sangon 12.5% SDS-PAGE Preparation Kit
• Electrophoresis Buffer (Tris-Glycine)
• Protein Molecular Weight Standard
• 6×Protein Loading Buffer
• Vertical Electrophoresis System
• Coomassie Brilliant Blue Staining Solution
• Destaining Solution

![Figure 6 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/6.webp)

Figure 6 Experimental record

**Procedures for protein crude extraction and SDS-PAGE:**

**1. Crude Protein Extraction**

• Transfer 500 µL of bacterial suspension from each induction condition to a centrifuge tube and centrifuge at 12,000 rpm for 1 minute.
• Discard the supernatant and resuspend the bacterial pellet in 50 µL of 1×protein loading buffer (6×loading buffer diluted with ddH2O).
• Heat the resuspension in a PCR thermocycler at 95℃ for 15 minutes to lyse the cells and denature the proteins.
• After completion, cool briefly or proceed immediately to SDS-PAGE analysis.

![Figure 7 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/7.webp)

Figure 7 Experimental record

**2. SDS-PAGE Gel Preparation**

• Combine 2.2 mL of 2×separating gel solution, 2.2 mL of 2×separating gel buffer, and 44 µL of catalyst in a centrifuge tube. Mix gently.
• Slowly pour the separating gel into the gel mold, avoiding the formation of bubbles.
• Add 1 mL of ddH2O to the top of the separating gel and let it stand for approximately 8 minutes to allow the gel to fully polymerize.
• After the gel solidifies, discard the top layer of water.
• Combine 825 µL of 2×stacking gel solution, 825 µL of 2×stacking gel buffer, and 11 µL of catalyst. Mix gently.
• Pour the stacking gel into the mold until full and slowly insert the comb to avoid creating bubbles.
• Let it stand for approximately 12 minutes to solidify, then carefully remove the comb and rinse the wells with running buffer.

**3. Sample Loading and Electrophoresis**

• Add 10 µL of protein molecular weight standard to the first well, followed by an appropriate amount of protein sample to each well.
• Place the gel in a vertical electrophoresis tank and add running buffer.
• Set the electrophoresis condition to 120 V and run for approximately 90 minutes until the protein bands are clearly separated.

**4. Staining and Destaining**

• After electrophoresis, stain the gel in Coomassie Brilliant Blue solution for approximately 10 minutes.
• Rinse the gel repeatedly in destaining solution until the background becomes transparent and protein bands are clearly visible.

**Notes:**

• Be gentle when preparing the separating and stacking gels to avoid creating bubbles, which can affect protein band resolution.
• Load a moderate amount of sample to avoid excessive sample loading, which can cause band smearing.
• Adjust the staining and destaining times based on band appearance to achieve optimal contrast.

![Figure 8 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/8.webp)

Figure 8 Experimental record

![Figure 9 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/9.webp)

Figure 9 Experimental record

##### Optimal free enzyme activity reaction conditions

**Goal:**

Determine the optimal reaction conditions for three chitin-related enzymes, AsCDA, Cts2p, and FjChiB, including optimal pH, temperature, pH stability, temperature stability, and the effects of metal ions on enzyme activity, to provide experimental basis for subsequent enzymatic applications and optimization.

**Materials:**

• Purified AsCDA, Cts2p, and FjChiB enzyme samples (0.5 mg)
• Colloidal chitin solution (1% w/v)
• Buffer system:
• 0.1 M glycine-HCl (pH 2.0–3.0)
• 0.1 M citric acid-Na2HPO4 (pH 3.0–8.0)
• 0.1 M Tris-HCl (pH 8.0–9.0)
• 0.1 M glycine-NaOH (pH 9.0–11.0)
• 50 mM Na2PO4-citric acid buffer (pH 4.0–6.0)
• 50 mM Na2PO4 buffer (pH 6.0–8.0)
• 50 mM Tris-HCl buffer (pH 8.0–9.0)
• 50 mM Glycine-NaOH buffer (pH 9.0–11.0)
• Metal ion solution (final concentration 10 mM): Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Na⁺, K⁺, Mn²⁺, Zn²⁺
• Spectrophotometer (OD540, for DNS assay of reducing sugars)
• Centrifuge tube (1.5 mL)
• Water bath

![Figure 10 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/10.webp)

Figure 10 Experimental record

**Procedures:**

**a. Determination of Optimal pH**

1. Centrifuge 1% colloidal chitin, discard the supernatant, and resuspend in an equal volume of buffer covering the pH range of 2.0–11.0 (glycine-HCl, citric acid-Na2HPO4, Tris-HCl, glycine-NaOH).
2. Add each enzyme to the colloidal chitin system in different pH buffers and incubate at 37℃ for 1 hour.
3. After the reaction, determine the reducing sugar yield using the DNS method. The highest enzyme activity was set as 100%. A relative enzyme activity versus pH curve was plotted to determine the optimal pH.

**b. Optimal Temperature Determination**

1. Dissolve the enzyme in the optimal pH buffer.
2. Set a temperature gradient (20, 30, 40, 50, 60, 70, and 80℃) and add colloidal chitin for a 1-hour reaction.
3. Determine reducing sugar production by DNS. Plot a curve of relative enzyme activity versus temperature to determine the optimal reaction temperature.

**c. Effect of Metal Ions on Enzyme Activity**

1. At the optimal pH, add a single metal ion (final concentration of 10 mM, Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Na⁺, K⁺, Mn²⁺, or Zn²⁺) to the enzyme solution.
2. Incubate at 37℃ for 1 hour, measure reducing sugar production, calculate relative enzyme activity, and analyze the promoting or inhibiting effect of each metal ion on enzyme activity.

![Figure 11 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/11.webp)

Figure 11 Experimental record

![Figure 12 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/12.webp)

Figure 12 Experimental record

**Notes**

• When conducting pH or temperature gradient experiments, perform at least three replicates per group.
• When determining reducing sugars using the DNS method, ensure that the sample temperature is consistent to prevent non-enzymatic factors from influencing the results.
• For metal ion experiments, it is recommended to include a blank control (without metal ions) to correct for background.
• The optimal pH and temperature for each enzyme are determined based on the highest relative enzyme activity and are then used for optimization in subsequent experiments.

![Figure 13 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/13.webp)

Figure 13 Experimental record

![Figure 14 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/14.webp)

Figure 14 Experimental record

![Figure 15 Experimental record](https://static.igem.wiki/teams/5721/img/notebook/15.webp)

Figure 15 Experimental record

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block extra\_css %}

{% endblock %}
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Engineering

![shrimp](https://static.igem.wiki/teams/5721/img/desc-xia.webp)

[Overview](#content1)

[Part 1 pET28a-AsCDA (BBa\_25VIQJQP)](#content2)

[Design](#content2-1)

[Build](#content2-2)

[Part 2 pET28a\_Cts2p (BBa\_25OCX0JU)](#content3)

[Design](#content3-1)

[Build](#content3-2)

[Part 3 pET28a-FjChiB (BBa\_250AYN60)](#content4)

[Design](#content4-1)

[Build](#content4-2)

[1 Protein expression test](#content5)

[1.1 Protein expression](#content5-1)

[1.2 Protein His-tag purification](#content5-2)

[2. Functional test](#content6)

[2.1 Free enzyme activity test](#content6-1)

[2.2 Optimal free enzyme activity reaction conditions](#content6-2)

[2.3 Optimal free enzyme synergistic ratio and reaction conditions](#content6-3)

[2.4 Preparation and immobilized enzyme efficiency](#content6-4)

[2.5 Number of reuses of immobilized enzymes](#content6-5)

[Learn](#content7)

[References](#content8)

### Overview

Chitin, a linear polymer composed of N-acetyl-D-glucosamine units, is the second most abundant natural polysaccharide after cellulose[1]. Chitinases degrade chitin (a major component of fungal cell walls and insect exoskeletons) into N-acetylglucosamine oligomers and monomers[2]. These enzymes, due to their ability to catalyze chitin hydrolysis, play a vital role in various fields, including agriculture, medicine, and industrial biotechnology[3].
In this project, we identified and screened three chitinases and chitin deacetylases from microbial sources. After in vitro gene synthesis, the target genes were cloned into expression plasmids and transformed into Escherichia coli to establish a recombinant expression system. We constructed a genetic system for expressing these chitin-degrading enzymes in E. coli and optimized protein yield and enzyme activity by varying reaction parameters. These enzymes effectively degrade chitin-rich wastes and promote the conversion of chitin into high-value-added products such as amino acids, calcium lactate, and high-purity chitin oligosaccharides, thereby improving the utilization efficiency of biomass resources.
We also explored the synergistic effects of exo- or endo-chitinases with chitin deacetylases to enhance chitin degradation efficiency. The most effective enzyme combinations were immobilized on epoxy resin supports to improve enzyme reusability, stability, and overall performance. This immobilized enzyme system offers a promising bioremediation tool for the efficient degradation of chitin-containing marine pollutants and a sustainable enzymatic solution for environmental remediation.

### Part 1 pET28a-AsCDA (BBa\_25VIQJQP)

**Design:**
The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene AsCDA was synthesized by a biotechnology company. The AsCDA fragment was homologously recombined with the linearized plasmid to construct pET28a-AsCDA.
![The plasmid map of pET28a-AsCDA](https://static.igem.wiki/teams/5721/img/engineering/1.webp)
Figure 1. The plasmid map of pET28a-AsCDA

**Build:**
We constructed pET28a-AsCDA by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5431 bp. Figure 2B shows a band consistent with the target size. The AsCDA gene was amplified by PCR, with a length of 975 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. After agarose gel electrophoresis and gel recovery, the recombinant plasmid was obtained by homologous recombination.
![Agarose gel electrophoresis of target genes and linearized pET-28a(+) vector](https://static.igem.wiki/teams/5721/img/engineering/2.webp)
Figure 2. Agarose gel electrophoresis of target genes and linearized pET-28a(+) vector
After ligating the target gene AsCDA with the vector, we first transformed the ligation product into E. coli DH5α, a cloning strain that helps confirm the successful transformation of the recombinant plasmid. As shown in Figure 3A, numerous colonies grew on the monoclonal plate after transformation of DH5α, indicating a high probability of successful transformation. However, further verification was required through colony PCR, so 5-6 colonies were selected from the monoclonal plate for verification. After colony PCR amplification, the amplified products were subjected to agarose gel electrophoresis, and the results are shown in Figure 3B. The length of pET-28a(+)-AsCDA was consistent with our expected size of 1000 bp, confirming that the transformation plasmid was successful.
![Verification of the transformation results of pET-28a(+)-AsCDA positive colony](https://static.igem.wiki/teams/5721/img/engineering/3.webp)
Figure 3. Verification of the transformation results of pET-28a(+)-AsCDA positive colony. A: The single colony on the plate; B: The amplification results of colony PCR.
Finally, we used universal primer T7-Forward to verify the positive colonies by Sanger sequencing. Figure 4 showed the correct sequences of recombination plasmids. We chose correct colony for further IPTG induction.
![Sanger sequencing results of pET-28a(+)-AsCDA positive colony](https://static.igem.wiki/teams/5721/img/engineering/4.webp)
Figure 4. Sanger sequencing results of pET-28a(+)-AsCDA positive colony.
We transformed the pET28a-AsCDA recombinant plasmid into DH5αcells. After verification by colony PCR, colonies were amplified and the plasmid was extracted and sent to a biotechnology company for sequencing. This series of verifications confirmed that the recombinant plasmid contained no ligation errors, base deletions, or mutations. Next, the correct plasmid was transformed into the E. coli expression strain BL21(DE3). As shown in Figure 5A, numerous single colonies grew overnight after transformation into BL21(DE3). However, colony PCR was still required to confirm that the transformed colonies contained the correct recombinant plasmid. Since the correct recombinant plasmid was obtained, agarose gel electrophoresis was performed on the products from colony PCR. Electrophoresis results showed that the amplified fragment in pET28a-AsCDA was 1202 bp in length, which was consistent with the expected length. Therefore, transformation into BL21(DE3) was successful. Based on this, we were able to culture the correct single colony and proceed with protein expression.
![Monoclonal plate of BL21 (DE3) transformed with recombinant plasmid](https://static.igem.wiki/teams/5721/img/engineering/5.webp)
Figure 5. A: Monoclonal plate of BL21 (DE3) transformed with recombinant plasmid, B: PCR electrophoresis results of colonies on monoclonal plate after transformation.

### Part 2 pET28a\_Cts2p (BBa\_25OCX0JU)

**Design:**
The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene Cts2p was synthesized by a biotechnology company. The Cts2p fragment was homologously recombined with the linearized plasmid to construct pET28a-Cts2p.
![The plasmid map of pET28a-Cts2p](https://static.igem.wiki/teams/5721/img/engineering/6.webp)
Figure 6. The plasmid map of pET28a-Cts2p

**Build:**
We constructed pET28a-Cts2p by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5431 bp. Figure 2B shows a band consistent with the target size. The Cts2p gene was amplified by PCR, with a length of 1536 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. The recombinant plasmid was obtained by homologous recombination after agarose gel electrophoresis and gel recovery.
The recombinant plasmid was introduced into E. coli DH5α and BL21, and different numbers of colonies were selected for colony PCR verification. Figure 3/5 B shows that the length is consistent with the target fragment, Figure 3/5 A is the clone on the plate, and the results in Figure 4 show that the Cts2p gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a-Cts2p was successfully constructed.

