Difference between revisions of "Part:BBa K5520012"

Revision as of 06:35, 30 September 2024

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

Plasmid construction

The recombinant plasmid PET-28a-CsnB was generated and utilized as the template DNA for reverse PCR using the primers V-F-Y and V-R-Y, targeting a 6073bp fragment. Subsequently, DpnI enzyme treatment was employed to remove the template DNA, following which the digested products were transformed. To confirm successful transformation, colony PCR was conducted on the transformed colonies into E. coli BL21 (DE3) using the primers CSNB-CX-F and CSNB-CX-R to amplify a 928-bp fragment. Positive colonies displaying the expected PCR product were selected for transfection. After plasmid extraction from these colonies, sequencing was performed to validate the correct insertion, resulting in the confirmation of the recombinant plasmid PET-28a-CsnBD78Y.

Test of CsnB-V186Y protein

1. SDS-PAGE

Recombinant mutant strain with PET-28a-CsnB-V186Y was successfully expressed in E. coli BL21(DE3) following IPTG induction. Purification of CsnB-V186Y 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 5 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 5 closely resembled that of CsnB, with a single lane detected at a position consistent with the unpurified enzyme solution.

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 V186Y mutant is reduced by 43.2%.

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.

4. Analysis of Enzyme Activity and Product Change Mechanisms

In the V186Y mutant, 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. The increased steric hindrance obstructs the binding of the enzyme to the substrate, reducing the enzymatic activity of this mutant.