Chitin, composed of N-acetyl-D-glucosamine, is the second largest polymer in nature after cellulose[1]. Chitinases, which catalyze the degradation of chitin, have the ability to degrade fungal cell walls and insect exoskeletons and produce N-acetylglucosamine oligomers or monomers[2]. Therefore, they play an important role and hold great potential in agriculture[3], medicine[4], and industry[5]. In this project, we screened three chitinases and chitin deacetylases from microbial sources. We then constructed a genetic system expressing chitinases by recombining the in vitro synthesized genes into plasmids and expressing them in Escherichia coli. By testing chitin expression and enzyme activity, as well as the optimal reaction conditions for enzyme activity, we optimized the constructed E. coli genetic system and adjusted the expression conditions to increase chitinase production and activity. By degrading these chitin wastes, chitin can be recovered into valuable products such as amino acids, calcium lactate, and high-purity chitin, thereby improving the utilization of biomass energy. Furthermore, we combined chitin exonucleases or endonucleases with chitin deacetylases to explore the synergistic effect of enzymes on chitin degradation, and immobilized the optimal ratio of free enzymes onto epoxy resin to further improve the catalytic efficiency and usage of the enzymes, ultimately providing a new decontamination enzyme preparation solution for the degradation of marine pollutants containing chitin.
During our experiment, we added some new parts for iGEM part and new information to an existing part (Table 1), for instance, Chitin deacetylase (AsCDA, BBa_25NJJMRZ)、Chitin exonuclease (Cts2p, BBa_25XXGWVY)、Chitin endonuclease (FjChiB, BBa_25S1GVYQ), There are three other recombinant plasmids, as follows：
| Part number | Part name | Contribution type | Part type |
|---|---|---|---|
| BBa_25NJJMRZ | AsCDA | New part | BBa_25NJJMRZ |
| BBa_25XXGWVY | Cts2p | New part | BBa_25XXGWVY |
| BBa_25S1GVYQ | FjChiB | New part | BBa_25S1GVYQ |
| BBa_25VIQJQP | pET28a_AsCDA | New part | BBa_25VIQJQP |
| BBa_25OCX0JU | pET28a_Cts2p | New part | BBa_25OCX0JU |
| BBa_250AYN60 | pET28a_FjChiB | New part | BBa_250AYN60 |
1. Add a basic part (AsCDA, BBa_25NJJMRZ)
Name: Chitin deacetylase (AsCDA)
Base Pairs: 975 bp
Origin: Acinetobacter baylyi
Properties:
Chitin deacetylase (AsCDA) is an enzyme that catalyzes the removal of acetyl groups from chitin, producing chitosan or partially deacetylated chitin. The coding sequence of AsCDA is 975 base pairs in length, corresponding to a polypeptide of approximately 325 amino acids. 37.5kDa, AsCDA belongs to the carbohydrate esterase family and functions by specifically cleaving the N-acetyl groups of β-(1→4)-linked N-acetylglucosamine units in chitin polymers. This enzymatic activity plays a crucial role in modifying the structural properties of chitin, affecting solubility, crystallinity, and bioavailability. The enzyme is typically active under neutral to slightly alkaline pH and shows optimal activity at moderate temperatures. Clinically and industrially, chitin deacetylases like AsCDA are of interest for producing bioactive chitosan, which has applications in biomedicine, agriculture, and food technology.
Figure 1. Gene maps of AsCDA
Usage and Biology
Chitin deacetylase (AsCDA) is widely used in both research and industrial contexts to convert naturally occurring chitin into chitosan or partially deacetylated derivatives. In biological systems, AsCDA catalyzes the hydrolysis of N-acetyl groups from chitin polymers, a process that alters the polymer's chemical and physical properties, making it more soluble and amenable to further enzymatic or chemical processing. The enzyme is highly specific for chitin and can act on various chitinous substrates, including crustacean shells, fungal cell walls, and insect exoskeletons.
