Difference between revisions of "Part:BBa K5520012"

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pT7-LacO-His- CsnBV186Y

This part contains BBa_K2406020, BBa_B0034, BBa_K5520008, BBa_K731721. We placed the mutant gene CsnBV186Y into the plasmid we described above.

Sequence and Features

Usage and Biology

Part Collection Description

There are 11 parts related to chitosanase (CsnB). The part list is as below. All of these parts contribute to expressing CsnB or CsnB mutant protein. So they make up a part collection.

Chitosanases play a crucial role in the hydrolysis of chitosan to produce chitooligosaccharides (COS), which have significant applications in medicine, agriculture, and food industries. In this study, we aimed to enhance the activity and product specificity of chitosanase CsnB from Bacillus sp. BY01. We heterologously expressed CsnB in Escherichia coliBL21(DE3) and observed low hydrolysis efficiency and a mixture of COS products. Using protein engineering guided by AlphaFold3 and molecular docking, we designed four mutants: D78Y, P115A, V186Y, and K260Y.

1 Recombinant expression plasmids for CsnB

We obtained chitosanase (CsnB) gene from Marine Bacterium Bacilius SP. BY01 by PCR amplification. Then, we used the PET-28a plasmids kept in the laboratory as a template and primers were designed to ligate the CsnB gene to the PET-28a linearized vector by Gibson assembly method to obtain recombinant expression plasmids PET-28a-CsnB. The plasmid mapping was constructed as follows:

2 Heterologous expression and characterization of CsnB

We transformed PET-28a-CsnB recombinant plasmids into E.coli BL21 (DE3) for overexpression of CsnB. As shown in Figure 2a, 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 2a).

The soluble sugar hydrolyzed by CsnB from chitosan was analyzed by TLC. As shown in the Figure 2b, 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.

From the above results, we found that 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.

3 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 3, 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. 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).

4 Recombinant expression plasmids for CsnB mutants

We will perform site-directed mutagenesis on the amino acid sequence to obtain four mutant variants. Next, we analyzed 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.

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 5b-c, 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.

5 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 6a). The enzyme activity of CsnB and its mutants was determined as shown in the figure 6b. 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.

6 Product 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.

7 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.

8 Synergistic Catalysis with CDA

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 9. 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.

9 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. Under optimized conditions: - Temperature: 50°C - pH: 7.0 - Enzyme Ratio (CDA：(D78Y)): 1:2

The synergistic catalysis resulted in a (GlcN)₂ concentration of 1.76 g/L from 2 g/L colloidal chitin substrate, exceeding the yield obtained with the single-enzyme system. The dual-enzyme system demonstrated higher degradation efficiency compared to stepwise catalysis.

Conclusion

This work demonstrates that protein engineering of chitosanase CsnB can effectively improve both enzymatic activity and product specificity. The mutants developed, particularly D78Y and P115A, offer valuable insights for industrial applications aiming to produce specific COS. The synergistic catalytic system with CDA presents a promising approach for efficient chitin degradation. Future studies could focus on in-depth mechanistic analysis, exploring additional mutation sites, and scaling up the process for industrial applications.