### Part 3 pET28a-FjChiB (BBa\_250AYN60)

**Design:**
The pET28a(+) plasmid was provided by the strain library of our unit, and the target gene FjChiB was synthesized by a biotechnology company. The FjChiB fragment was homologously recombined with the linearized plasmid to construct pET28a-FjChiB.
![The plasmid map of pET28a-FjChiB](https://static.igem.wiki/teams/5721/img/engineering/7.webp)
Figure 7. The plasmid map of pET28a-FjChiB

**Build:**
We constructed pET28a-FjChiB by homologous recombination. The pET28a vector backbone was obtained by enzyme digestion, with a length of 5431 bp. Figure 2B shows a band consistent with the target size. The FjChiB gene was amplified by PCR, with a length of 1023 bp. Figure 2A shows a band consistent with the target size, indicating that both the target gene and the vector were successfully amplified. The recombinant plasmid was obtained by homologous recombination after agarose gel electrophoresis and gel recovery.
The recombinant plasmid was introduced into E. coli DH5α and BL21, and different numbers of colonies were selected for colony PCR verification. Figure 3/5 B shows that the length is consistent with the target fragment, Figure 3/5 A is the clone on the plate, and the results in Figure 4 show that the FjChiB gene has been successfully connected to the pET28a vector without obvious mutations, confirming that the recombinant plasmid pET28a-FjChiB was successfully constructed.

### 1 Protein expression test

**1.1 Protein expression**
After confirming that the three recombinant plasmids were successfully transformed into the expression strain BL21, we can perform protein expression tests. First, we need to induce the expression of a small amount of protein without setting different parameter conditions. Specifically, we set different temperatures (16 degrees Celsius and 37 degrees Celsius) and time (0-12h). Then, the recombinant BL21 bacterial solution containing the positive was inoculated into 5mL LB (containing kanamycin) liquid culture medium at a rate of 1/100, and shaken overnight. The 5mL overnight bacterial solution was inoculated into 250mL LB (pET-28a is Kan+ resistant) liquid culture medium and shaken at 37℃ until the OD600 was about 0.6 (about 3~4 hours, adding IPTG when OD was 0.6 had the best effect, and fewer protein bands were induced). 1ml of bacterial solution was retained as a control; 1M sterile IPTG was added. To a final concentration of 1 mM, culture at different temperatures, collect bacterial liquid at different time points, and centrifuge at 3000g for 10 minutes, collect 1 mL of bacteria, and retain the bacterial liquid without inducer as the control group. Then use 100 μL of 1x protein loading buffer to resuspend and boil, and after cooling, perform protein electrophoresis identification.
Figure 8 shows the protein electrophoresis results (SDS-PAGE gel) of three recombinant chitinases (AsCDA, Cts2p, and FjChiB) expressed in E. coli using the pET28a-6xHis vector. Experimental conditions included induction with 1 mM IPTG at 16℃ and 37℃, with samples collected at different time points (0 h, 0.5 h, 1 h, 3 h, 6 h, and 12 h). Markers (M) serve as molecular weight references, and protein bands are indicated by orange arrows. Based on the theoretical molecular weights, the three recombinant proteins are: AsCDA-6xHis: approximately 37.5 kDa, Cts2p-6xHis: approximately 59.1 kDa, and FjChiB-6xHis: approximately 38.1 kDa. Specific protein bands clearly appear near the corresponding molecular weights, increasing over time, demonstrating that all three target proteins were successfully expressed in E. coli. At 16℃, a band near 37 kDa was observed to gradually increase in intensity for AsCDA (37.5 kDa), but the overall intensity was not as strong as at 37℃. The 37.5 kDa band significantly intensified with induction time, becoming particularly pronounced after 3 hours and peaking at 6 and 12 hours. The band was very clear. AsCDA protein expression levels were higher at high temperatures, suggesting that the protein's structure is more stable, making it suitable for induction at 37℃ and less susceptible to misfolding or degradation.
Cts2p (59.1kDa) showed a more gradual increase in intensity at 16℃, with overall expression levels being low. However, the protein band was clear, indicating more stable expression at low temperatures. At 37℃, the band near 59 kDa significantly intensified, particularly after 3 hours, rapidly deepening and reaching very high intensity at 6 and 12 hours. This indicates that Cts2p is a large molecular weight protein. High temperatures can yield higher yields, but this also carries the potential risk of inclusion bodies. Low-temperature expression, while yielding lower amounts, is beneficial for proper protein folding and soluble expression.
FjChiB (38.1kDa) showed a weak band at 16℃, which slightly intensified at 6 and 12 hours of induction. At 37℃, the 38 kDa band gradually intensified after induction, becoming prominent at 1 hour and reaching peak expression at 6 and 12 hours. The significantly higher expression efficiency of FjChiB at high temperatures compared to low temperatures suggests its structural stability is suitable for rapid high-temperature induction. However, the overall difference was not significant. Therefore, based on literature, we controlled the subsequent large-scale fermentation of this protein at an IPTG induction concentration of 0.3 mM and a temperature of 27℃.
![SDS-PAGE analysis for small amount of target proteins expression](https://static.igem.wiki/teams/5721/img/engineering/8.webp)
Figure 8. SDS-PAGE analysis for small amount of target proteins expression.

**1.2 Protein His-tag purification**
After confirming the fermentation conditions, we performed large-scale fermentation of the three proteins, inducing them at 1 mM IPTG and 16℃ for 16 h to achieve heterologous expression of the chitin deacetylase AsCDA. SDS-PAGE electrophoresis (Figure 9) revealed a distinct band around 37.5 kDa in the whole-cell lysate, demonstrating efficient expression of AsCDA under induced conditions. Further comparison of the soluble and precipitated fractions revealed a strong band of the target protein in the supernatant, with a certain amount of protein also present in the precipitate. This indicates good soluble expression of AsCDA under these conditions, although some protein was still present as inclusion bodies, a common finding in heterologous expression.
As the imidazole concentration increased, distinct and intense target protein bands appeared in the elution fractions between 20 and 100 mM, indicating gradual elution of the target protein. The band intensity was significantly reduced in the elution fractions at higher concentrations of 250 mM and 500 mM, indicating that the primary target protein was completely released under low and medium imidazole concentrations. This result demonstrates that the purification process can obtain high-purity AsCDA, and the elution peak is relatively concentrated, facilitating subsequent collection and application.
![The expression of a large amount of target protein AsCDA was analyzed by SDS-PAGE](https://static.igem.wiki/teams/5721/img/engineering/9.webp)
Figure 9. The expression of a large amount of target protein AsCDA was analyzed by SDS-PAGE.
Cts2p (59.1 kDa) chitinase exonuclease was expressed using 1 mM IPTG at 16℃ for 16 h. This low-temperature, long-term induction strategy was chosen to slow cellular metabolism, facilitate folding of the target protein into a soluble form, and avoid the formation of numerous inclusion bodies.
SDS-PAGE electrophoresis results (Figure 10) showed a distinct band corresponding to Cts2p at 59.1 kDa in the whole-cell lysate, indicating successful induced expression of the target protein. Further comparison of the supernatant and precipitate revealed the presence of the target protein in the soluble supernatant fraction, demonstrating that Cts2p has a certain degree of soluble expression under these conditions. As the imidazole concentration gradually increased, a 59.1 kDa band was clearly observed in the 20–50 mM elution fractions, while the band gradually weakened or even disappeared in the higher 100–500 mM elution fractions, indicating that the primary target protein can be effectively eluted under low to moderate imidazole concentrations.
Overall, these results demonstrate that under the induction conditions of 1 mM IPTG, 16℃, and 16 hours, Cts2p can be expressed at high levels and in a partially soluble form, facilitating efficient purification by Ni-NTA affinity chromatography.
![The expression of a large amount of target protein Cts2p was analyzed by SDS-PAGE](https://static.igem.wiki/teams/5721/img/engineering/10.webp)
Figure 10. The expression of a large amount of target protein Cts2p was analyzed by SDS-PAGE.
The chitinase FjChiB was fermented at 27℃ using 0.3 mM IPTG as an inducer for 16 hours. Electrophoresis results (Figure 11) demonstrate successful induced expression and purification. In the whole-cell lysate, a dense new band was clearly observed slightly above the 34 kD marker, consistent with the theoretical molecular weight of the target protein FjChiB (38.1 kD). Finally, stepwise elution was performed using different imidazole concentrations. Clear target protein bands were observed in the elution lanes from 20 mM to 500 mM imidazole, with the most intense bands at approximately 20mM concentrations, confirming successful elution and purification of the target protein.
The chitinase FjChiB was efficiently expressed under the induction conditions of 0.3 mM IPTG at 27℃ for 16 hours. The expression product exhibited a mixture of soluble and insoluble properties, a common finding in recombinant protein expression. The purification process was successfully designed, and imidazole gradient elution results demonstrated good binding specificity to the nickel column.
![The expression of a large amount of target protein FjChiB was analyzed by SDS-PAGE](https://static.igem.wiki/teams/5721/img/engineering/11.webp)
Figure 11. The expression of a large amount of target protein FjChiB was analyzed by SDS-PAGE.

### 2. Functional test

**2.1 Free enzyme activity test**
After obtaining a large amount of purified protein, we first conducted preliminary measurements of the enzyme activities of the three proteins. For the determination of chitin deacetylase, we used the p-nitroacetanilide method, and for chitin exonuclease and endonuclease, we used the N-acetylglucosamine method. Both methods require the preparation of standard curves.
![N-acetylglucosamine standard curve](https://static.igem.wiki/teams/5721/img/engineering/12.webp)
Figure 12. N-acetylglucosamine standard curve
Figure 12 shows the standard curve used for quantitative analysis. The image above shows the color gradient produced by the reaction of N-acetylglucosamine (GlcNAc) standard solutions with different concentrations and DNS. The solution color changes gradually from light yellow to dark red as the concentration increases (from left to right), indicating increasing absorbance. The fitted curve below accurately quantifies this color change. The horizontal axis (X-axis) represents the N-acetylglucosamine standard concentration (in mg/mL), and the vertical axis (Y-axis) represents the absorbance measured at a wavelength of 540 nm. The curve exhibits a good linear positive correlation. The fitted linear equation is y = 2.0439x - 0.0288, with a squared correlation coefficient R2 = 0.9849.
![p-Nitroacetanilide standard curve](https://static.igem.wiki/teams/5721/img/engineering/13.webp)
Figure 13. p-Nitroacetanilide standard curve
This standard curve (Figure 13) serves as a quantitative scale for chitin deacetylase (AsCDA) activity assays. Its core function is to convert measured absorbance values into precise product concentrations, thereby calculating enzyme activity. Chitin deacetylase catalyzes the removal of the acetyl group (-COCH3) from chitin or its derivative substrates (such as glycol chitin), generating free amino groups and acetic acid. In this assay, a synthetic chromogenic substrate, such as p-nitroacetanilide, is typically used. Upon enzyme action, this substrate hydrolyzes to form p-nitroaniline. p-Nitroaniline is a compound with a strong yellow absorption around 400 nm (as indicated by the color gradient in the upper test tube). Therefore, the more p-nitroaniline produced by the enzyme reaction, the higher the absorbance of the solution at 400 nm. Based on the calculated parameters such as product concentration, reaction time, enzyme solution volume and cuvette path length, the enzyme activity unit can be finally calculated.
According to the formula corresponding to the standard curve, we calculated that the chitin exonuclease (Cts2p) activity was 115 U/mL, the chitin endonuclease (FjChiB) activity was 116 U/mL, and the chitin deacetylase (AsCDA) activity was 41 U/mL.

**2.2 Optimal free enzyme activity reaction conditions**
After preliminarily measuring the activity of the three enzymes, we need to explore the optimal reaction conditions for the three enzymes, starting with the optimal pH value.
![The optimal pH values of three proteins were investigated](https://static.igem.wiki/teams/5721/img/engineering/14.webp)
Figure 14. The optimal pH values of three proteins were investigated, where A represents chitin deacetylase AsCDA, C represents chitin exonuclease Cts2p, and F represents chitin endonuclease FjChiB, the same below.
The results in Figure 14 show that chitin deacetylase (AsCDA, A), chitin exonuclease (Cts2p, C), and chitin endoenuclease (FjChiB, F) all exhibit a typical pH-dependent relationship in enzyme activity, with their optimum pH values around 6. The activities of all three enzymes reach peak values at pH 7, indicating that this pH value is the optimal condition for their catalytic reactions.
Enzyme activity generally decreases under conditions deviating from the optimal pH. In particular, in extremely acidic (pH 2-3) or alkaline (pH 10-11) environments, the activities of all three enzymes decrease significantly, or even become completely inactivated. Notably, all three enzymes maintain relatively high activity over a wide pH range of 4-8, demonstrating a certain degree of acid-base stability. This provides flexibility in the selection of buffer systems for practical applications.
The discussion suggests that the similarity in the optimum pH values of the three enzymes may be related to the ecological environment of their source microorganisms or the characteristics of the substrates they act on. Furthermore, this highly efficient catalytic performance under neutral to slightly acidic conditions is consistent with the general properties of chitin-degrading enzymes. This result suggests that when using these enzymes for chitin bioconversion, the pH of the reaction system should be controlled around 6 to achieve optimal catalytic efficiency. Furthermore, the enzyme's stability over a wide pH range offers potential advantages for its application in complex industrial environments.
![The optimal temperature values of three proteins were investigated](https://static.igem.wiki/teams/5721/img/engineering/15.webp)
Figure 15. The optimal temperature values of three proteins were investigated, where A represents chitin deacetylase AsCDA, C represents chitin exonuclease Cts2p, and F represents chitin endonuclease FjChiB.
The bar graph and line chart in Figure 15 show significant differences in the optimal temperatures of the three chitinases. The chitin deacetylase AsCDA (A) exhibits maximum activity in the 40-50℃ range, reaching its peak at 40℃. The exochitinase Cts2p (C) exhibits an optimal temperature of approximately 50℃. The endochitinase FjChiB (F) exhibits a higher optimal temperature, maintaining high activity between 40-50℃. The activity of all enzymes decreased significantly at low temperatures (20℃) and high temperatures (70℃).
This difference in optimal temperature may be related to the microbial ecosystems from which the enzymes originate and the molecular structure of the enzymes. The mesophilic properties of AsCDA and Cts2p suggest that they may originate from mesophilic microorganisms, while the higher optimal temperature of FjChiB suggests that it may originate from a thermotolerant strain or possess a more stable spatial structure. The generally high activity of the enzymes around 50℃ suggests that this temperature may be the common optimal condition for chitinase-catalyzed reactions. These findings provide an important basis for optimizing the application conditions of chitinase. It is recommended that in practical applications, a reaction temperature of 40℃ be selected according to the enzyme type to obtain the best catalytic efficiency.
![The optimal metal ions for three proteins were investigated](https://static.igem.wiki/teams/5721/img/engineering/16.webp)
Figure 16. The optimal metal ions for three proteins were investigated, where A represents chitin deacetylase AsCDA, C represents chitin exonuclease Cts2p, and F represents chitin endonuclease FjChiB.
The bar graph and line chart in Figure 16 clearly demonstrate the responses of three chitinases (AsCDA, Cts2p, and FjChiB) to different metal ions. the chitin deacetylase AsCDA (A) exhibits the highest activity under Mn2+ conditions; the exochitinase Cts2p (C) exhibits the strongest response to Mn2+(nearly 300 U/mL); and the endochitinase FjChiB (F) exhibits optimal activity under Mn2+ conditions (approximately 280 U/mL).
This result suggests that the effects of metal ions on enzyme activity are highly specific, potentially related to the enzyme's spatial structure, active site characteristics, and catalytic mechanism. AsCDA's preference for Mn2+ may stem from the ion's stabilizing effect on its substrate-binding domain; Cts2p's specific binding to Mn2+ suggests that the ion may be directly involved in the conformational regulation of its catalytic center; and FjChiB's compatibility with Mn2+ may be related to the ion's ability to maintain its structural stability.