In research, AsCDA is used to study enzymatic deacetylation mechanisms and structure–function relationships of carbohydrate-active enzymes. Its stability and activity under moderate temperatures and neutral pH make it suitable for in vitro assays and protein engineering studies. Mutagenesis and structural analysis of AsCDA have provided insights into substrate binding, catalytic residues, and the influence of active site architecture on enzymatic efficiency.
Industrially, AsCDA enables the production of chitosan with controlled degrees of deacetylation, which is critical for its biological activity, solubility, and functional properties. Chitosan derived from AsCDA-mediated deacetylation is used in wound dressings, drug delivery systems, biodegradable films, and agricultural treatments.
Cultivation
PCR was used to amplify the AsCDA gene, and its length was 975 bp. A band with the same size as the target gene appeared in Figure 2A, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-AsCDA.
Figure 2 A: The results of amplification of different target genes, B: The results of linearized vectors
2. Add a basic part (Cts2p, BBa_25XXGWVY)
Name: Chitin exonuclease (Cts2p)
Base Pairs: 1536 bp
Origin: Saccharomyces cerevisiae
Properties:
Chitin exonuclease (Cts2p) is an enzyme encoded by a 1536 bp gene from Saccharomyces cerevisiae, corresponding to a protein of about 512 amino acids with a predicted molecular mass of 59.1 kDa. Cts2p functions as an exo-acting glycosidase that hydrolyzes chitin polymers by releasing N-acetylglucosamine units from the chain ends. As part of the yeast chitin metabolism system, it contributes to cell-wall remodeling and degradation of chitin-containing structures. Like other secreted fungal enzymes, Cts2p is expected to possess a catalytic domain characteristic of glycosyl hydrolases and may contain additional regions for substrate binding. Its enzymatic activity is typically compatible with the physiological conditions of S. cerevisiae, such as moderate temperature and slightly acidic to neutral pH.
Figure 3. Gene maps of Cts2p
Usage and Biology
Biologically, Cts2p plays an important role in S. cerevisiae by participating in chitin turnover, especially during cell-wall remodeling, bud separation, and sporulation. By trimming chitin polymers into smaller oligosaccharides or monomers, it helps regulate the structure and dynamics of the fungal cell wall. In research, Cts2p is studied to understand the mechanisms of chitin degradation, enzyme–substrate interactions, and the role of chitin-modifying enzymes in fungal physiology.
From a biotechnological perspective, Cts2p is useful for producing well-defined chitooligosaccharides, which have applications in agriculture, food technology, and biomedicine. It can also be employed in enzymatic cascades with other chitin-active enzymes, such as chitin deacetylases, to generate chitosan derivatives with controlled properties. Since the enzyme is encoded by yeast, a generally recognized BSL-1 organism, it is safe for use under standard laboratory conditions.
Cultivation
The Cts2p gene was amplified by PCR, and its length was 1536 bp. As shown in Figure 2A, a band with the same size as the target gene appeared, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-Cts2p.
3. Add a basic part (FjChiB, BBa_25S1GVYQ)
Name: Chitin endonuclease (FjChiB)
Base Pairs: 522 bp
Origin: Flavobacterium johnsoniae
Properties:
Chitin endonuclease (FjChiB) is an enzyme encoded by a 522 bp gene from Flavobacterium johnsoniae, corresponding to a protein of about 174 amino acids with a predicted molecular weight near 38.1 kDa. Unlike exonucleases that act progressively from chain ends, FjChiB functions as an endo-acting chitinase, cleaving internal β(1→4)-glycosidic bonds within chitin polymers to generate chitooligosaccharides of various lengths. This catalytic property enables rapid depolymerization of crystalline chitin, an otherwise highly recalcitrant polysaccharide.
Structurally, FjChiB is expected to contain a catalytic domain characteristic of glycoside hydrolase families active on N-acetylglucosamine polymers. The relatively small size of the protein suggests it may represent a single-domain enzyme optimized for catalytic efficiency without additional carbohydrate-binding modules. Its bacterial origin indicates adaptation to mesophilic conditions, with activity likely optimal at moderate temperatures and neutral to slightly alkaline pH, consistent with the natural environment of F. johnsoniae, a soil and freshwater bacterium.
