Model

Analysis

In our project, modeling played a critical role. We utilized engineering techniques to predict protein structures, analyze the interactions between proteins and small molecules, and apply rational design to inform protein engineering strategies, aiding in experimental planning. Modeling also provided mechanistic insights into experimental results. The P115A mutant exhibited higher enzymatic activity than the wild type, while the D78Y and K260Y mutants retained some activity in chitosan hydrolysis, producing DP2 products after prolonged reactions. This further validated the success of our modeling efforts.

Structural Biology Predictive Analysis

After an extensive review and analysis of existing data, we have identified site-directed mutagenesis as a strategy for producing chitosan oligosaccharides with uniform degrees of polymerization and optimizing enzyme catalytic efficiency. However, to date, there are no reports on the structure of CsnB through X-ray crystallography (XRD), and there is a lack of data on binding sites. To address this issue, we predicted the protein structure using the AlphaFold3 platform and identified potential binding sites for the interaction between CsnB and chito-hexaose through molecular docking techniques.

AlphaFold3

AlphaFold3 is an AI-based protein folding simulation algorithm that we employ. Its powerful capabilities stem from its next-generation architecture and training, which encompasses all molecules in life. At the core of AlphaFold3 is an improved Evoformer module. After processing the input, AlphaFold3 uses a diffusion network for predictions, starting the diffusion process from a cloud of atoms and, through numerous steps, eventually generating the most accurate molecular structure. Its accuracy in predicting molecular interactions surpasses all existing systems. In many practical applications, AlphaFold3 has achieved recognized levels of high precision. Through the AlphaFold3 platform, we obtained the predicted structure of the CsnB protein([1]). PyMOL is used for structural visualization, analysis, and the creation of relevant images([2]).

AutoDock-Vina

AutoDock-Vina Chitohexaose was used as the ligand, with its structure sourced from the PDB database (PDB ID: 4OLT)[3], and docking positions based on this structure. Docking simulations were performed using AutoDock Vina. The substrate and protein structures were preprocessed, including removing water from the protein, and adding hydrogen atoms to both the protein and the small molecule. AutoDock Vina employs an empirical scoring function that considers van der Waals forces, Coulomb repulsion, hydrogen bonding, and hydrophobic effects. The most stable and likely binding modes were identified by minimizing the scoring function's value. During the docking process, Vina uses a fast and efficient simulated annealing algorithm, performing random searches and local optimizations through multiple iterations to generate potential binding modes, with each conformation receiving a score. By default, nine conformations are generated for each substrate[4].

Selection of Mutation Sites

To obtain chito-oligosaccharide products with specific degrees of polymerization (DP), various methods have been developed to improve the distribution of DP in the final enzymatic hydrolysis products, such as protein engineering of enzymes( [5]), immobilization( [6]), and the application of coupled membrane reactors( [7]). However, research has verified that intervening in the substrate's binding at the active site and altering the binding affinity of the substrate remains the most effective strategy for enhancing the specificity of enzymatic products. Therefore, rational design of the chitosanase gene is crucial for producing chito-oligosaccharides with specific DP and for better understanding the functional mechanisms of chito-oligosaccharides.In this study, chitosanase CsnB was classified as a member of the GH46 family and confirmed to employ an endo-type degradation mechanism. Its main hydrolysis products are chito-dimer (GlcN)₂ and chito-trimer (GlcN)₃, without producing the monomer (GlcN). Additionally, CsnB belongs to subclass II of GH46 chitosanases, capable of cleaving GlcN-GlcN and GlcN-GlcNAc bonds. Based on the structure of the protein-ligand complex and previous successful mutation studies that altered product DP, mutation sites were selected.

In the CsnB protein structure, Pro115 (P), Val186 (V), Asp78 (D), and Lys260 (K) are located near the (+3) and (-3) subsites of chitohexaose. Notably, Asp78 corresponds to the highly conserved Ser27 residue in chitosanase OU01, which is involved in substrate binding and whose mutation has some effect on substrate binding. Val186 (V) is a conserved amino acid in CsnB located between structural domains, while Pro115 is one of the residues with a rigid, irregularly coiled structure near the (-3) subsite, potentially interfering with correct substrate interaction. The extension of the carbonyl group of Lys260 might cause steric hindrance due to the N-acetyl group, possibly affecting enzyme-substrate binding and catalytic efficiency. To assess the effects of these residues on CsnB’s enzyme activity and substrate specificity, the following mutations were designed: Val186 to Tyr (V186Y), Asp78 to Tyr (D78Y), Lys260 to Trp (K260W), and Pro115 to Ala (P115A).

Analysis and Discussion of Experimental Results

Induced expression of primitive and mutant enzymes

All recombinant mutant strains were successfully expressed in E. coli BL21(DE3) after IPTG induction. The crude enzyme solution was purified using Ni-NTA, and both the crude enzyme and purified proteins were analyzed by SDS-PAGE. Distinct bands appeared between 25 and 35 kDa in the crude enzyme samples, matching the theoretical molecular weight, indicating that the mutant enzymes were successfully expressed in a soluble form. After purification, the mutant bands were consistent with those of CsnB, with a single distinct band appearing at the same position as in the crude enzyme solution.

Enzyme Activity Assay for Chitosanase

(1) Determination of the Glucosamine Standard Curve

Prepare a 1 mg/mL glucosamine hydrochloride standard solution by weighing 1 g of glucosamine hydrochloride and dissolving it in a volumetric flask to a final volume of 1 L. Add varying volumes of the glucosamine hydrochloride standard solution, distilled water, and DNS solution.