**2.3 Optimal free enzyme synergistic ratio and reaction conditions**
After exploring the optimal reaction conditions of the three enzymes, we need to explore the enzyme synergy. Specifically, we first mix them in the same volume ratio to determine the optimal effect of the enzyme combination, and then explore the ratio of enzyme synergy. In order to determine which combination has a better effect, we used the chitinase detection kit for exploration. Before the determination, we need to specify the standard curve.
![Chitinase activity assay kit standard curve](https://static.igem.wiki/teams/5721/img/engineering/17.webp)
Figure 17. Chitinase activity assay kit standard curve, A: standard curve preparation process, B: standard curve
The results in Figure 18 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; 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.
![The enzyme activity of different types of enzymes is determined by synergy](https://static.igem.wiki/teams/5721/img/engineering/18.webp)
Figure 18. 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 bar chart in Figure 19 shows the trends in reducing sugar yields in the reaction systems of the AsCDA+Cts2p and AsCDA+FjChiB enzyme combinations at different ratios. For the AsCDA+Cts2p combination, the highest reducing sugar yield was achieved at a 1:1 ratio, significantly higher than at other ratios. As the ratio deviated from 1:1 (e.g., 0.5:1 or 1:0.5), reducing sugar yields decreased significantly, indicating a strict stoichiometric dependence between the two. For the AsCDA+FjChiB combination, reducing sugar yields were higher at ratios of 1:0.5 and 1:1, with the 1:0.5 ratio showing the highest yield. This suggests that this combination is less sensitive to changes in ratio and has a wider range of adaptability.
This result may stem from differences in the synergistic mechanisms of the two enzymes: AsCDA and Cts2p may require precise molecular matching to achieve efficient synergy. For example, the exolytic activity of Cts2p depends on the specific substrate conformation generated by AsCDA deacetylation. In contrast, the synergy between AsCDA and FjChiB may be achieved through more relaxed functional complementarity, with FjChiB's endolysis generating more termini that provide substrates for AsCDA. Notably, the difference in peak yield between the two combinations (AsCDA+Cts2p outperforming AsCDA+FjChiB) suggests that Cts2p and AsCDA exhibit superior synergy, providing important insights into optimizing enzyme combinations.
![Study on the content of reducing sugar products in different synergistic ratios](https://static.igem.wiki/teams/5721/img/engineering/19.webp)
Figure 19. Study on the content of reducing sugar products in different synergistic ratios of AsCDA+Cts2p and AsCDA+FjChiB
Since the AsCDA+Cts2p combination has the highest reducing sugar yield when the ratio is 1:1, and the AsCDA+FjChiB combination has the highest reducing sugar yield when the ratio is 1:0.5, we also explored the optimal reaction conditions for the two ratios of complex enzymes.
The bar graph in Figure 20 shows significant differences in the optimal pH values for the two enzyme complexes, AC (AsCDA+Cts2p) and AF (AsCDA+FjChiB). The AF complex exhibits peak activity (approximately 170 U/mL) at pH 5 and maintains a high activity level within the pH range of 3-6. The AC complex exhibits an optimal pH of 7, with a peak activity of approximately 120 U/mL, and its activity is relatively stable within the pH range of 6-8. The activity of both enzymes decreases significantly under extreme pH conditions (pH ≤ 3 or ≥ 9).
Notably, the overall activity of the AF complex is significantly higher than that of the AC complex (by an average of approximately 40%), and its optimal pH range is more acidic. This phenomenon may be due to the stronger synergistic effect between FjChiB (endonuclease) and AsCDA (deacetylase): the endonuclease cleaves chitin chains to produce more ends, providing more sites for the deacetylase to target, and deacetylation further enhances the substrate's sensitivity to the endonuclease. In contrast, the synergistic efficiency of Cts2p (exonuclease) and AsCDA is low, which may lead to the poor overall activity of the AC complex enzyme.
![Exploration of the optimal pH value for the activity of two complex enzymes](https://static.igem.wiki/teams/5721/img/engineering/20.webp)
Figure 20. Exploration of the optimal pH value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB
The bar graph in Figure 21 shows that the two enzyme complexes, AC (AsCDA+Cts2p) and AF (AsCDA+FjChiB), exhibit significant differences in their optimal temperatures. The AC enzyme reaches peak activity (approximately 120 U/mL) at 40℃. The activity at 20℃-30℃ and 50℃-70℃ is similar, stable at around 100U/mL, The AF enzyme complex exhibits a completely different temperature response, its activity peaks at 50℃ (over 200 U/mL) and remains (150U/mL) within the 20-30℃ range, demonstrating better temperature tolerance than the AC enzyme.
This phenomenon may be due to the different temperature adaptabilities of the different enzyme components in the two enzyme complexes. The Cts2p (exonuclease) in the AC complex may be sensitive to high temperatures, leading to its rapid inactivation above 40℃. However, the FjChiB (endonuclease) in the AF complex possesses inherently good thermostability (optimum temperature around 50℃), and its synergistic effect with AsCDA further enhances the complex's thermostability. Notably, the AF complex's activity at 50℃ is nearly 1.7 times that of the AC complex, indicating that the synergistic effect of AsCDA and FjChiB is more pronounced at high temperatures.
![Exploration of the optimal temperature value for the activity of two complex enzymes](https://static.igem.wiki/teams/5721/img/engineering/21.webp)
Figure 21. Exploration of the optimal temperature value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB
The bar graph in Figure 22 shows that different metal ions significantly and regularly affect the activity of the AC (AsCDA+Cts2p) and AF (AsCDA+FjChiB) enzyme complexes. For the AC enzyme, Mn2+ had the most significant activation effects, reaching approximately 135 U/mL and 110 U/mL, respectively, significantly higher than those treated with other ions.
The AF enzyme complex was more sensitive to metal ions, with Mn2+ exhibiting the strongest activation effect (approximately 150 U/mL), followed by Zn2+ (approximately 140 U/mL). while Ni2+ still exhibited inhibitory effect.
![Exploration of the optimal Metal ions value for the activity of two complex enzymes](https://static.igem.wiki/teams/5721/img/engineering/22.webp)
Figure 22. Exploration of the optimal Metal ions value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB
Since we discovered that the synergistic catalytic activity of AsCDA+Cts2p and AsCDA+FjChiB was higher than that of the former, we selected the latter for the next step of enzyme immobilization. First, we obtained purified AsCDA and FjChiB proteins through heterologous expression in E. coli. Both proteins were then immobilized with epoxy resin. The supernatants before and after fixation were collected for SDS-PAGE analysis (samples can be diluted 5-fold). ImageJ software was used to calculate the grayscale value changes of the bands and the immobilization efficiency: Immobilization efficiency (%) = (grayscale value of the sample before fixation − grayscale value of the sample after fixation) / grayscale value of the sample before fixation \* 100%, As shown in Figure 23, the next step is to calculate the grayscale value using imageJ software.
![Purified proteins of AsCDA and FjChiB and SDS-page images of proteins before and after immobilization](https://static.igem.wiki/teams/5721/img/engineering/23.webp)
Figure 23. Purified proteins of AsCDA and FjChiB and SDS-page images of proteins before and after immobilization

**2.4 Preparation and immobilized enzyme efficiency**
Before calculating the grayscale value, for accurate grayscale value analysis, we first convert the image to 8-bit grayscale image, and then we measure the mean grayscale value and integrated density. The higher the protein expression, the larger the value of Integrated Density.
The results in Figure 24 show significant differences between the two proteins before and after immobilization. Grayscale analysis showed that the immobilization efficiency of AsCDA was 17.84%; the immobilization efficiency of FjChiB was 24.59%. This indicates that both proteins experienced some loss during the immobilization process, with FjChiB having a relatively higher immobilization efficiency.
SDS-PAGE electrophoresis patterns show that the band positions of the two proteins (approximately 37.5 kDa for AsCDA and approximately 38.1 kDa for FjChiB) remained unchanged before and after immobilization, indicating that the immobilization process did not cause protein degradation or molecular weight changes. However, the band intensity decreased significantly after immobilization, consistent with the decreasing grayscale value trend.
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.
![Calculation of grayscale values of the two proteins before and after immobilization](https://static.igem.wiki/teams/5721/img/engineering/24.webp)
Figure 24. Calculation of grayscale values of the two proteins before and after immobilization**2.5 Number of reuses of immobilized enzymes**
After the enzyme immobilization treatment, we added the two together according to the ratio and explored the number of times of repeated use.
![Number of times the immobilized enzyme can be reused](https://static.igem.wiki/teams/5721/img/engineering/25.webp)
Figure 25. Number of times the immobilized enzyme can be reused, abcde represent the first to fifth repeated experiments
Figure 25 shows the relationship between the number of times the immobilized enzyme was reused and its activity. The results show that enzyme activity shows a continuous downward trend with increasing reuse. The enzyme activity reached its highest level (approximately 43 U/g) during the first use, declining to approximately 22 U/g by the fifth use, with activity retention approximately 60% of the initial value.
This activity decay trend may be due to several factors. The immobilized enzyme may experience physical loss with each use, with enzyme molecules gradually falling off the support surface due to mechanical agitation or washing. Furthermore, the enzyme protein may undergo structural denaturation during repeated use, particularly conformational changes in the active center, leading to reduced catalytic efficiency, the accumulation of reaction products or impurities on the support surface may hinder substrate-enzyme contact.
Notably, despite the gradual decrease in enzyme activity, a significant percentage of activity was retained after five reuses, indicating that immobilization significantly improved enzyme stability. The activity decrease was most pronounced from the first to the second use, Then the rate of descent accelerated.
Therefore, the reusability of immobilized enzymes can significantly reduce the cost of enzyme preparations, but the optimal replacement cycle needs to be determined according to specific process requirements.

### Learn

1. The three enzymes (AsCDA, Cts2p, and FjChiB) exhibit significant differences in their optimal pH, temperature, and metal ion responses, demonstrating that even within the same chitinase family, their catalytic properties and structural stability exhibit wide diversity. Importantly, the complex enzymes exhibited significantly superior synergistic degradation efficiency compared to single enzymes or other combinations under multiple conditions, demonstrating that enzyme co-design is an effective strategy for improving substrate conversion efficiency.
2. While immobilization significantly improved operational stability and reusability (60% activity retained after five cycles), the immobilization efficiency was relatively low (17-25%), suggesting that further optimization of support selection, coupling chemistry, and surface modification techniques is needed to balance immobilization efficiency and enzyme activity retention.
3. The chitinase system can efficiently convert marine waste (such as shrimp and crab shells) into high-value-added products (such as N-acetylglucosamine and calcium lactate), aligning with the development of a circular bioeconomy. Future applications include wastewater treatment, marine plastic co-degradation, and crustacean biomass refining.
4. Although the immobilization efficiency needs to be improved, the reusability of the enzyme has been verified, significantly reducing the cost of single use. If it can be combined with reactor design (such as microfluidics and packed beds), it is expected to develop a continuous flow biocatalytic process suitable for large-scale production.

### References

1.Ahmedd S. Polysaccharides: Chitin and Chitosan[J]. Chitin and Chitosan: Physical and Chemical Properties, 2025, 1.
2.Gaderer R, Seidl-Seiboth V, Kappel L. Chitin and N-acetylglucosamine metabolism in fungi-a complex machinery harnessed for the design of chitin-based high value products[J]. Current Biotechnology, 2017, 6(3): 178-193.
3.Dave U, Somanader E, Baharlouei P, et al. Applications of chitin in medical, environmental, and agricultural industries[J]. Journal of Marine Science and Engineering, 2021, 9(11): 1173.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
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Experiments

![shrimp](https://static.igem.wiki/teams/5721/img/desc-xia.webp)

[1. Preparation of LB culture medium](#content1)

[2. The construction of plasmid](#content2)

[3. Protein expression and purification](#content3)

[4. Functional test](#content4)

### 1. Preparation of LB (Luria-Bertani) culture medium

**Goal:** To formulate a nutrient-rich medium capable of supporting the growth, activation, and fermentation of Escherichia coli and other microorganisms. Both liquid and solid forms can be prepared depending on experimental needs.

**Materials:**
**Required Materials and Reagents：**
1)Glass conical flask (≥1 L capacity)
2)High-pressure steam sterilizer (autoclave)
3)Tryptone (protein hydrolysate, nitrogen source)
4)Yeast extract (vitamin and growth factor source)
5)Sodium chloride (NaCl, osmotic stabilizer)
6)Double-distilled water (ddH2O)
7)Agar powder (only for solid medium preparation)

**Procedures:**
**1.Weighing of components,** For each liter of medium, prepare:
a.10 g tryptone
b.5 g yeast extract
c.10 g NaCl
d.For solid medium: additionally weigh 15 g agar powder (final concentration 1.5%).