Figure 4. Gene maps of FjChiB
Usage and Biology
Biologically, FjChiB contributes to the degradation of chitin in the environment, supporting nutrient cycling by converting insoluble chitin into soluble chitooligosaccharides and monomers. In F. johnsoniae, this activity provides a carbon and nitrogen source and may also facilitate interactions with other organisms in chitin-rich habitats, such as insect remains, crustacean shells, and fungal biomass.
In research, FjChiB is valuable for investigating endo-acting chitinolytic mechanisms, enzyme kinetics, and substrate specificity. Its relatively small coding sequence makes it an attractive candidate for heterologous expression and protein engineering. FjChiB can be combined with chitin deacetylases or exo-chitinases in enzymatic cascades to produce defined oligomers and chitosan derivatives with tailored properties.
From an applied perspective, FjChiB is of interest in biotechnology for the bioconversion of chitin waste into value-added products. The chitooligosaccharides generated by its activity have documented roles in plant defense induction, antimicrobial action, and biomedical applications such as wound healing and drug delivery.
Cultivation
PCR was used to amplify the FjChiB gene, and its length was 1023 bp. As shown in Figure 2A, a band with the same size as the target gene appeared, indicating that the target gene was successfully amplified. After agarose gel electrophoresis and gel recovery, homologous recombination was performed to obtain the recombinant plasmid pET28a(+)-FjChiB.
4. Add a Composite part (pET28a_AsCDA, BBa_25VIQJQP)
Composition: pET-28a(+) backbone; AsCDA gene fragment.
Apparatus used: pET-28a(+) plasmid, AsCDA gene fragment, restriction endonucleases (EcoRI, XhoI), Homologous recombination enzyme.
Figure 5. Plasmid map of pET28a- AsCDA
Engineering Principle: The pET-28a-AsCDA expression vector was constructed to achieve efficient expression of AsCDA in E.coli using the T7 promoter. The system is regulated based on the lac operon. When IPTG is added, the T7 RNA polymerase is activated, which in turn initiates the transcription of the AsCDA gene. In addition, by fusing the His tag with AsCDA, efficient purification can be achieved by affinity chromatography, which not only ensures the efficient production of recombinant AsCDA, but also facilitates subsequent functional detection, such as protein degradation, etc.
In the construction process, we first obtained the AsCDA gene fragment ( 975 bp ) by PCR amplification. Subsequently, the AsCDA gene and the pET-28a ( + ) vector were digested with EcoRI and XhoI restriction endonucleases, respectively, to generate complementary viscous ends, followed by the next connection operation.
In general, the purified insert fragment was ligated with the backbone sequence using homologous recombinase. Subsequently, the ligation product was subjected to heat shock treatment and transformed into E.coli BL21 ( DE3 ) competent cells to obtain a recombinant plasmid. After the transformation was completed, the bacteria were cultured at 37 °C for 1 hour, and then inoculated onto LB agar plates containing kanamycin for overnight culture. After that, a single colony was selected to extract the plasmid, which was verified by gel electrophoresis and Sanger sequencing. After homologous recombination, the recombinant plasmid was transformed into chemically competent E.coli BL21 ( DE3 ) by heat shock method. After overnight culture on LB agar plates containing corresponding antibiotics, multiple well-separated colonies were observed ( see Figure 6A ), indicating that the transformation process was successful. In order to verify whether the target gene was successfully inserted, several independent colonies were randomly selected for colony PCR detection. The results of gel electrophoresis ( see Figure 6B ) showed that each colony produced a DNA band of the expected size, which confirmed that the plasmid was successfully constructed and the target gene was correctly inserted.
Figure 6. A: Monoclonal plate of BL21 (DE3) transformed with recombinant plasmid, B: PCR electrophoresis results of colonies on monoclonal plate after transformation.
In order to further ensure the accuracy of plasmid construction, the recombinant pET28a plasmid was sent to a commercial sequencing agency for Sanger sequencing. The results of sequence alignment (Figure 7) showed that the inserted gene fragment was completely consistent with the designed sequence, and no gene mutation occurred, which proved that the cloning of the target gene was correct.
