Engineering Success

Design 1.0

Recombinant expression plasmids for CDA and CsnB

We obtained chitin deacetylase (CDA) gene and chitosanase (CsnB) gene from Bacillus pumilus and Marine Bacterium Bacilius SP. BY01 by DNA extraction and PCR amplification, respectively. Then, we used the PET-28a plasmids kept in the laboratory as a template and primers were designed to ligate the CDA gene and CsnB gene to the PET-28a linearized vector by Gibson assembly method to obtain recombinant expression plasmids PET-28a-CDA and PET-28a-CsnB.

The plasmid mapping was constructed as follows:

Build 1.0

Heterologous expression and characterization of CDA and CsnB

We transformed PET-28a-CDA and PET-28a-CsnB recombinant plasmids into E.coli BL21 (DE3) for overexpression of CDA and CsnB. As shown in Figure 2, it can be seen that there are obvious protein bands around 30 kDa, which is consistent with the theoretical size of CDA protein 31.5kDa, indicating successful expression verification. And the purified crude enzyme liquid (Figure 2b, Lane 3) washed away the miscellaneous proteins, leaving only the CDA enzyme (Figure 2b, Lane 2). The results indicated that the purification was successful and the enzymatic properties of the produced enzyme could be studied.

As shown in Figure 3, a distinct protein lane can be observed around 30 kDa, which corresponds to the theoretical size of the CsnB protein at 31.7 kDa, confirming successful expression. The figure also shows that the crude enzyme solution after purification has removed the contaminating proteins, leaving only the CsnB enzyme (Figure 3b).

Test 1.0

1. Enzymatic activity determination of CDA

The enzyme activity of CDA was determined by chromogenic substrate method. The substrate p-nitroacetanilide can be hydrolyzed by CDA to p-nitroaniline. The concentration of p-nitroaniline can be measured by the characteristic absorption at 400 nm. Figure 4 showed the standard curve of p-nitroaniline. Calculate the CDA enzyme activity based on the formula (1) in DESIGN 3.1. The CDA enzyme activity was obtained as 51.56 U/mL.

2. Analysis of the degree of deacetylation of the CDA hydrolysis product

According to the method of product deacetylation analysis mentioned in DESIGN 3.1, the product chitosan was obtained with a deacetylation degree of 65.24% while 1% colloidal chitin was hydrolyzed by CDA.

3. Product Analysis of CsnB

The soluble sugar hydrolyzed by CsnB from chitosan was analyzed by TLC. As shown in the Figure 5, the hydrolysis of CsnB was incomplete at 30 min and 150 min, and the main hydrolyzed products were (GlcN)3 and (GlcN)4. After 13 h, the hydrolyzed products were (GlcN)2 and (GlcN)3. No GlcN was produced after 17 h, indicating that it was also endo-chitanase.

Learn 1.0

From the above results, we found that CDA enzyme showed high enzyme activity in catalyzing the deacetylation reaction, but the degree of deacetylation of the product was only 65.24%, which did not reach the expected high purity, and needs to be further improved. The CsnB enzyme catalyzed the generation of chitosan from chito-oligosaccharides with a mixture of chitobiose and chitotriose, which did not reach our initial goal (to obtain chito-oligosaccharides with a single degree of polymerization), and we found that the hydrolysis efficiency of CsnB was low, and decomposition catalysis should take at least 13 h. Comprehensive analyses, in particular, the deficiencies of CsnB in terms of enzyme activity and product purity prompted us to realize that it is essential to carry out enzyme engineering for its modification. The necessity of enzyme engineering for CsnB.

In order to clarify the direction of the next optimization step, 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.

1. CsnB amino acid sequence analysis

The chitosanase gene CsnB of Bacilius sp. BY01 was looked up from the NCBI database, and the structure of CsnB was predicted as follows using AlphaFold3 for modeling:

As can be seen from Figure 6, the structure of the target protein consists of a large and a small region, with two curved helices in the middle connecting the upper and lower structural domains, which is typical of GH46 chitosanase. Valine (Val186, V) and tyrosine (Tyr148, Y) located between the structural domains are conserved amino acids, and the groove in the middle is the catalytic cleft, which is the location for binding the substrate chitosan.