**2.Dissolution of ingredients**
a.Add all dry components into a beaker or flask containing ~800 mL ddH2O.
b.Stir continuously until completely dissolved.
c.Adjust the final volume to 1 L with ddH2O.

**3.Distribution into containers**
a.Transfer the prepared solution into a 1 L Erlenmeyer flask (leave 1/3 volume empty to prevent overflow during sterilization).
b.If required, dispense into smaller bottles or tubes according to downstream applications.

**4.Sterilization process**
a.Place the prepared medium into an autoclave.
b.Sterilize at 121℃ under 15 psi pressure for 20 minutes.
c.Allow the medium to cool naturally after sterilization.

**5.Final preparation**
a.For liquid LB: use directly after cooling.
b.For solid LB: keep the agar medium warm (~50℃), pour into sterile Petri dishes, and let it solidify under aseptic conditions.

### 2. The construction of plasmid

**pET-28a(+)-AsCDA/Cts2p/FjChiB**

**Ⅰ. Obtain target gene fragments by PCR**
**Goal:** PCR technology is used to amplify the target gene fragment to provide DNA fragments for subsequent infusion recombination.

**Materials:**
1)2x PrimeStarMix
2)Gene template
3)Double distilled water (ddH2O)
4)Primer-F
5)Primer-R
6)PCR tube
7)PCR thermal cycler
PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.

**Procedures:**
1)Aseptically prepare the PCR reaction system. The recommended total reaction volume is 50 µL: add 25 µL of 2×PrimeStar Mix, 1 µL of template DNA, 1 µL of Primer-F, and 1 µL of Primer-R. Add ddH2O to the remaining volume to 50 µL. After preparing the reaction solution, briefly centrifuge to remove any bubbles.
2)Place the reaction tube in a PCR instrument and set the reaction program:
3)Initial denaturation: 95℃ for 3 minutes to fully unwind the double-stranded structure of the template DNA;
4)Cycling: Perform 35 cycles, each cycle consisting of:
5)Denaturation at 95℃ for 30 seconds
6)Annealing at 55℃ for 30 seconds (the specific temperature can be adjusted based on primer design);
7)Extension at 72℃ for 2 minutes (the PrimeStar Mix has an extension rate of approximately 1000 bp/10 seconds; the time can be adjusted based on the target fragment size);
8)End extension: 72℃ for 1 minute to ensure complete extension of all fragments.
9)After the reaction, if not used immediately, the product can be stored at 4℃ for a short period of time.
10)To obtain purified DNA fragments, separate the PCR products by electrophoresis on a 1%–1.2% agarose gel. Cut out the target band and use a DNA recovery kit to extract the fragment for subsequent infusion recombination.

**Ⅱ. Linearize pET-28a(+) backbone by restriction endonucleases**
**Goal:** The pET-28a(+) plasmid was double-digested with restriction endonucleases EcoRI and XhoI to obtain a linearized vector fragment that can be used for subsequent infusion cloning.

**Materials:**
1)pET-28a(+) plasmid DNA (approximately 5 µg)
2)10×digestion buffer (matching the enzymes used)
3)EcoRI restriction enzyme
4)XhoI restriction enzyme
5)Sterile double-distilled water (ddH2O)
6)PCR tubes or 1.5 mL centrifuge tubes
7)Micropipette and tips
8)Benchtop low-speed centrifuge
9)PCR instrument or thermostatted metal bath

**Procedures:**
1. Prepare a 50 µL total reaction mixture on ice. Typical ratios are:
a.Plasmid DNA: approximately 5 µg
b.10×digestion buffer: 5 µL
c.EcoRI: 5 µL
d.XhoI: 5 µL
e.ddH2O: make up to 50 µL
2. Gently pipette the reaction mixture to mix thoroughly, avoiding violent shaking and creating bubbles. If bubbles are present, briefly centrifuge at low speed to allow the liquid to settle to the bottom of the tube.
3. Place the reaction mixture in a PCR instrument or metal bath at 37℃ for 30 minutes to allow the plasmid to be completely digested by both enzymes.
4. After digestion, remove a small sample for electrophoresis to confirm that the plasmid has been linearized. Subsequently, separate the entire reaction product on a 1% agarose gel and extract the corresponding band.
5. Purify the linearized pET-28a(+) fragment using a DNA purification kit according to the kit instructions. Dissolve the fragment in sterile ddH2O or low-salt buffer and set aside.

**Notes:**
• EcoRI and XhoI must maintain good activity in the same buffer system to avoid incomplete cleavage due to buffer incompatibility.
• During electrophoresis, use a DNA marker to accurately distinguish linearized plasmid from uncut supercoiled forms.
• Avoid prolonged UV exposure during gel excision and DNA recovery to minimize DNA damage.

**Ⅲ. Agarose gel construction, deployment and recycle**
**Goal:** Gels suitable for DNA electrophoresis are prepared by dissolving agarose in a buffer and adding a nucleic acid dye.

**Materials:**
1)Agarose powder (analytical grade, Agarose M is recommended)
2)1×TAE buffer
3)10,000×nucleic acid dye (such as GelRed, GelSafe, etc.)
4)Microwave oven
5)Erlenmeyer flask (≥ 200 mL)
6)Gel casting tray
7)Gel well comb
8)Gloves, pipette, and sterile tips

**Procedures:**
1. Weigh 1.5 g of agarose powder and add it to a 200 mL Erlenmeyer flask.
2. Add 100 mL of 1×TAE buffer to the flask. Gently swirl to evenly distribute the agarose powder.
3. Heat the mixture in a microwave oven. Heat for 30 seconds at a time, then gently shake the mixture to ensure the agarose is completely dissolved. Avoid excessive boiling and bubbles.
4. Once the agarose is completely dissolved and the solution is clear, cool it to approximately 60℃.
5. Add approximately 3 µL of 10,000×nucleic acid dye to the solution and mix thoroughly. Wear gloves to avoid contact with skin.
6. Pour the prepared agarose solution into the gel casting well. Pour the gel slowly along the sides of the well to reduce bubbles.
7. Insert a comb to create sample wells and let the gel stand at room temperature for approximately 20–30 minutes to allow the gel to completely solidify.
8. After the gel solidifies, gently remove the comb and carefully remove the casting template. This will yield an agarose gel with electrophoresis wells.
9. Place the prepared gel in the electrophoresis tank and cover it with 1×TAE buffer until the gel is completely submerged. Sample loading and electrophoresis can then begin.

**Notes:**
• Observe the agarose solution carefully as it dissolves to ensure there are no visible particles.
• If bubbles appear in the solution, remove them with a pipette before pouring the gel.
• When adding the dye, ensure the temperature is not too high to prevent inactivation of the dye.

**Ⅳ. Agarose gel electrophoresis**
**Goal:** Use agarose gel electrophoresis to detect PCR or enzyme digestion products to confirm whether the DNA band size is as expected, providing a basis for subsequent gel recovery.

**Procedures:**
1. Take approximately 50 µL of each sample to be tested and add 5 µL of 10× loading buffer. Pipette repeatedly to mix until the solution is uniform in color and to ensure that the sample is fully incorporated into the buffer.
2. Place the solidified agarose gel into a horizontal electrophoresis tank and add enough 1×TAE buffer to completely submerge the gel surface.
3. Before loading the sample, add a DNA molecular weight marker to the first sample well on the gel to facilitate subsequent band length comparison.
4. Carefully pipette the mixed DNA sample into the sample wells of the gel one by one, adding approximately 50 µL to each well. Slowly move the pipette tip close to the bottom of the well to avoid puncturing the gel or spilling the liquid.
5. Connect the electrophoresis instrument, set the voltage to 120 V, and run for approximately 20 minutes. Observe the dye migration at the sample front during electrophoresis to ensure the correct electrophoresis direction (DNA migration toward the positive electrode).
6. After electrophoresis, turn off the power and carefully remove the gel. Then, place the gel under a UV or blue light transilluminator to observe the positions of DNA bands and compare the fragment sizes to molecular weight standards.
7. Based on the size of the target fragment, determine whether gel excision and recovery are necessary and proceed to the subsequent DNA extraction step.

**Notes:**
• The loading buffer increases sample density, allowing it to sink into the gel wells. It also contains an indicator dye to facilitate monitoring of migration progress.
• Electrophoresis time that is too short will result in unclear DNA separation, while too long may cause small fragments to escape the gel. Therefore, adjustments should be made based on gel concentration and fragment size.
• When observing bands, minimize UV exposure time to avoid DNA damage, which could affect subsequent recovery efficiency.

**Ⅴ. DNA gel extraction**
**Goal:** The target DNA fragments (target gene and linearized pET-28a(+) vector) separated by electrophoresis were cut out from the agarose gel and purified to obtain high-purity DNA for subsequent cloning.

**Materials:**
• Agarose gel containing the target band
• 1.5 mL sterile centrifuge tube (EP tube)
• Agarose gel DNA recovery kit (including gel elution buffer, elution buffer, purification column, etc.)
• Pipette and tips
• Constant temperature water bath or metal bath (50℃)
• Benchtop high-speed centrifuge (supporting ≥12,000 rpm)

**Procedures:**
1)Confirm the location of the target DNA band using a blue light or UV gel exciter, minimizing prolonged UV exposure to the DNA.
2)Use a sterile scalpel or blade to cut the gel block containing the target band, cutting as close to the edge of the band as possible to avoid any excess agarose. Place the gel block in a 1.5 mL EP tube and record the approximate weight of the gel block (usually 100 mg = 100 µL).
3)Add an appropriate amount of dissolution buffer to the gel block (generally, approximately 3–5 times the volume of the gel block, 500 µL is common). Place the centrifuge tube in a 50℃ water bath, heating with intermittent gentle shaking until the gel block is completely dissolved.
4)Transfer the dissolved mixture to a DNA purification column, place it in a collection tube, and centrifuge at 12,000 rpm for 30 seconds to allow the DNA to bind to the silica membrane. Discard the flow-through.
5)Add 500 µL of wash buffer (containing 70% ethanol) to the column and centrifuge again for 30 seconds. Discard the flow-through.
6)Repeat the wash step to ensure removal of residual impurities and salts.
7)Centrifuge again at 12,000 rpm for 2 minutes to completely remove any residual ethanol from the column. Then, leave the purification column uncapped for approximately 1 minute to allow the ethanol to evaporate completely.
8)Transfer the purification column to a new, clean 1.5 mL EP tube. Slowly add 50 µL of sterile double-distilled water (ddH2O) or low-salt elution buffer to the center of the column membrane and let it sit for 1 minute to facilitate DNA elution.
9)Centrifuge again at 12,000 rpm for 1 minute. Collect the supernatant, which is the recovered DNA fragments.
10)Discard the purification column and store the DNA solution at 4℃ for short-term storage or -20℃ for long-term storage.

**Notes:**
• Avoid contaminants when cutting the gel. Smaller gel fragments improve purification efficiency.
• Ensure sufficient gel dissolution buffer, otherwise the gel fragments may not be completely dissolved.
• The final elution volume of DNA can be adjusted as needed. A smaller volume results in a higher concentration, but the total amount may be slightly lower.
• During the elution step, add water precisely to the center of the column membrane to ensure maximum recovery.

**Ⅵ. Homologous recombination of linearized pET-28a(+) backbone with target gene fragments**
**Goal:** To assemble the recombinant plasmid pET-28a(+) carrying the target gene (AsCDA/Cts2p/FjChiB) through homologous recombination using a linearized vector backbone and gene fragments.

**Materials:**
• CloneExpress Mix (2×reaction buffer and enzyme mix)
• Purified PCR fragments of target genes (AsCDA, Cts2p, FjChiB)
• Linearized pET-28a(+) backbone DNA
• PCR tubes suitable for thermal cycler use

**Procedure:**
**1.Quantification of DNA**
• After gel purification of both the linearized vector backbone and the PCR-amplified gene inserts, measure the DNA concentration of each sample using a spectrophotometer (e.g., NanoDrop) or equivalent method.
• Record concentrations to allow accurate adjustment of fragment ratios in the recombination reaction.

**2.Assembly Reaction Setup**
• In a sterile PCR tube, prepare the following reaction mixture for each construct:
• 5 µL of CloneExpress Mix (2×)
• 2.5 µL of the linearized pET-28a(+) backbone DNA
• 2.5 µL of the purified gene fragment (either AsCDA, Cts2p, or FjChiB)
• Gently pipette to mix and briefly centrifuge to collect the liquid at the bottom of the tube.

**3.Recombination Reaction**
• Place the PCR tubes into a thermal cycler.
• Run the recombination program: maintain at 50 ℃ for 30 minutes to allow ligation-free homologous recombination between the overlapping ends of the vector and insert.
• After completion, hold the reaction at 4 ℃ until transformation.

**Notes**
• Ensure equimolar ratios of insert and vector DNA for optimal recombination efficiency.
• Use freshly prepared, high-purity DNA fragments to maximize success rate.
• The resulting recombination products are directly suitable for transformation into competent E. coli cells.

**Ⅶ. Transformation**
**Materials:**
• Recombinant plasmid DNA
• LB liquid medium (without antibiotics)
• LB plates (with kanamycin)
• Competent E. coli BL21
• Ice
• Micropipette and tips
• Centrifuge
• 37℃ incubator

**Procedures:**
1. Place 50–100 µL of freshly prepared competent E. coli BL21 culture on ice. Add an appropriate amount of recombinant plasmid (approximately 10–50 ng) to the culture and mix gently.
2. Incubate the mixture on ice for 20 minutes to allow the plasmid to fully contact the cells.
3. Quickly transfer the tube to a 42℃ water bath for 45 seconds to heat shock the cells. This will instantly permeabilize the cell membrane and facilitate DNA entry into the cells. Immediately return the tube to ice for 2–3 minutes to stabilize the cell membrane.
4. Add 400 µL of LB liquid medium (without antibiotics) to the cells and incubate at 37℃ with shaking or allow the mixture to stand for 30 minutes to promote bacterial recovery and plasmid expression.
5. Centrifuge the culture at 5,000 rpm for 3 minutes, discard approximately 300 µL of supernatant (reserve a portion if desired), and gently resuspend the pelleted cells.
6. Spread the resuspended cells evenly onto an LB plate containing kanamycin, ensuring even distribution.
7. Invert the plate and incubate in a 37℃ incubator for 12–16 hours, until a single colony appears.
8. After transformation is complete, single colonies can be selected for liquid culture and subsequent plasmid extraction.