Figure 7. Sanger sequencing results of pET-28a(+)-AsCDA/Cts2p/FjChiB positive colony.
Cultivation, Purification and SDS-PAGE:
In E.coli BL21 ( DE3 ), the protein expression was induced by 1 mM IPTG, and the cells were lysed by ultrasonic disruptor at low temperature to maintain the activity of the enzyme. Subsequently, the 6 × His tag of the recombinant protein was purified by nickel affinity chromatography. During the purification process, the protein was washed with low concentration and high concentratio imidazole. After SDS-PAGE analysis of the collected components, it was found that obvious protein bands appeared in all lysis samples. A protein band of about 37.5 kDa was detected in the strain expressing AsCDA, which was consistent with the expected molecular weight. These results indicate that the AsCDA has been successfully expressed in E.coli.
Figure 8. The expression of a large amount of target protein AsCDA was analyzed by SDS-PAGE.
5. Add a Composite part (pET28a_Cts2p, BBa_25OCX0JU)
Composition: pET-28a(+) backbone; Cts2p gene fragment.
Apparatus used: pET-28a(+) plasmid, Cts2p gene fragment, restriction endonucleases (EcoRI, XhoI), Homologous recombination enzyme.
Figure 9. Plasmid map of pET28a- Cts2p
Engineering Principle: In this study, the pET28a-Cts2p plasmid was constructed to express Cts2p enzyme in E.coli. The gene was placed downstream of the T7 promoter, and precise regulation and efficient expression were achieved by the lac manipulation subsystem under IPTG induction. The carrier design contains an N-terminal His tag, which is convenient for simplified purification by nickel affinity chromatography. This engineering scheme can efficiently prepare functional Cts2 p protease, which provides an experimental basis for evaluating its application value in the preparation of chitooligosaccharides. The Cts2p gene ( 1536 bp ) plays an important role in S.cerevisiae and can be successfully amplified by PCR. The PCR product and pET-28a ( + ) vector were treated with appropriate endonucleases to obtain complementary ends. Figure 6 shows the agarose gel electrophoresis results of PCR, and the DNA fragment length of Cts2p is about 1536 bp, which is completely consistent with the theoretical prediction. This indicates that the target gene has been successfully amplified. Similarly, the sequencing results are shown in Figure 7.
Cultivation, Purification and SDS-PAGE: The verified plasmids were transformed into E.coli BL21 ( DE3 ) for protein expression. After IPTG induction overnight, the cells were collected and lysed by low temperature ultrasonication. The recombinant protein was purified by Ni-NTA affinity chromatography, which included a washing step using an imidazole buffer step by step. As shown in Figure 10, obvious protein bands were observed in the lysate samples, which were consistent with the expected molecular weight. These results confirmed that the recombinant enzyme had been successfully expressed in E.coli.
Figure 10. The expression of a large amount of target protein Cts2p was analyzed by SDS-PAGE.
6. Add a Composite part (pET28a_FjChiB, BBa_250AYN60)
Composition: pET-28a(+) backbone; FjChiB gene fragment.
Apparatus used: pET-28a(+) plasmid, FjChiB gene fragment, restriction endonucleases (EcoRI, XhoI), Homologous recombination enzyme.
Figure 11. Plasmid map of pET28a- FjChiB
Engineering Principle: The pET28a _ FjChiB vector was constructed to express chitinase B under the control of T7 promoter in E.coli. The system uses the lac operon to achieve IPTG-induced expression to ensure precise regulation and high yield of recombinant proteins. The N-terminal His tag facilitates nickel affinity chromatography purification. The carrier can efficiently produce and analyze enzyme activity, aiming to evaluate its potential to promote chitin degradation in the environment. The target gene ( 1023bp ) was obtained by PCR amplification using codon-optimized synthetic templates. The inserted fragment and the pET-28a ( + ) backbone were digested with EcoRI and XhoI, respectively, to produce a sticky end, and the plasmid pET28a _ FjChiB was generated by homologous recombination enzyme. Figure 6 shows the results of PCR agarose gel electrophoresis, which is consistent with the predicted value, indicating that the target gene was successfully amplified.
