Part:BBa_K5520010

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We placed the mutant gene CsnBD78Y into PET-28a vector to obtain enzyme of higher activity under the control of the T7 promoter and terminated transcription by the T7 promoter. We used RBS and 6×His tag to ensure the expression of CsnBD78Y.

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Plasmid construction

The constructed recombinant plasmid PET-28a-CsnB was used as a template, and the template was subjected to reverse PCR (6073 bp) using D-F-Y and D-R-Y as primers. The template DNA was eliminated by digesting the template using DpnI enzyme, and the digested system was transformed into E. coli BL21 (DE3). Colony PCR (928 bp) was performed on the transformed colonies with primers CSNB-CX-F and CSNB-CX-R. Positive colonies with correct colony PCR were transfected, plasmid extracted, and verified by sequencing to obtain the recombinant plasmid PET-28a-CsnBD78Y.

Test of CsnB-D78Y protein

1. SDS-PAGE

Recombinant mutant strain with PET-28a-CsnB-D78Y was successfully expressed in E. coli BL21(DE3) following IPTG induction. Purification of CsnB-D78Y enzyme was accomplished by Ni-NTA affinity chromatography, and both the unpurified and purified proteins were verified via SDS-PAGE. As illustrated in Figure below, distinct lane 4 was observed in the unpurified enzyme sample within the molecular weight range of 30 kDa, which corresponds to the expected theoretical value. After purification, the mutant lane 4 closely resembled that of CsnB, with a single lane detected at a position consistent with the unpurified enzyme solution.

Figure (a) Lane 4 represents CsnB-D78Y unpurified protein and Figure (b) lane 4 represents CsnB-D78Y purified protein.

2. Enzymatic activity determination of CsnB mutant

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 a). 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 D78Y mutant is reduced by 57.0%, respectively.

Figure (a) Glucosamine standard curve and (b) enzyme activity determination of CsnB and CsnB-D78Y.

3. 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.

Figure Analysis of enzymolysis products of enzyme CsnB and its mutantS

4. Analysis of Enzyme Activity and Product Change Mechanisms

In the D78Y mutant, the original residues was 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.

5. 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 below. 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.

Figure Residue of substrate after 10 h reaction with double enzyme step and synergistic catalysis

6. 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, the optimal reaction temperature of the dual-enzyme co-catalytic system is 50 ℃.

Figure Effect of temperature on double enzyme co-catalytic system

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 , the optimal reaction pH for the dual-enzyme co-catalytic system is 7.

Figure Effect of pH on dual-enzyme co-catalytic system

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 below illustrates that the most effective enzyme combination for the synergistic catalysis system is achieved with a 1:2 ratio of CDA to CsnB(D78Y).

Figure Effect of double enzyme addition ratio on double enzyme co-catalytic system

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.