Selection of Mutation Sites

In this study, chitosanase CsnB was classified as a member of the GH46 family and confirmed to use an endo-type degradation mechanism. Its primary hydrolysis products are chitobiose ((GlcN)2) and chitotriose ((GlcN)3), and it does not produce the monomer (GlcN). Additionally, CsnB belongs to subclass II of the GH46 family chitosanases, capable of cleaving both GlcN-GlcN and GlcN-GlcNAc bonds. Based on the protein-ligand complex structure and previous successful mutation studies that altered product polymerization, 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).

Design 2.0

Based on the results of the predictive analysis in Learn 1.0, we will perform site-directed mutagenesis on the amino acid sequence to obtain four mutant variants. Next, we analyzed the mutant enzyme activity and hydrolysis products. We chose E. coli as the host for protein expression, and using plasmid pET-28a as the vector, we designed plasmids pET-28a-D78Y, pET-28a-K260Y, pET-28a-P115A, and pET-28a-V186Y containing the mutant genes, and the plasmid maps of the four mutants are shown in Figure 8.

Build 2.0

1.Construction of CsnB mutant plasmid

We used the previously constructed recombinant plasmid PET-28a-CsnB as a template. Primers specific to each desired mutation were employed to perform inverse PCR (6073 bp) on this template to introduce the mutations D78Y, P115A, V186Y, and K260Y into the CsnB gene. Transformed colonies were screened using colony PCR (928 bp) with primers CSNB-CX-F and CSNB-CX-R. Colonies yielding the correct PCR products were cultured, and plasmids were extracted for sequencing verification. Through this process, we successfully obtained the mutant recombinant plasmids PET-28a-CsnB-D78Y, PET-28a-CsnB-P115A, PET-28a-CsnB-V186Y, and PET-28a-CsnB-K260Y.

The construction strategy of the recombinant plasmid and its validation are as follows:

2. Induced expression of mutant enzymes

All recombinant mutant strains were successfully expressed in E. coli BL21(DE3) following IPTG induction. Purification of enzymes was accomplished by Ni-NTA affinity chromatography, and both the unpurified and purified proteins were verified via SDS-PAGE. As illustrated in Figure 10, distinct bands were observed in the unpurified enzyme sample within the molecular weight range of 25 to 35 kDa, which corresponds to the expected theoretical value. After purification, the mutant lanes closely resembled those of CsnB, with a single lane detected at a position consistent with the unpurified enzyme solution.

Test 2.0

1. Enzymatic activity determination of CsnB mutants

The DNS method was used to detect the enzyme activity of CsnB. The standard curve was plotted using the concentration of glucosamine and OD540 as the horizontal and vertical coordinates, respectively (Figure 11a). The enzyme activity of CsnB and its mutants was determined as shown in the figure 11b. 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.

2. Products analysis of mutant enzymes

CsnB and its mutants were incubated with 0.5% colloidal chitosan in an acetic acid-sodium acetate buffer at 50°C and pH 6 for 24 hr. The enzymatic reactions of both the V186Y mutant and the wild-type CsnB resulted in a mixture of chitosan((GlcN)2 and chitotriose((GlcN)3) as the products. However, the final productios of the D78Y and K260Y mutants were primarily composed of (GlcN)2, with minimal (GlcN)3. Due to the higher activity of the D78Y mutant compared to the K260Y mutant, we intend to explore the optimal conditions for the synergistic preparation of chitosan using the D78Y mutant in conjunction with chitin deacetylase. We expected to obtain chitosan with higher concentration and purity in that way.

3. 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 chito-oligosaccharides 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.

4. Analysis of dual enzyme catalytic system

4.1 Comparison of dual-enzyme stepwise and synergistic catalysis

In order to study the mechanism of action of CDA and CsnB(D78Y) in the degradation of colloidal chitin, the effect of CDA and CsnB(D78Y) in stepwise or synergistic catalysis for 10 h was compared and analyzed with 1% colloidal chitin as the substrate, and the results were shown in the Figure 14. Under the same reaction conditions, CDA and CsnB(D78Y) co-catalyzed for 10 h can continuously degrade 60% of colloidal chitin, and the degradation rate under synergistic catalysis was higher than that of the stepwise catalysis of the two enzymes.