Cultivation, Purification and SDS-PAGE: The constructed plasmid was transformed into E.coli BL21 ( DE3 ) for protein expression. After adding IPTG to induce expression, the bacteria were collected after 20 hours of culture. In order to prevent protease denaturation, cell lysis was performed using an ultrasonic disruptor at low temperature. Purification was performed by nickel affinity chromatography. The column was first washed with low imidazole and then eluted with high imidazole. The collected lysates, eluents and elution components were analyzed by SDS-PAGE. As shown in Figure 12, there are obvious protein bands in the lysate samples. A band of ~ 38.1 kDa was detected in the strain expressing FjChiB, which was consistent with the expected molecular weight of FjChiB. These results confirmed that the recombinant enzyme had been successfully expressed in E.coli.
Figure 12. The expression of a large amount of target protein FjChiB was analyzed by SDS-PAGE.
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.
Figure 13. N-acetylglucosamine standard curve
Figure.13 shows the standard curve for quantitative analysis. The above picture depicts the color gradient change of N-acetylglucosamine ( GlcNAc ) standard solution after reacting with different concentrations of DNS reagent. As the concentration increases ( from left to right ), the color of the solution gradually changes from light yellow to deep red, indicating that the absorbance also gradually increases. The fitting curve below accurately quantifies this color change process. In this standard curve, the horizontal axis ( X axis ) represents the standard concentration of N-acetylglucosamine ( unit : mg / mL ), and the vertical axis ( Y axis ) represents the absorbance value measured at 540 nm wavelength. This curve showed a significant linear positive correlation, indicating that there was a direct linear correlation between N-acetylglucosamine concentration and absorbance.
Figure 14. p-Nitroacetanilide standard curve
The standard curve (Figure.14) is used as a quantitative scale for the determination of chitosan deacetylase ( CDA ) activity. Its core function is to convert the measured absorbance value into an accurate product concentration, thereby calculating the enzyme activity. Chitosan deacetylase catalyzes the removal of acetyl ( -COCH3 ) from chitin or its derivative substrates ( such as ethanol chitin ) to produce free amino groups and acetic acid. In this detection, synthetic chromogenic substrates such as p-nitroacetanilide are usually used. After enzyme action, the substrate was hydrolyzed to p-nitroaniline. P-nitroaniline is a compound with strong yellow absorption near the wavelength of 400 nm ( as shown in the color gradient of the upper test tube ). Therefore, the more p-nitroaniline produced by the enzyme reaction, the higher the absorbance of the solution at 400 nm. By calculating the parameters such as product concentration, reaction time, enzyme liquid volume and cuvette light path, the enzyme activity unit can be finally obtained. According to the formula corresponding to the standard curve, we calculated that the activity of chitin exonuclease ( Cts2p ) was 115 U / mL, the activity of chitin endonuclease ( FjChiB ) was 116 U / mL, and the activity of chitin deacetylase ( AsCDA ) was 41 U / mL.
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 results in Figure 15 showed that the enzyme activities of chitin deacetylase ( AsCDA, A ), chitinase ( Cts2p, C ) and chitinase ( FjChiB, F ) showed a typical pH-dependent relationship, and the optimum pH values were all about 6. The activity of these three enzymes reached a peak at pH 7, indicating that the pH value was the best condition for their catalytic reaction.
Enzyme activity is usually reduced under conditions that deviate from the optimal pH. Especially in extremely acidic ( pH 2-3 ) or alkaline ( pH 10-11 ) environments, the activity of these three enzymes will decrease significantly or even completely inactivated. It is worth noting that these three enzymes can still maintain relatively high activity in a wide pH range of 4-8, showing a certain acid-base stability. This provides flexibility for the selection of buffer system in practical applications.
The discussion shows that the similarity of the optimal pH values of the three enzymes may be related to the ecological environment of the source microorganisms or the characteristics of the substrates. In addition, the high catalytic performance under neutral to weakly acidic conditions is consistent with the general characteristics of chitin degrading enzymes. This result indicates that when these enzymes are used for chitin biotransformation, the pH value of the reaction system should be controlled at about 6 to achieve the best catalytic efficiency. In addition, the stability of the enzyme in a wide pH range provides a potential advantage for its application in complex industrial environments.