**Ⅷ. Colony PCR verification**
**Goal:** Colony PCR was used to identify whether the transformed E. coli BL21 single colonies contained the correctly constructed recombinant plasmid, thereby screening positive clones for subsequent experiments.

**Materials:**
• Taq DNA polymerase master mix (2×Master Mix)
• Double-distilled water (ddH2O)
• LB plates containing transformed bacteria
• PCR tubes
• Sterile pipette tips
• Specific primers (forward and reverse)
• PCR thermocycler
• Gel electrophoresis equipment and agarose gel

**Procedures:**

1. PCR Reaction Setup
• Add the following to each PCR tube:
• 10 µL 2×Taq Master Mix
• 1 µL Forward Primer
• 1 µL Reverse Primer
• 8 µL ddH2O
• Gently mix and briefly centrifuge to collect the liquid at the bottom of the tube.

2. Single Colony Sampling
• Use a sterile 10 µL pipette tip to pick up a single colony from the LB plate.
• Gently pipette the colony into the liquid in the PCR tube and mix by pipetting up and down several times to ensure that the colony is fully exposed to the reaction solution. Be careful not to introduce too much agar.

3. PCR Amplification
• Place the PCR tube in a thermal cycler and perform amplification according to the manufacturer's protocol or the temperature cycle designed for the target fragment:
• Initial denaturation
• Cycling of denaturation-annealing-extension
• Final extension
• The number of cycles and temperature parameters are determined by the primers and fragment length.

4. PCR Product Detection
• After amplification, load the PCR product onto an agarose gel for electrophoresis analysis.
• Determine the band size against a molecular weight standard. If a band of the expected size appears, the colony is positive and contains the correctly constructed recombinant plasmid.

### 3. Protein expression and purification

**3.1 Induction of protein expression for 6xHis-AsCDA/Cts2p/FjChiB**

**Ⅰ. Amplification culture for correct colony**
**Goal:** The pET-28a(+)-AsCDA/Cts2p/FjChiB recombinant protein was efficiently expressed in Escherichia coli BL21, and bacterial samples were collected at different times and temperatures for SDS-PAGE analysis of protein expression.

**Materials:**
• LB liquid medium
• E. coli BL21 transformed with pET-28a(+)-AsCDA/Cts2p/FjChiB
• Kanamycin solution
• Isopropyl β-D-thiogalactopyranoside (IPTG) inducer
• Iso-thermic shaker
• Spectrophotometer (for OD600 measurement)
• 1.5 mL centrifuge tubes and sterile pipette tips

**Procedures:**
1) Inoculation and Preculture
2) Add 100 µL of the recombinant bacterial culture to 100 mL of LB liquid medium. Also add an appropriate amount of kanamycin (e.g., 100 µg/mL) to ensure selective pressure.
3) Incubate the culture flask at 37℃ on a constant-temperature shaker, shaking vigorously to ensure aeration.
4) Growth Monitoring
5) Measure the OD600 value of the culture every 30 minutes until the culture reaches the logarithmic growth phase (OD600 ≈ 0.4–0.6), which usually takes approximately 2–3 hours.
6) At this stage, the bacteria are actively growing and suitable for IPTG induction.
7) Protein Induction
8) Add IPTG to the culture to a final concentration of 1 mM and gently mix to initiate recombinant protein expression.
9) Parallel cultures at different temperatures (e.g., 25℃ and 37℃) can be set up to compare expression efficiency and solubility.
10) Sampling and Monitoring
11) At 0.5 h, 1 h, 3 h, and 6 h after induction, collect equal volumes of bacterial culture samples (e.g., 1–2 mL) and place them in centrifuge tubes.
12) Samples can be frozen immediately or added to lysis buffer for subsequent SDS-PAGE analysis of protein expression.

**Notes:**
• IPTG solution should be prepared fresh or stored frozen in aliquots to avoid repeated freeze-thaw cycles.
• The induction temperature affects protein solubility and folding. Low temperatures (25℃) generally favor soluble protein expression.
• When sampling, perform the procedure as quickly as possible to prevent continued bacterial growth or protein degradation.

**Ⅱ: Protein crude extraction and SDS-PAGE**
**Goal:** Crude protein was extracted from the induced expression E. coli BL21 culture medium, and the expression and size of the target protein were analyzed by SDS-PAGE.

**Materials for protein crude extraction:**
• Induced bacterial suspension (approximately 500 µL per induction condition)
• Centrifuge
• Double-distilled water (ddH2O)
• Sangon 12.5% SDS-PAGE Preparation Kit
• Electrophoresis Buffer (Tris-Glycine)
• Protein Molecular Weight Standard
• 6×Protein Loading Buffer
• Vertical Electrophoresis System
• Coomassie Brilliant Blue Staining Solution
• Destaining Solution

**Procedures for protein crude extraction and SDS-PAGE:**
1. Crude Protein Extraction
• Transfer 500 µL of bacterial suspension from each induction condition to a centrifuge tube and centrifuge at 12,000 rpm for 1 minute.
• Discard the supernatant and resuspend the bacterial pellet in 50 µL of 1×protein loading buffer (6×loading buffer diluted with ddH2O).
• Heat the resuspension in a PCR thermocycler at 95℃ for 15 minutes to lyse the cells and denature the proteins.
• After completion, cool briefly or proceed immediately to SDS-PAGE analysis.

2. SDS-PAGE Gel Preparation
• Combine 2.2 mL of 2×separating gel solution, 2.2 mL of 2×separating gel buffer, and 44 µL of catalyst in a centrifuge tube. Mix gently.
• Slowly pour the separating gel into the gel mold, avoiding the formation of bubbles.
• Add 1 mL of ddH2O to the top of the separating gel and let it stand for approximately 8 minutes to allow the gel to fully polymerize.
• After the gel solidifies, discard the top layer of water.
• Combine 825 µL of 2×stacking gel solution, 825 µL of 2×stacking gel buffer, and 11 µL of catalyst. Mix gently.
• Pour the stacking gel into the mold until full and slowly insert the comb to avoid creating bubbles.
• Let it stand for approximately 12 minutes to solidify, then carefully remove the comb and rinse the wells with running buffer.

3. Sample Loading and Electrophoresis
• Add 10 µL of protein molecular weight standard to the first well, followed by an appropriate amount of protein sample to each well.
• Place the gel in a vertical electrophoresis tank and add running buffer.
• Set the electrophoresis condition to 120 V and run for approximately 90 minutes until the protein bands are clearly separated.

4. Staining and Destaining
• After electrophoresis, stain the gel in Coomassie Brilliant Blue solution for approximately 10 minutes.
• Rinse the gel repeatedly in destaining solution until the background becomes transparent and protein bands are clearly visible.

**Notes:**
• Be gentle when preparing the separating and stacking gels to avoid creating bubbles, which can affect protein band resolution.
• Load a moderate amount of sample to avoid excessive sample loading, which can cause band smearing.
• Adjust the staining and destaining times based on band appearance to achieve optimal contrast.

**3.2 Protein His-tag purification**
**Goal:** Utilizing the His tag on the recombinant protein, the target protein is recovered through Ni-NTA agarose affinity purification technology to provide high-purity protein for subsequent antigen preparation.

**Materials:**
• E. coli culture containing the target protein
• 50 mL centrifuge tube
• Ice-filled container for cold operation
• High-speed centrifuge
• Sonicator
• Beyotime Ni-NTA Agarose Affinity Purification Kit
• Pipette and sterile tips

**Procedures:**
1) Bacterial Cell Collection
2) Transfer the target protein-expressing bacterial suspension to a 50 mL centrifuge tube.
3) Centrifuge at 4000 rpm for 20 minutes to pellet the cells.
4) Bacterial Cell Lysis
5) Chill the centrifuge tube in an ice-water bath to maintain a low temperature and prevent protein denaturation.
6) Insert an ultrasonic probe and sonicate the cells. Each sample should be processed for approximately 15 minutes, with multiple intervals to avoid overheating.
7) All samples should be processed identically to ensure consistency.
8) Cell Debris Removal
9) After sonication, centrifuge the sample at 10,000 rpm for 30 minutes.
10) Discard the cell debris at the bottom and transfer the supernatant (containing the target protein) to a fresh centrifuge tube for later use. 4.Ni-NTA Agarose Pretreatment
11) Add 500 µL of Ni-NTA agarose to a 2 mL centrifuge tube and centrifuge at low speed (approximately 500×g for 5 minutes). Discard the supernatant.
12) Wash the agarose three times with 1.5 mL of PBS buffer, centrifuging at 500×g for 5 minutes each time to ensure removal of impurities from the storage solution.
13) Protein Binding
14) Add the pretreated Ni-NTA agarose to the protein supernatant and incubate at 4℃ with gentle shaking for 60 minutes to allow the His-tagged protein to fully bind to the agarose.
15) Column Packing and Washing
16) Transfer the incubated protein-agarose mixture to the adsorption column and let it stand for approximately 2 minutes to promote binding. Discard the flow-through.
17) Wash the agarose in the column with 5 mL of wash buffer five times to remove non-specifically bound proteins.
18) Protein Elution
19) Elute the target protein five times using elution buffer containing 250 mM imidazole (500 µL/wash).
20) Collect each eluate as a purified protein sample and combine for subsequent analysis.
21) SDS-PAGE Detection
22) Take a 50 µL sample of the eluate, add 6×protein loading buffer, and mix thoroughly.
23) Load the sample onto an SDS-PAGE gel and separate by electrophoresis.
24) Detect protein bands using Coomassie Brilliant Blue staining to verify the expression and purity of the target protein.

**Notes**
• Maintain low temperature throughout the entire process to avoid protein degradation.
• Intermittent sonication can be used with cooling intervals to prevent protein denaturation.
• The imidazole concentration in the wash and elution buffers can be adjusted based on the binding strength of the target protein to improve purity and recovery.
• The collected eluate can be further concentrated or dialyzed to meet the needs of subsequent antigen preparation or functional experiments.

### 4. Functional test

**4.1 Free enzyme activity test**
**Goal:** The enzyme activities of AsCDA, Cts2p, and FjChiB were determined to provide data support for subsequent comparison of enzyme performance and optimization of application conditions.

**Materials:**
**AsCDA enzyme activity assay:**
• p-Nitroacetanilide substrate (p-NPAc, 1 mg/mL)
• 50 mM phosphate buffer (pH 7.4)
• AsCDA enzyme solution
• Ice-water bath or stop solution (e.g., 1 M Na2CO3)
• Spectrophotometer (400 nm)
• 1.5 mL centrifuge tube

**Cts2p and FjChiB enzyme activity assay:**
• Colloidal chitin solution (1% w/v)
• Enzyme solution (Cts2p or FjChiB)
• DNS reagent
• Boiling water bath
• Centrifuge
• 1.5 mL centrifuge tube
• Spectrophotometer (OD540)

**Procedures:**
**a. AsCDA Enzyme Activity Assay**
1. Prepare a 200 μL total reaction system consisting of: 150 μL p-nitroacetanilide substrate solution, 40 μL buffer, and 10 μL AsCDA enzyme solution.
2. After mixing, incubate the reaction tube in a 37℃ water bath for 30 minutes.
3. Upon completion of the reaction, immediately terminate the reaction by placing the tube in ice water or adding an equal volume of stop solution.
4. Measure the absorbance at 400 nm using a spectrophotometer and calculate the concentration of p-nitroaniline produced using a standard curve.
5. Calculate enzyme activity according to the formula:
![formula](https://static.igem.wiki/teams/5721/img/experiments/formula.webp)
Where C is the p-nitroaniline concentration (μg/mL), V\_total is the total reaction volume (0.2 mL), V\_enzyme is the volume of enzyme solution added (0.01 mL), and t is the reaction time (30 minutes).
6. Set up a blank control (substrate + buffer only) and a negative control (inactivated enzyme or irrelevant protein + substrate), measure the absorbance, and subtract the background value from the sample.

**b. Cts2p and FjChiB Enzyme Activity Assay**
1. Combine 250 μL of 1% colloidal chitin solution with 250 μL of enzyme solution and mix thoroughly.
2. Incubate the mixture at 42℃ for 1 hour to allow the enzyme to catalyze the degradation of the substrate and produce reducing sugars.
3. After the reaction is complete, add 2 mL of DNS solution and immediately heat in a boiling water bath for 5 minutes.
4. Remove the mixture and cool the 50 + 50 + 400 μL DNS mixture to room temperature.
5. Centrifuge at 12,000 rpm for 5 minutes. Collect the supernatant and measure the OD540 absorbance.
6. Convert the OD540 value to reducing sugar concentration using a standard curve. Calculate the enzyme activity using defined enzyme activity units: the amount of enzyme required to produce 1 μmol/L reducing sugar per minute.
7. Similarly, set up a blank control (colloidal chitin + buffer, no enzyme) and a negative control (substrate + heat-inactivated enzyme or unrelated protein) to subtract the background absorbance.

**Notes**
• Maintain a constant temperature throughout the incubation process: Cts2p and FjChiB should be incubated at 42℃, and AsCDA should be incubated at 37℃ in a water bath.
• Controls are essential to correct for spontaneous substrate degradation or nonspecific reactions.
• DNS treatments must be cooled quickly after heating to avoid further degradation of reducing sugars.
• All assays should be repeated at least three times to ensure data reliability.

**4.2 Optimal free enzyme activity reaction conditions**
**Goal:** Determine the optimal reaction conditions for three chitin-related enzymes, AsCDA, Cts2p, and FjChiB, including optimal pH, temperature, pH stability, temperature stability, and the effects of metal ions on enzyme activity, to provide experimental basis for subsequent enzymatic applications and optimization.