Target concentration/%	glucosamine hydrochloride solution/µL	Deionized water / µL	DNS/µL
0	0	400	600
10	100	300	600
12.5	125	275	600
15	150	250	600
17.5	175	225	600
20	200	200	600
22.5	225	175	600
25	250	150	600

Boil the mixture in a water bath for 10 minutes, then rapidly cool it. Measure the absorbance at 540 nm. Perform three parallel replicates for each measurement and take the average value. Plot a standard curve with glucosamine hydrochloride concentration on the x-axis and OD540 on the y-axis.

(2) Determination of enzyme activity of the strain using the DNS method

Subsequently, take 50 μL of enzyme solution diluted to a certain multiple and mix it with 350 μL of 1% colloidal chitosan. React in a 50°C water bath for 30 minutes, then boil to inactivate the enzyme and terminate the reaction. Next, add 600 μL of DNS solution, boil for 10 minutes, and centrifuge at 12,000 r/min for 5 minutes. After the supernatant cools to room temperature, measure the absorbance at 540 nm. Each group of experiments is done in triplicate. The blank group consists of 50 μL of inactivated enzyme. Calculate the reducing sugar content based on the glucosamine hydrochloride standard curve.

(3) Definition of chitosanase activity unit

The amount of enzyme required to catalyze the production of 1 μmol of reducing sugar from colloidal chitosan per minute under certain conditions.

(4) Formula for calculating enzyme activity

Enzyme activity is usually expressed as the amount of enzyme required to produce 1 micromole of reducing sugar per unit time. The specific formula is as follows:

(5) Measurement of enzyme activity of mutant enzymes

The enzyme activity of CsnB and its mutants was determined as shown in the figure below. The wild-type enzyme exhibits an activity of 28.8 (U/mL), while the K260Y mutant shows the lowest enzyme activity, with a 69.7% decrease compared to the wild type. The enzyme activities of the V186Y and D78Y mutants are reduced by 43.2% and 57.0%, respectively. On the other hand, the P115A mutant shows a 15.2% increase in chitosanase activity.

Product Analysis

Method: Thin Layer Chromatography, TLC

Mechanism: Because of the different distribution coefficients between the fixed phase and the mobile phase, the result is that they move at different speeds on the thin layer plate. The more polar group adsorbs more strongly to the polar stationary phase and moves slowly. However, the less polar group will dissolve more in the mobile phase and move faster. By comparing the spot location of the sample with that of the known reference material, the presence and relative amount of each component in the sample can be determined.

Material and reagent:

After terminating the enzymatic hydrolysis reaction under optimal reaction conditions for a certain period, sampling is conducted. Standard samples (GlcN)2 and (GlcN)3 are used as controls. 15 μL of each sample is spotted onto a capillary with a distance of 1 cm between each spot, and a development distance of 80 cm is used. The samples are developed in a chromatography tank using the ascending method for 1.5 to 2 hours. After development, the developing solvent is dried by blowing, and the TLC color reagent is sprayed onto the silica gel plate for staining. A hairdryer is then used to heat the silica.

Analysis of Enzyme Activity and Product Change Mechanisms

In the V186Y and D78Y mutants, the original residues were replaced with tyrosine (Tyr, Y). The phenyl ring of tyrosine can form π-π interactions with the sugar chain, enhancing the stability of the sugar chain in the substrate-binding cleft. However, this makes it more difficult for substrates with a higher degree of polymerization to be positioned and bound within the catalytic cleft, as the degree of cleft closure increases. Only chitooligosaccharides with a low degree of polymerization can pass through smoothly and be released, while those with a higher degree of polymerization struggle to be released from the cleft. As a result, the D78Y mutant exhibits a single polymerization degree product, chitobiose. The increased steric hindrance obstructs the binding of the enzyme to the substrate, reducing the enzymatic activity of these two mutants. For the K260Y mutant, the extension of the carbonyl group of K260 may cause steric hindrance with the N-acetyl group. After mutating it to tyrosine, the π-π interactions between tyrosine and the sugar chain further stabilize the substrate. Meanwhile, the larger phenyl ring structure of tyrosine introduces new steric hindrance, which limits the binding and release of chitooligosaccharides with a higher degree of polymerization. The K260Y mutant mainly produces chitobiose with a single degree of polymerization, and the enzyme activity is significantly reduced. As for the P115A mutant, the original proline residue, due to its strong rigidity, may interfere with the proper binding of the substrate. Mutating P115 to alanine increases the flexibility of the unstructured coil region, bringing the enzyme closer to the substrate. This mutation improves the enzyme's ability to bind to the substrate, thereby enhancing its catalytic activity.

Conclusion

Based on the protein-ligand complex structure and previous mutation studies that successfully altered product polymerization, mutations were introduced at different subsites of CsnB (Val186, K260, Asp78, Pro115). A total of four mutants were designed, all of which were successfully expressed solubly and purified. The enzyme activities of the four mutants were compared with the wild-type enzyme. The results showed that the P115A mutant exhibited a 15.2% increase in chitosanase activity compared to the wild-type enzyme. The enzyme activities of the other mutants decreased due to residue mutations. The D78W and K260Y mutants successfully achieved the intended mutation goals. After prolonged hydrolysis, the mutant enzymes still retained some chitosanase activity, and the main product of these mutants was chitobiose.

References

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[2] Schrödinger, L., & DeLano, W. (2020). PyMOL. Retrieved from http://www.pymol.org/pymol

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[4] Eberhardt, J., Santos-Martins, D., Tillack, A.F., Forli, S. (2021). AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. Journal of Chemical Information and Modeling.

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