Figure 15. 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 bar graph and line chart in Figure 16 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℃).
Figure 16. 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.
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.
Figure 17. 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 17 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. Optimal free enzyme synergistic ratio and reaction conditions
After determining the optimal reaction conditions of the three enzymes, we need to further explore the enzyme synergistic effect. Specifically, the enzyme preparation was first mixed in equal volume ratio to evaluate the combination effect, and then the enzyme synergy ratio was analyzed. In order to determine which combination is more effective, we used a chitinase detection kit for verification. Before the experiment, it is necessary to establish a standard curve as a reference.
Figure 18. Chitinase activity assay kit standard curve, A: standard curve preparation process, B: standard curve
The results in Figure 19 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.
Figure 19. 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 column diagram in Figure 20 shows the trend of reducing sugar yield in the reaction system of AsCDA+Cts2 p and AsCDA+FjChiB enzyme combination at different ratios. For AsCDA + Cts2p combination, when the ratio was 1 : 1, the reducing sugar yield was the highest, which was significantly higher than other ratios. When the ratio deviated from 1 : 1 ( such as 0.5 : 1 or 1 : 0.5 ), the reducing sugar yield decreased significantly, indicating that there was a strict stoichiometric dependence between the two. The AsCDA + FjChiB combination had higher yield at 1 : 0.5 and 1 : 1 ratios, and the 1 : 0.5 ratio performed best. This shows that the combination is less sensitive to proportional changes and has a wider range of adaptability.
This result may be due to the difference in the synergistic mechanism of the two enzymes : AsCDA and Cts2p may require precise molecular matching to achieve efficient synergy. For example, the exonuclease activity of Cts2p depends on the specific substrate conformation produced by AsCDA deacetylation. In contrast, the synergistic effect between AsCDA and FjChiB may be achieved through more relaxed functional complementarity - the endo-cleavage of FjChiB will produce more terminal structures and provide substrates for AsCDA. It is worth noting that the difference in peak yield between the two combinations ( AsCDA + Cts2p is superior to AsCDA + FjChiB ) indicates that Cts2p and AsCDA show better synergistic effects, which provides important enlightenment for optimizing enzyme combinations.
Figure 20. 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 histogram in Figure 21 shows that the optimal pH values of the two enzyme complexes AC ( AsCDA + Cts2p ) and AF ( AsCDA + FjChiB ) are significantly different. The AF complex showed a peak activity at pH 5 ( about 170 U / mL ) and maintained a high activity level in the pH range of 3-6. The optimal pH value of AC complex was 7, and the peak activity was about 120 U / mL. The activity was relatively stable in the range of pH 6-8. The activities of both enzymes decreased significantly under extreme pH conditions (pH ≤ 3 or ≥ 9 ).
It is worth noting that the overall activity of AF complex is significantly higher than that of AC complex ( about 40 % higher on average ), and its optimal pH range is more acidic. This phenomenon may be due to the stronger synergistic effect between FjChiB ( endonuclease ) and AsCDA ( deacetylase ) : endonucleases produce more ends by cleaving chitin chains, providing more action sites for deacetylases, and deacetylation further enhances the sensitivity of substrates to endonucleases. In contrast, the synergistic efficiency of Cts2p ( exonuclease ) and AsCDA is low, which may lead to poor overall activity of AC complex enzyme.
Figure 21. Exploration of the optimal pH value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB
The bar graph in Figure 22 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.
Figure 22. Exploration of the optimal temperature value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB
The bar graph in Figure 23 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.