**Materials:**
• Purified AsCDA, Cts2p, and FjChiB enzyme samples (0.5 mg)
• Colloidal chitin solution (1% w/v)
• Buffer system:
• 0.1 M glycine-HCl (pH 2.0–3.0)
• 0.1 M citric acid-Na2HPO4 (pH 3.0–8.0)
• 0.1 M Tris-HCl (pH 8.0–9.0)
• 0.1 M glycine-NaOH (pH 9.0–11.0)
• 50 mM Na2PO4-citric acid buffer (pH 4.0–6.0)
• 50 mM Na2PO4 buffer (pH 6.0–8.0)
• 50 mM Tris-HCl buffer (pH 8.0–9.0)
• 50 mM Glycine-NaOH buffer (pH 9.0–11.0)
• Metal ion solution (final concentration 10 mM): Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Na⁺, K⁺, Mn²⁺, Zn²⁺
• Spectrophotometer (OD540, for DNS assay of reducing sugars)
• Centrifuge tube (1.5 mL)
• Water bath

**Procedures:**
**a. Determination of Optimal pH**
1. Centrifuge 1% colloidal chitin, discard the supernatant, and resuspend in an equal volume of buffer covering the pH range of 2.0–11.0 (glycine-HCl, citric acid-Na2HPO4, Tris-HCl, glycine-NaOH).
2. Add each enzyme to the colloidal chitin system in different pH buffers and incubate at 37℃ for 1 hour.
3. After the reaction, determine the reducing sugar yield using the DNS method. The highest enzyme activity was set as 100%. A relative enzyme activity versus pH curve was plotted to determine the optimal pH.

**b. Optimal Temperature Determination**
1. Dissolve the enzyme in the optimal pH buffer.
2. Set a temperature gradient (20, 30, 40, 50, 60, 70, and 80℃) and add colloidal chitin for a 1-hour reaction.
3. Determine reducing sugar production by DNS. Plot a curve of relative enzyme activity versus temperature to determine the optimal reaction temperature.

**c. Effect of Metal Ions on Enzyme Activity**
1. At the optimal pH, add a single metal ion (final concentration of 10 mM, Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Na⁺, K⁺, Mn²⁺, or Zn²⁺) to the enzyme solution.
2. Incubate at 37℃ for 1 hour, measure reducing sugar production, calculate relative enzyme activity, and analyze the promoting or inhibiting effect of each metal ion on enzyme activity.

**Notes**
• When conducting pH or temperature gradient experiments, perform at least three replicates per group.
• When determining reducing sugars using the DNS method, ensure that the sample temperature is consistent to prevent non-enzymatic factors from influencing the results.
• For metal ion experiments, it is recommended to include a blank control (without metal ions) to correct for background.
• The optimal pH and temperature for each enzyme are determined based on the highest relative enzyme activity and are then used for optimization in subsequent experiments.

**4.3 Optimal free enzyme synergistic ratio and reaction conditions**
**Goal:** Evaluate the synergistic effects of three chitin-related enzymes: AsCDA, Cts2p, and FjChiB. Compare the total enzyme activity of different pairwise and trio combinations to identify the most active combination. Further optimize the volume ratio of the two enzymes (2:1, 1:1, 0.5:1, 1:0.5, and 1:2) to ensure optimal conditions for subsequent synergistic reactions and substrate degradation efficiency.

**Materials:**
• Purified AsCDA, Cts2p, and FjChiB enzyme samples
• Colloidal chitin solution (1% w/v)
• Buffer (optimal pH buffer, based on previous measurements)
• DNS reducing sugar assay reagent or commercial reducing sugar assay kit
• 1.5 mL centrifuge tube
• Micropipette and tips
• Water bath or constant temperature incubator
• Spectrophotometer (OD540 or according to kit instructions)

**Procedures:**
**a. Pairwise and Triple Enzyme Combination Testing**
1. Prepare the following combinations of the three enzymes in equal amounts (e.g., 20 μL per combination):
• AsCDA + Cts2p
• AsCDA + FjChiB
• Cts2p + FjChiB
• AsCDA + Cts2p + FjChiB
2. Add an equal volume of the optimal pH buffer to each combination and add colloidal chitin (1%) to the system to a uniform total volume (e.g., 500 μL).
3. Incubate at the optimal temperature for 1 hour.
4. After the reaction, determine the amount of reducing sugar produced in the system using the DNS method or a kit and record the absorbance or measured value.
5. Convert the reaction activity of each group to U/mL (or relative enzyme activity percentage) and compare the combination with the highest enzyme activity.

**b. Optimizing the Volume Ratio of the Two Best Enzymes**
1. Select the two enzyme combinations with the highest activity from the first step.
2. Prepare different volume ratio systems (keeping the total enzyme volume constant):
• 2:1; 1:1; 0.5:1; 1:0.5; 1:2
3. Mix each enzyme ratio with colloidal chitin and add the optimal pH buffer, maintaining the same total reaction volume.
4. Incubate at the optimal temperature for 1 hour.
5. After the reaction, measure reducing sugar production using the DNS method or a kit.
6. Compare enzyme activity at different ratios to determine the optimal synergistic volume ratio.

**c. Data Processing**
• Perform at least three replicates for each experiment.
• Calculate relative enzyme activity, taking the highest reducing sugar production or highest U/mL value as 100%.
• Create a bar chart or line graph to display the changes in enzyme activity at different combinations and ratios.

**4.4 Preparation and immobilized enzyme efficiency**
**Goal:** Prepare immobilized chitinases (including exo-chitinase, endo-chitinase, and chitin deacetylase), characterize the immobilization efficiency by SDS-PAGE, and evaluate the binding of the enzymes to the epoxy support LX-1000EP.

**Materials:**
• Epoxy carrier LX-1000EP (0.5 g/tube)
• Purified enzyme
• PBS buffer (0.1 M, pH 7.0)
• 15 mL empty centrifuge tube
• Centrifuge
• Mixer
• SDS-PAGE reagents and equipment
• ImageJ software (for band grayscale analysis)

**Procedures:**
**a. Resin Pretreatment**
1. Transfer 0.5 g of LX-1000EP epoxy support to a 15 mL empty centrifuge column tube.
2. Add 3 mL of PBS buffer, gently mix, and wash three times. Centrifuge at 4000 rpm for 1 minute each time, and discard the supernatant.

**b. Enzyme Immobilization**
1. Adjust the purified enzyme solution to 5 mg/mL with PBS buffer. Add 10 mg of enzyme/g of resin to the resin. Add 2 mL of enzyme solution, which is the pre-immobilization supernatant.
2. Place the centrifuge tube horizontally on a shaker to allow the resin to shake freely. Incubate at 25°C for 12 hours.

**c. Collecting the Post-Immobilization Supernatant**
1. After the reaction is complete, transfer the resin-enzyme mixture to a 15 mL centrifuge tube and centrifuge at 4000 rpm for 1 minute. Collect the supernatant and record it as the post-immobilization supernatant.

**d. Resin Washing**
1. Add 2 mL of PBS buffer to a centrifuge tube containing 0.5 g of resin. Gently mix. Centrifuge at 4000 rpm for 1 minute. Discard the supernatant.
2. Repeat the wash twice to remove unbound enzyme from the surface.

**e. SDS-PAGE Analysis and Calculation of Immobilization Efficiency**
1. Take the pre- and post-immobilization supernatants, dilute them 5-fold if necessary, and perform SDS-PAGE analysis.
2. Calculate the grayscale value of each sample band using ImageJ software.
3. Calculate the Immobilization Efficiency

**4.5 Number of reuses of immobilized enzymes**
**Goal:** Evaluate the reusability of immobilized chitinase by measuring the amount of reducing sugars produced during each reaction cycle to determine the enzyme's activity retention after multiple reactions.

**Materials:**
• Immobilized chitinase (immobilized on LX-1000EP resin)
• 1% colloidal chitin solution
• PBS buffer (0.1 M, pH 7.0)
• DNS reducing sugar assay reagent or commercial reducing sugar assay kit
• Centrifuge tubes
• 37°C water bath or incubator
• Centrifuge
• Spectrophotometer or microplate reader
• Pipette and tips

**Procedures:**
**a. Enzyme Cycling Reaction**
1. Add an appropriate amount of immobilized chitinase (e.g., 0.5 g resin) to a reaction system containing 1% colloidal chitin solution (total volume adjusted according to experimental requirements, e.g., 2 mL).
2. Incubate the reaction in a 37°C water bath or on a constant-temperature shaker for 1 hour.
3. After the reaction, remove the immobilized enzyme from the reaction solution with a pipette and gently wash twice with PBS buffer to remove any residual substrate or product.
4. Collect the reaction supernatant for reducing sugar determination.

**b. Reducing Sugar Determination**
1. Take an appropriate amount of the reaction supernatant (e.g., 200 μL) and add an equal volume of DNS reagent. Incubate in a boiling water bath for 5 minutes. After the reaction, cool to room temperature.
2. Alternatively, use a commercial reducing sugar kit according to the instructions.
3. Measure the absorbance at 540 nm (or the wavelength recommended by the kit) using a spectrophotometer or microplate reader and calculate the reducing sugar concentration in the product.

**c. Recycling**
1. Add the washed immobilized enzyme back to fresh 1% colloidal chitin solution and repeat steps 1-2.
2. Record the amount of reducing sugar in the product after each cycle and calculate the relative enzyme activity (assuming the yield of the first reaction is 100%).
3. Continue until the enzyme activity significantly decreases. Plot a curve comparing the number of times the immobilized enzyme is reused versus the relative enzyme activity.

{% endblock %}

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{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block extra\_css %}

{% endblock %}
{% block page\_content %}

Results

![shrimp](https://static.igem.wiki/teams/5721/img/desc-xia.webp)

[Overview](#content1)

[1. The construction of plasmid](#content2)

[1.1 Construction for pET-28a(+)-AsCDA/Cts2p/FjChiB](#content2-1)

[2. Protein expression and purification](#content3)

[2.1 Protein expression](#content3-1)

[2.2 Protein His-tag purification](#content3-2)

[3. Functional test](#content4)

[3.1 Free enzyme activity test](#content4-1)

[3.2 Optimal reaction conditions](#content4-2)

[3.3 Synergistic ratio and conditions](#content4-3)

[3.4 Immobilized enzyme efficiency](#content4-4)

[3.5 Number of reuses of immobilized enzymes](#content4-5)

[References](#content5)

### Overview

Chitin, a polymer composed of repeating units of N-acetyl-D-glucosamine, is the second largest natural macromolecule after cellulose[1]. It is widely distributed in the exoskeletons of insects, the cell walls of fungi, and marine organisms such as shrimp and crabs. Due to its structural stability and insolubility, chitin is difficult to directly utilize[2]. Chitinases, including chitin degrading enzymes and chitin deacetylases, catalyze the hydrolysis of chitin into oligomers and monomers, such as N-acetylglucosamine, which are highly valuable bioactive compounds. These products have a wide range of biological activities and industrial applications, including antifungal and insecticide properties in agriculture, bioactive oligosaccharides in pharmaceuticals, and high-purity chitin derivatives in food, cosmetics, and biotechnology. Therefore, chitinases hold great potential in agriculture, medicine, environmental protection, and industrial biotechnology[3-5].

In this project, we characterized three chitinases from microorganisms, including endo- and exo-enzymes, as well as a chitin deacetylase. To achieve high enzyme production, we synthesized the corresponding gene in vitro, recombined it into a plasmid vector, and then heterologously expressed it in E. coli.

To optimize enzyme performance, we evaluated protein yield and enzyme activity. We systematically investigated key parameters such as the optimal reaction temperature, pH, and the effects of metal ions. The optimized E. coli system provides a more comprehensive platform for large-scale production of functional chitinase.

Furthermore, we explored a synergistic degradation strategy by combining chitinase (exo- or endo-acting) with chitin deacetylase. By testing different enzyme ratios, we evaluated the impact of synergistic catalysis on chitin hydrolysis efficiency. The most effective enzyme ratio was then immobilized on an epoxy resin support. Enzyme immobilization not only improved catalytic efficiency but also prolonged enzyme reusability and operational stability, which are critical for industrial applications.

Ultimately, the project resulted in the development of a stable chitinase-based system that effectively degrades chitin-containing marine debris. This provides a novel, environmentally friendly enzymatic solution for marine pollution control and also offers the opportunity to produce high-value biochemicals, thereby combining environmental sustainability with industrial biotechnology.

### The construction of plasmid

**1.1 Construction for pET-28a(+)-AsCDA/Cts2p/FjChiB**

We used homologous recombination to connect AsCDA/Cts2p/FjChiB to pET-28a(+). First, we obtained our target gene AsCDA/Cts2p/FjChiB by PCR. After amplification, we performed agarose gel electrophoresis on the amplified products to observe whether our target gene amplification was successful. In Figure 1A, we can see that the length of the amplified target gene FjChiB is 1023bp, Cts2p is 1536bp, and AsCDA is 975bp, which is consistent with our expected length. Therefore, the fragment amplification was successful. In addition, we also obtained our pET-28a(+) linearized vector by double enzyme digestion and EcoRI. After enzyme digestion with +XhoI, we also subjected the digestion products to agarose gel electrophoresis and found that the linearized vector after digestion was 5431 bp in length. We can also see clear bands from Figure 1B, which shows that our vector acquisition was also successful. We then recovered the target genes and vectors from the agarose gel and used homologous recombination to connect the three target genes and vectors respectively.

![Agarose gel electrophoresis of target genes and linearized pET-28a(+) vector](https://static.igem.wiki/teams/5721/img/results/1.webp)

Figure 1. Agarose gel electrophoresis of target genes and linearized pET-28a(+) vector

After connecting the three target genes and the vector together, we first transformed the ligation product into Escherichia coli DH5α, which is a cloning strain that can help us confirm whether the recombinant plasmid is successful. We can see from Figure 2A that many colonies grew on the monoclonal plate grown overnight after transformation into DH5α, which indicates that the transformation is likely successful. However, we still need to further verify it by colony PCR. Therefore, we selected 5-6 colonies on each monoclonal plate for verification. After colony PCR amplification, we subjected the amplified products to agarose gel electrophoresis. The results are shown in Figure 2B. For pET-28a(+)-AsCDA, the length of the amplified fragment is 1000bp, the length of the amplified fragment is 600bp for pET-28a(+)-Cts2p, and the length of the amplified fragment is 800bp for pET-28a(+)-FjChiB. The lengths are all in line with our expected sizes. Therefore, it can be preliminarily judged that the transformed plasmid is successful.