4.2 Optimization of conditions for double enzyme synergistic catalysis

To further explore and optimize the effects of temperature, pH, and the ratio of dual enzyme addition on catalytic activity, corresponding condition optimization experiments were set up.

1. Effect of temperature on dual enzyme synergistic catalysis system

In order to explore the effect of temperature on the dual-enzyme collaborative catalytic system, under the condition of pH 8, different temperatures (40, 50, 60 ℃) and colloidal chitin of 2 g/L, 50 U/mL CDA and CsnB(D78Y) were added to the pure enzyme reaction for 10 h, and then the mass concentration of chitosaccharides was detected by HPLC. As shown in Figure 15, the optimal reaction temperature of the dual-enzyme co-catalytic system is 50 ℃.

2. The effect of pH value on the dual-enzyme synergistic catalysis system

For the effect of pH value on the dual-enzyme co-catalytic system, we added the substrate colloidal chitin of 2 g/L, 50 U/mL CDA and CsnB(D78Y) to the pure enzyme reaction for 10 h under the optimal temperature of 50 ℃, different pH of 6, 7, 8, and detected the mass concentration of the product chitosaccharide by HPLC. As shown in Figure 16, the optimal reaction pH for the dual-enzyme co-catalytic system is 7.

3. The effect of double enzyme addition ratio on dual-enzyme synergistic catalysis system

To examine the influence of the CDA-to-CsnB(D78Y) addition ratio on the collaborative catalytic efficiency of the dual-enzyme system, we conducted enzymatic reactions using varying mixtures of CDA and CsnB(D78Y) at a 1:2, 1:1, and 2:1 ratio. These reactions were carried out under standardized optimal conditions of 50°C and pH 7, utilizing 2 g/L of colloidal chitin as the substrate. The reaction was allowed to proceed for 10 hr, after which the concentration of the produced chitobiose was quantified by HPLC. Figure 17 illustrates that the most effective enzyme combination for the synergistic catalysis system is achieved with a 1:2 ratio of CDA to CsnB(D78Y).

In conclusion, the optimal reaction temperature for the dual-enzyme synergistic catalysis system is 50°C, and the optimal pH value is 7. The addition ratio of the two enzymes has a minor effect on the catalytic system, with the optimal ratio being 1:2. Under the above-mentioned optimal reaction conditions with 2 g/L of colloidal chitin as the substrate, the concentration of the product (GlcN)2 is 1.76 g/L.

Learn 2.0

1.Utilizing AlphaFold3

For protein structure prediction, four mutants were successfully designed. Mutation analyses revealed alterations in CsnB enzymatic activity, with noteworthy enhancements in COS yield observed for CsnBP115A upon hydrolysis. Notably, the enzymatic products of the D78Y and K260Y mutants were predominantly chitobiose, indicating significantly increased product purity compared to the control. The synergistic reaction between the D78Y mutant and CDA demonstrated a substantial enhancement in catalytic efficiency. Under optimal conditions of temperature, pH, and substrate-to-enzyme ratio, the (GlcN)2 was produced at a concentration of 1.76 g/L, yielding chito-oligosaccharides at a higher concentration than that achieved by single-enzyme systems.

2.Future optimization directions:

Based on the above achievements and experiences, our future research directions may include the following:

1) In-depth mechanism research: Through molecular dynamics simulation, explore how mutation sites affect the structural stability and substrate binding ability of enzymes. Explore the deep mechanism between enzyme activity and product purity.

2) Design new mutation sites, especially amino acid residues in the active center and substrate channel region, hoping to obtain enzymes with higher activity or higher purity of synthetic products.

3) Product purification and functional evaluation: Purify and characterize the generated chitosan oligosaccharides, analyze their molecular weight distribution and purity, and evaluate their biological activity and application potential in medicine, food and other fields.

4) Explore new synergistic systems: multi-enzyme synergy: Introduce other related enzymes to cooperate with the optimized CsnB mutant and CDA to build a more efficient biocatalytic system.

5) Development of green treatment methods for shrimp and crab shell waste, and further transformation and application