Figure 23. Exploration of the optimal Metal ions value for the activity of two complex enzymes, AC: AsCDA+Cts2p, AF: AsCDA+FjChiB
Since we found that the synergistic catalytic activity of AsCDA + Cts2p and AsCDA + FjChiB was higher than that of the former, the latter was selected for enzyme immobilization. Firstly, the purified AsCDA and FjChiB proteins were obtained by heterologous expression in E.coli, and then the two proteins were immobilized with epoxy resin. The supernatant before and after fixation was collected for SDS-PAGE analysis ( the sample could be diluted 5 times ). ImageJ software was used to calculate the gray value change and fixation efficiency of the strip : fixation efficiency ( % ) = ( gray value of sample before fixation − gray value of sample after fixation ) / gray value of sample before fixation × 100 %. As shown in Figure 24, the next step is to use ImageJ software to calculate the gray value.
4. Preparation and immobilized enzyme efficiency
Figure 24. Purified proteins of AsCDA and FjChiB and SDS-page images of proteins before and after immobilization
Before calculating the gray value, in order to accurately analyze the gray value, we first convert the image into an 8-bit gray image, and then measure the average gray value and the integral density. The higher the protein expression, the greater the value of the integral density.
The results in Figure 25 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.
The discussion shows that the difference in the immobilization efficiency of the two proteins may be related to their characteristics : FjChiB, as an endonuclease, may contain more surface active groups ( such as amino groups ), which makes it easier to bind to the carrier ; the spatial structure of AsCDA may limit its effective binding. The immobilization efficiency of both proteins did not exceed 25 %, indicating that the current immobilization methods ( possibly epoxy or amino coupling ) still have room for optimization, such as adjustable carrier pore size, activation conditions or reaction time. In addition, the retained protein activity after immobilization still needs to be further verified, because the gray value only reflects the protein content rather than the activity level.
Figure 25. Calculation of grayscale values of the two proteins before and after immobilization
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.
Figure 26. Number of times the immobilized enzyme can be reused, abcde represent the first to fifth repeated experiments
Figure 26 shows the relationship between the number of reuses of the immobilized enzyme and its activity. The results show that enzyme activity shows a continuous downward trend with increasing reuse. The enzyme activity reaches its highest level (approximately 43 U/g) after the first use and drops to approximately 22 U/g after the fifth use, remaining at approximately 60% of its initial value. The activity decay of the immobilized enzyme is primarily caused by physical loss of the support surface, structural denaturation, and impurity accumulation. Despite this, immobilization significantly improves the enzyme's stability, allowing it to retain a significant proportion of its activity after five reuses. The reusability of immobilized enzymes can significantly reduce the cost of enzyme preparations, but the optimal replacement cycle needs to be determined based on specific process requirements.
In our iGEM project, we focused on the construction of chitinase expression genetic systems and their applications in biomass resource degradation and conversion, contributing to at least three key areas:
1. Successful Expression of Chitin-Degrading Enzymes
In our project, we identified and expressed three microbial chitin-active enzymes: chitin deacetylase (AsCDA)、chitin exonuclease (Cts2p) and chitin endonuclease (FjChiB), By synthesizing the corresponding coding sequences and cloning them into an Escherichia coli expression system, we established a genetic platform capable of producing these enzymes in vitro. This achievement represents a key step toward creating a reliable source of chitin-modifying proteins, enabling us to systematically test their enzymatic activity and optimize production conditions.
2. Innovation in Chitin Bioconversion Strategies
Our work also provides an innovative approach to the valorization of chitin waste. By combining enzymes with distinct catalytic modes, AsCDA for deacetylation, Cts2p for progressive exocleavage, and FjChiB for endocleavage, we demonstrated the potential for synergistic degradation of crystalline chitin into soluble, bioactive products. This multi-enzyme strategy provides a more efficient and controllable degradation process compared to using individual enzymes alone. Furthermore, we optimized reaction parameters such as pH, temperature, and enzyme ratio to enhance activity and explored immobilizing the enzyme mixture on epoxy resin to improve stability and reusability.
3. Foundation for Future Applications
These contributions collectively lay a solid foundation for future applied research in chitin degradation. The recombinant enzymes we generated can be further engineered or modified for industrial applications, such as converting marine chitin waste into high-purity chitosan, chitooligosaccharides, or other valuable derivatives. Beyond waste valorization, our designed enzyme system can serve as a model for developing novel biocatalytic solutions to address marine pollution and generate sustainable biomaterials for agriculture, medicine, and biotechnology.