![Verification of the transformation results of pET-28a(+)-AsCDA/Cts2p/FjChiB positive colony](https://static.igem.wiki/teams/5721/img/results/2.webp)

Figure 2. Verification of the transformation results of pET-28a(+)-AsCDA/Cts2p/FjChiB positive colony. A: The single colony on the plate; B: The amplification results of colony PCR.

Finally, we used universal primer T7-Forward to verify the positive colonies by Sanger sequencing. Figure 3 showed the correct sequences of recombination plasmids. We chose correct colony for further IPTG induction.

![Sanger sequencing results of pET-28a(+)-AsCDA/Cts2p/FjChiB positive colony](https://static.igem.wiki/teams/5721/img/results/3.webp)

Figure 3. Sanger sequencing results of pET-28a(+)-AsCDA/Cts2p/FjChiB positive colony.

We transformed the three recombinant plasmids into DH5α and expanded the colonies after colony PCR verification. We extracted the plasmids and sent them to a biological company for sequencing. After this series of verifications, we confirmed that the recombinant plasmids had no connection errors, base deletions or mutations. Then we needed to transform the correct plasmids into the E. coli expression strain BL21 (DE3). As shown in Figure 4A, many monoclonal colonies grew overnight after transformation into BL21 (DE3), but colony PCR was still needed to confirm that the transformed colonies contained Since we have the correct recombinant plasmid, we performed agarose gel electrophoresis on the products after colony PCR. The results of electrophoresis showed that the length of the fragment amplified in pET28a-AsCDA was 1202bp, the length of the fragment amplified in pET28a-Cts2p was 843bp, and the length of the fragment amplified in pET28a-FjChiB was 1027bp. The amplified lengths were consistent with our expected lengths. Therefore, the transformation to BL21 (DE3) was successful. We can subsequently culture the correct monoclonal colonies and express proteins on this basis.

![Monoclonal plate of BL21 (DE3) transformed with recombinant plasmid](https://static.igem.wiki/teams/5721/img/results/4.webp)

Figure 4. A: Monoclonal plate of BL21 (DE3) transformed with recombinant plasmid, B: PCR electrophoresis results of colonies on monoclonal plate after transformation.

### Protein expression and purification

**2.1 Protein expression**

After confirming that the three recombinant plasmids were successfully transformed into the expression strain BL21, we can perform protein expression tests. First, we need to induce the expression of a small amount of protein without setting different parameter conditions. Specifically, we set different temperatures (16 degrees Celsius and 37 degrees Celsius) and time (0-12h). Then, the recombinant BL21 bacterial solution containing the positive was inoculated into 5mL LB (containing kanamycin) liquid culture medium at a rate of 1/100, and shaken overnight. The 5mL overnight bacterial solution was inoculated into 250mL LB (pET-28a is Kan+ resistant) liquid culture medium and shaken at 37℃ until the OD600 was about 0.6 (about 3~4 hours, adding IPTG when OD was 0.6 had the best effect, and fewer protein bands were induced). 1ml of bacterial solution was retained as a control.

1)1M sterile IPTG was added.

2)To a final concentration of 1 mM.

3)Culture at different temperatures.

4)Collect bacterial liquid at different time points.

5)Centrifuge at 3000g for 10 minutes.

6)Collect 1 mL of bacteria

And retain the bacterial liquid without inducer as the control group. Then use 100 μL of 1x protein loading buffer to resuspend and boil, and after cooling, perform protein electrophoresis identification.

Figure 5 shows the protein electrophoresis results (SDS-PAGE gel) of three recombinant chitinases (AsCDA, Cts2p, and FjChiB) expressed in E. coli using the pET28a-6xHis vector. Experimental conditions included induction with 1 mM IPTG at 16℃ and 37℃, with samples collected at different time points (0 h, 0.5 h, 1 h, 3 h, 6 h, and 12 h). Markers (M) serve as molecular weight references, and protein bands are indicated by orange arrows. Based on the theoretical molecular weights, the three recombinant proteins are: AsCDA-6xHis: approximately 37.5 kDa, Cts2p-6xHis: approximately 59.1 kDa, and FjChiB-6xHis: approximately 38.1 kDa. Specific protein bands clearly appear near the corresponding molecular weights, increasing over time, demonstrating that all three target proteins were successfully expressed in E. coli.

At 16℃, a band near 37 kDa was observed to gradually increase in intensity for AsCDA (37.5 kDa), but the overall intensity was not as strong as at 37℃. The 37.5 kDa band significantly intensified with induction time, becoming particularly pronounced after 3 hours and peaking at 6 and 12 hours. The band was very clear. AsCDA protein expression levels were higher at high temperatures, suggesting that the protein's structure is more stable, making it suitable for induction at 16℃ and less susceptible to misfolding or degradation.

Cts2p (59.1kDa) showed a more gradual increase in intensity at 16℃, with overall expression levels being low. However, the protein band was clear, indicating more stable expression at low temperatures. At 37℃, the band near 59 kDa significantly intensified, particularly after 3 hours, rapidly deepening and reaching very high intensity at 6 and 12 hours. This indicates that Cts2p is a large molecular weight protein. High temperatures can yield higher yields, but this also carries the potential risk of inclusion bodies. Low-temperature expression, while yielding lower amounts, is beneficial for proper protein folding and soluble expression.

FjChiB (38.1kDa) showed a weak band at 16℃, which slightly intensified at 6 and 12 hours of induction. At 37℃, the 38 kDa band gradually intensified after induction, becoming prominent at 1 hour and reaching peak expression at 6 and 12 hours. The significantly higher expression efficiency of FjChiB at high temperatures compared to low temperatures suggests its structural stability is suitable for rapid high-temperature induction. However, the overall difference was not significant. Therefore, based on literature, we controlled the subsequent large-scale fermentation of this protein at an IPTG induction concentration of 0.3 mM and a temperature of 27℃.

![SDS-PAGE analysis for small amount of target proteins expression](https://static.igem.wiki/teams/5721/img/results/5.webp)

Figure 5. SDS-PAGE analysis for small amount of target proteins expression.

**2.2 Protein His-tag purification**

After confirming the fermentation conditions, we performed large-scale fermentation of the three proteins, inducing them at 1 mM IPTG and 16℃ for 16 h to achieve heterologous expression of the chitin deacetylase AsCDA.

SDS-PAGE electrophoresis (Figure 6) revealed a distinct band around 37.5 kDa in the whole-cell lysate, demonstrating efficient expression of AsCDA under induced conditions. Further comparison of the soluble and precipitated fractions revealed a strong band of the target protein in the supernatant, with a certain amount of protein also present in the precipitate. This indicates good soluble expression of AsCDA under these conditions, although some protein was still present as inclusion bodies, a common finding in heterologous expression.

As the imidazole concentration increased, distinct and intense target protein bands appeared in the elution fractions between 20 and 100 mM, indicating gradual elution of the target protein. The band intensity was significantly reduced in the elution fractions at higher concentrations of 250 mM and 500 mM, indicating that the primary target protein was completely released under low and medium imidazole concentrations. This result demonstrates that the purification process can obtain high-purity AsCDA, and the elution peak is relatively concentrated, facilitating subsequent collection and application.

![The expression of a large amount of target protein AsCDA was analyzed by SDS-PAGE](https://static.igem.wiki/teams/5721/img/results/6.webp)

Figure 6. The expression of a large amount of target protein AsCDA was analyzed by SDS-PAGE.

Cts2p (59.1 kDa) chitinase exonuclease was expressed using 1 mM IPTG at 16℃ for 16 h. This low-temperature, long-term induction strategy was chosen to slow cellular metabolism, facilitate folding of the target protein into a soluble form, and avoid the formation of numerous inclusion bodies.

SDS-PAGE electrophoresis results (Figure 7) showed a distinct band corresponding to Cts2p at 59.1 kDa in the whole-cell lysate, indicating successful induced expression of the target protein. Further comparison of the supernatant and precipitate revealed the presence of the target protein in the soluble supernatant fraction, demonstrating that Cts2p has a certain degree of soluble expression under these conditions. As the imidazole concentration gradually increased, a 59.1 kDa band was clearly observed in the 20–50 mM elution fractions, while the band gradually weakened or even disappeared in the higher 100–500 mM elution fractions, indicating that the primary target protein can be effectively eluted under low to moderate imidazole concentrations.

Overall, these results demonstrate that under the induction conditions of 1 mM IPTG, 16℃, and 16 hours, Cts2p can be expressed at high levels and in a partially soluble form, facilitating efficient purification by Ni-NTA affinity chromatography.

![The expression of a large amount of target protein Cts2p was analyzed by SDS-PAGE](https://static.igem.wiki/teams/5721/img/results/7.webp)

Figure 7. The expression of a large amount of target protein Cts2p was analyzed by SDS-PAGE.

The chitinase FjChiB was fermented at 27℃ using 0.3 mM IPTG as an inducer for 16 hours. Electrophoresis results (Figure 8) demonstrate successful induced expression and purification. In the whole-cell lysate, a dense new band was clearly observed slightly above the 34 kD marker, consistent with the theoretical molecular weight of the target protein FjChiB (38.1 kD). Finally, stepwise elution was performed using different imidazole concentrations. Clear target protein bands were observed in the elution lanes from 20 mM to 500 mM imidazole, with the most intense bands at approximately 20mM concentrations, confirming successful elution and purification of the target protein.

The chitinase FjChiB was efficiently expressed under the induction conditions of 0.3 mM IPTG at 27℃ for 16 hours. The expression product exhibited a mixture of soluble and insoluble properties, a common finding in recombinant protein expression. The purification process was successfully designed, and imidazole gradient elution results demonstrated good binding specificity to the nickel column.

![The expression of a large amount of target protein FjChiB was analyzed by SDS-PAGE](https://static.igem.wiki/teams/5721/img/results/8.webp)

Figure 8. The expression of a large amount of target protein FjChiB was analyzed by SDS-PAGE.

### Functional test

**3.1 Free enzyme activity test**

After obtaining a large amount of purified protein, we first conducted preliminary measurements of the enzyme activities of the three proteins. For the determination of chitin deacetylase, we used the p-nitroacetanilide method, and for chitin exonuclease and endonuclease, we used the N-acetylglucosamine method. Both methods require the preparation of standard curves.

![N-acetylglucosamine standard curve](https://static.igem.wiki/teams/5721/img/results/9.webp)

Figure 9. N-acetylglucosamine standard curve

Figure 9 shows the standard curve used for quantitative analysis. The image above shows the color gradient produced by the reaction of N-acetylglucosamine (GlcNAc) standard solutions with different concentrations and DNS. The solution color changes gradually from light yellow to dark red as the concentration increases (from left to right), indicating increasing absorbance. The fitted curve below accurately quantifies this color change. The horizontal axis (X-axis) represents the N-acetylglucosamine standard concentration (in mg/mL), and the vertical axis (Y-axis) represents the absorbance measured at a wavelength of 540 nm. The curve exhibits a good linear positive correlation. The fitted linear equation is y = 2.0439x - 0.0288, with a squared correlation coefficient R² = 0.9849.

![p-Nitroacetanilide standard curve](https://static.igem.wiki/teams/5721/img/results/10.webp)

Figure 10. p-Nitroacetanilide standard curve

This standard curve (Figure 10) serves as a quantitative scale for chitin deacetylase (CDA) activity assays. Its core function is to convert measured absorbance values into precise product concentrations, thereby calculating enzyme activity. Chitin deacetylase catalyzes the removal of the acetyl group (-COCH3) from chitin or its derivative substrates (such as glycol chitin), generating free amino groups and acetic acid. In this assay, a synthetic chromogenic substrate, such as p-nitroacetanilide, is typically used. Upon enzyme action, this substrate hydrolyzes to form p-nitroaniline. p-Nitroaniline is a compound with a strong yellow absorption around 400 nm (as indicated by the color gradient in the upper test tube). Therefore, the more p-nitroaniline produced by the enzyme reaction, the higher the absorbance of the solution at 400 nm. Based on the calculated parameters such as product concentration, reaction time, enzyme solution volume and cuvette path length, the enzyme activity unit can be finally calculated.

According to the formula corresponding to the standard curve, we calculated that the chitin exonuclease (Cts2p) activity was 115 U/mL, the chitin endonuclease (FjChiB) activity was 116 U/mL, and the chitin deacetylase (AsCDA) activity was 41 U/mL.

**3.2 Optimal free enzyme activity reaction conditions**

After preliminarily measuring the activity of the three enzymes, we need to explore the optimal reaction conditions for the three enzymes, starting with the optimal pH value.

![The optimal pH values of three proteins were investigated](https://static.igem.wiki/teams/5721/img/results/11.webp)

Figure 11. The optimal pH values of three proteins were investigated, where A represents chitin deacetylase AsCDA, C represents chitin exonuclease Cts2p, and F represents chitin endonuclease FjChiB, the same below.

The results in Figure 11 show that chitin deacetylase (AsCDA, A), chitin exonuclease (Cts2p, C), and chitin endoenuclease (FjChiB, F) all exhibit a typical pH-dependent relationship in enzyme activity, with their optimum pH values around 6. The activities of all three enzymes reach peak values at pH 7, indicating that this pH value is the optimal condition for their catalytic reactions.

Enzyme activity generally decreases under conditions deviating from the optimal pH. In particular, in extremely acidic (pH 2-3) or alkaline (pH 10-11) environments, the activities of all three enzymes decrease significantly, or even become completely inactivated. Notably, all three enzymes maintain relatively high activity over a wide pH range of 4-8, demonstrating a certain degree of acid-base stability. This provides flexibility in the selection of buffer systems for practical applications.

