Parts

Overview

The aim of our project is to establish a synergistic catalytic system of chitinase and chitosanase, which can realize the one-step reaction to degrade chitin for the single production of chitosan, and to explore the effects of different factors on the dual-enzyme synergistic catalytic system and the mechanism of the synergistic action of the dual-enzymes.

In this project, we successfully constructed 14 parts. We constructed BBa_K4885000 to enhance the expression of chitin deacetylase (CDA). We constructed BBa_K4885001 to enhance the expression of chitosanase (CsnB). In order to enhance the enzymatic activity of CsnB and to improve the uniqueness of the product, we analyzed the protein structure and mechanism of CsnB and modified the protein. By this approach, we directly or indirectly enhanced chitosan production.

Part Collection

There are 11 parts related to chitosanase (CsnB). The part numbers are BBa_K5520001, BBa_K5520003, BBa_K5520005, BBa_K5520006, BBa_K5520007, BBa_K5520008, BBa_K5520009, BBa_K5520010, BBa_K5520011, BBa_K5520012, BBa_K5520013. 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. 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. The enzyme activity of CsnB and its mutants was determined as shown in the figure. 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. 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.

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. 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 4. 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.

5. 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:

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.