The discussion suggests that the similarity in the optimum pH values of the three enzymes may be related to the ecological environment of their source microorganisms or the characteristics of the substrates they act on. Furthermore, this highly efficient catalytic performance under neutral to slightly acidic conditions is consistent with the general properties of chitin-degrading enzymes. This result suggests that when using these enzymes for chitin bioconversion, the pH of the reaction system should be controlled around 6 to achieve optimal catalytic efficiency. Furthermore, the enzyme's stability over a wide pH range offers potential advantages for its application in complex industrial environments.

![The optimal temperature values of three proteins were investigated](https://static.igem.wiki/teams/5721/img/results/12.webp)

Figure 12. The optimal temperature values of three proteins were investigated, where A represents chitin deacetylase AsCDA, C represents chitin exonuclease Cts2p, and F represents chitin endonuclease FjChiB.

The bar graph and line chart in Figure 12 show significant differences in the optimal temperatures of the three chitinases. The chitin deacetylase AsCDA (A) exhibits maximum activity in the 40-50℃ range, reaching its peak at 40℃. The exochitinase Cts2p (C) exhibits an optimal temperature of approximately 50℃. The endochitinase FjChiB (F) exhibits a higher optimal temperature, maintaining high activity between 40-50℃. The activity of all enzymes decreased significantly at low temperatures (20℃) and high temperatures (70℃).

This difference in optimal temperature may be related to the microbial ecosystems from which the enzymes originate and the molecular structure of the enzymes. The mesophilic properties of AsCDA and Cts2p suggest that they may originate from mesophilic microorganisms, while the higher optimal temperature of FjChiB suggests that it may originate from a thermotolerant strain or possess a more stable spatial structure. The generally high activity of the enzymes around 50℃ suggests that this temperature may be the common optimal condition for chitinase-catalyzed reactions. These findings provide an important basis for optimizing the application conditions of chitinase. It is recommended that in practical applications, a reaction temperature of 40℃ be selected according to the enzyme type to obtain the best catalytic efficiency.

![The optimal metal ions for three proteins were investigated](https://static.igem.wiki/teams/5721/img/results/13.webp)

Figure 13. The optimal metal ions for three proteins were investigated, where A represents chitin deacetylase AsCDA, C represents chitin exonuclease Cts2p, and F represents chitin endonuclease FjChiB.

The bar graph and line chart in Figure 13 clearly demonstrate the responses of three chitinases (AsCDA, Cts2p, and FjChiB) to different metal ions. the chitin deacetylase AsCDA (A) exhibits the highest activity under Mn2+ conditions; the exochitinase Cts2p (C) exhibits the strongest response to Mn2+(nearly 300 U/mL); and the endochitinase FjChiB (F) exhibits optimal activity under Mn2+ conditions (approximately 280 U/mL).

This result suggests that the effects of metal ions on enzyme activity are highly specific, potentially related to the enzyme's spatial structure, active site characteristics, and catalytic mechanism. AsCDA's preference for Mn2+ may stem from the ion's stabilizing effect on its substrate-binding domain; Cts2p's specific binding to Mn2+ suggests that the ion may be directly involved in the conformational regulation of its catalytic center; and FjChiB's compatibility with Mn2+ may be related to the ion's ability to maintain its structural stability.

**3.3 Optimal free enzyme synergistic ratio and reaction conditions**

After exploring the optimal reaction conditions of the three enzymes, we need to explore the enzyme synergy. Specifically, we first mix them in the same volume ratio to determine the optimal effect of the enzyme combination, and then explore the ratio of enzyme synergy. In order to determine which combination has a better effect, we used the chitinase detection kit for exploration. Before the determination, we need to specify the standard curve.

![Chitinase activity assay kit standard curve](https://static.igem.wiki/teams/5721/img/results/14.webp)

Figure 14. Chitinase activity assay kit standard curve, A: standard curve preparation process, B: standard curve

The results in Figure 15 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; 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.

![The enzyme activity of different types of enzymes is determined by synergy](https://static.igem.wiki/teams/5721/img/results/15.webp)

Figure 15. 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 bar chart in Figure 16 shows the trends in reducing sugar yields in the reaction systems of the A+C and A+F enzyme combinations at different ratios. For the A+C combination, the highest reducing sugar yield was achieved at a 1:1 ratio, significantly higher than at other ratios. As the ratio deviated from 1:1 (e.g., 0.5:1 or 1:0.5), reducing sugar yields decreased significantly, indicating a strict stoichiometric dependence between the two. For the A+F combination, reducing sugar yields were higher at ratios of 1:0.5 and 1:1, with the 1:0.5 ratio showing the highest yield. This suggests that this combination is less sensitive to changes in ratio and has a wider range of adaptability.

This result may stem from differences in the synergistic mechanisms of the two enzymes: AsCDA and Cts2p may require precise molecular matching to achieve efficient synergy. For example, the exolytic activity of Cts2p depends on the specific substrate conformation generated by AsCDA deacetylation. In contrast, the synergy between AsCDA and FjChiB may be achieved through more relaxed functional complementarity, with FjChiB's endolysis generating more termini that provide substrates for AsCDA. Notably, the difference in peak yield between the two combinations (AsCDA+Cts2p outperforming AsCDA+FjChiB) suggests that Cts2p and AsCDA exhibit superior synergy, providing important insights into optimizing enzyme combinations.

![Study on the content of reducing sugar products in different synergistic ratios of AsCDA+Cts2p and AsCDA+FjChiB](https://static.igem.wiki/teams/5721/img/results/16.webp)

Figure 16. Study on the content of reducing sugar products in different synergistic ratios of AsCDA+Cts2p and AsCDA+FjChiB

Since the AsCDA+Cts2p combination has the highest reducing sugar yield when the ratio is 1:1, and the AsCDA+FjChiB combination has the highest reducing sugar yield when the ratio is 1:0.5, we also explored the optimal reaction conditions for the two ratios of complex enzymes.

![Exploration of the optimal pH value for the activity of two complex enzymes](https://static.igem.wiki/teams/5721/img/results/17.webp)

Figure 17. Exploration of the optimal pH value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB

The bar graph in Figure 17 shows significant differences in the optimal pH values for the two enzyme complexes, AC (AsCDA+Cts2p) and AF (AsCDA+FjChiB). The AF complex exhibits peak activity (approximately 170 U/mL) at pH 5 and maintains a high activity level within the pH range of 3-6. The AC complex exhibits an optimal pH of 7, with a peak activity of approximately 120 U/mL, and its activity is relatively stable within the pH range of 6-8. The activity of both enzymes decreases significantly under extreme pH conditions (pH ≤ 3 or ≥ 9).

Notably, the overall activity of the AF complex is significantly higher than that of the AC complex (by an average of approximately 40%), and its optimal pH range is more acidic. This phenomenon may be due to the stronger synergistic effect between FjChiB (endonuclease) and AsCDA (deacetylase): the endonuclease cleaves chitin chains to produce more ends, providing more sites for the deacetylase to target, and deacetylation further enhances the substrate's sensitivity to the endonuclease. In contrast, the synergistic efficiency of Cts2p (exonuclease) and AsCDA is low, which may lead to the poor overall activity of the AC complex enzyme.

![Exploration of the optimal temperature value for the activity of two complex enzymes](https://static.igem.wiki/teams/5721/img/results/18.webp)

Figure 18. Exploration of the optimal temperature value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB

The bar graph in Figure 18 shows that the two enzyme complexes, AC (AsCDA+Cts2p) and AF (AsCDA+FjChiB), exhibit significant differences in their optimal temperatures. The AC enzyme reaches peak activity (approximately 120 U/mL) at 40℃. The activity at 20℃-30℃ and 50℃-70℃ is similar, stable at around 100U/mL, The AF enzyme complex exhibits a completely different temperature response, its activity peaks at 50℃ (over 200 U/mL) and remains (150U/mL) within the 20-30℃ range, demonstrating better temperature tolerance than the AC enzyme.

This phenomenon may be due to the different temperature adaptabilities of the different enzyme components in the two enzyme complexes. The Cts2p (exonuclease) in the AC complex may be sensitive to high temperatures, leading to its rapid inactivation above 40℃. However, the FjChiB (endonuclease) in the AF complex possesses inherently good thermostability (optimum temperature around 50℃), and its synergistic effect with AsCDA further enhances the complex's thermostability. Notably, the AF complex's activity at 50℃ is nearly 1.7 times that of the AC complex, indicating that the synergistic effect of AsCDA and FjChiB is more pronounced at high temperatures.

![Exploration of the optimal Metal ions value for the activity of two complex enzymes](https://static.igem.wiki/teams/5721/img/results/19.webp)

Figure 19. Exploration of the optimal Metal ions value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB

The bar graph in Figure 19 shows that different metal ions significantly and regularly affect the activity of the AC (AsCDA+Cts2p) and AF (AsCDA+FjChiB) enzyme complexes. For the AC enzyme, Mn2+ had the most significant activation effects, reaching approximately 135 U/mL and 110 U/mL, respectively, significantly higher than those treated with other ions.

The AF enzyme complex was more sensitive to metal ions, with Mn2+ exhibiting the strongest activation effect (approximately 150 U/mL), followed by Zn2+ (approximately 140 U/mL). while Ni2+ still exhibited inhibitory effects.

Since we discovered that the synergistic catalytic activity of AsCDA+Cts2p and AsCDA+FjChiB was higher than that of the former, we selected the latter for the next step of enzyme immobilization. First, we obtained purified AsCDA and FjChiB proteins through heterologous expression in E. coli. Both proteins were then immobilized with epoxy resin. The supernatants before and after fixation were collected for SDS-PAGE analysis (samples can be diluted 5-fold). ImageJ software was used to calculate the grayscale value changes of the bands and the immobilization efficiency: Immobilization efficiency (%) = (grayscale value of the sample before fixation − grayscale value of the sample after fixation) / grayscale value of the sample before fixation \* 100%，As shown in Figure 20, the next step is to calculate the grayscale value using imageJ software.

![Purified proteins of AsCDA and FjChiB and SDS-page images of proteins before and after immobilization](https://static.igem.wiki/teams/5721/img/results/20.webp)

Figure 20. Purified proteins of AsCDA and FjChiB and SDS-page images of proteins before and after immobilization

**3.4 Preparation and immobilized enzyme efficiency**

Before calculating the grayscale value, for accurate grayscale value analysis, we first convert the image to 8-bit grayscale image, and then we measure the mean grayscale value and integrated density. The higher the protein expression, the larger the value of Integrated Density.

The results in Figure 21 show significant differences between the two proteins before and after immobilization. Grayscale analysis showed that the immobilization efficiency of AsCDA was 17.84%; the immobilization efficiency of FjChiB was 24.59%. This indicates that both proteins experienced some loss during the immobilization process, with FjChiB having a relatively higher immobilization efficiency.

SDS-PAGE electrophoresis patterns show that the band positions of the two proteins (approximately 37.5 kDa for AsCDA and approximately 38.1 kDa for FjChiB) remained unchanged before and after immobilization, indicating that the immobilization process did not cause protein degradation or molecular weight changes. However, the band intensity decreased significantly after immobilization, consistent with the decreasing grayscale value trend.

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.

![Calculation of grayscale values of the two proteins before and after immobilization](https://static.igem.wiki/teams/5721/img/results/21.webp)

Figure 21. Calculation of grayscale values of the two proteins before and after immobilization

**3.5 Number of reuses of immobilized enzymes**

After the enzyme immobilization treatment, we added the two together according to the ratio and explored the number of times of repeated use.

![Number of times the immobilized enzyme can be reused](https://static.igem.wiki/teams/5721/img/results/22.webp)

Figure 22. Number of times the immobilized enzyme can be reused, abcde represent the first to fifth repeated experiments

Figure 22 shows the relationship between the number of times the immobilized enzyme was reused and its activity. The results show that enzyme activity shows a continuous downward trend with increasing reuse. The enzyme activity reached its highest level (approximately 43 U/g) during the first use, declining to approximately 22 U/g by the fifth use, with activity retention approximately 60% of the initial value.

This activity decay trend may be due to several factors. The immobilized enzyme may experience physical loss with each use, with enzyme molecules gradually falling off the support surface due to mechanical agitation or washing. Furthermore, the enzyme protein may undergo structural denaturation during repeated use, particularly conformational changes in the active center, leading to reduced catalytic efficiency. the accumulation of reaction products or impurities on the support surface may hinder substrate-enzyme contact.

Notably, despite the gradual decrease in enzyme activity, a significant percentage of activity was retained after five reuses, indicating that immobilization significantly improved enzyme stability. The activity decrease was most pronounced from the first to the second use, Then the rate of descent accelerated.

Therefore, the reusability of immobilized enzymes can significantly reduce the cost of enzyme preparations, but the optimal replacement cycle needs to be determined according to specific process requirements.

**Future plans**

1. Continue to utilize genetic engineering technology: Further optimize the chitinase gene through gene editing, mutation, and other methods to screen mutants with higher activity and yield.

2. Optimize the fermentation process: Combine advanced fermentation technology and equipment, such as optimized bioreactor design, to precisely control parameters during the fermentation process, such as temperature, pH, dissolved oxygen, etc., to increase yield.

3. Combine computer simulation: Use computer simulation technology to assist in enzyme design and modification, and predict mutation sites or structures that may have high activity.

### References

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2. Yang X, Liu J, Pei Y, et al. Recent progress in preparation and application of nano‐chitin materials[J]. Energy & Environmental Materials, 2020, 3(4): 492-515.
3. Mincea M, Negrulescu A, Ostafe V. Preparation, modification, and applications of chitin nanowhiskers: a review[J]. Rev. Adv. Mater. Sci, 2012, 30(3): 225-242.
4. Rathore A S, Gupta R D. Chitinases from bacteria to human: properties, applications, and future perspectives[J]. Enzyme research, 2015, 2015(1): 791907.
5. Barikani M, Oliaei E, Seddiqi H, et al. Preparation and application of chitin and its derivatives: a review[J]. Iranian Polymer Journal, 2014, 23(4): 307-326.

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