Part:BBa_K5520013
pT7-LacO-His- CsnBK260Y
This part consists of BBa_K2406020, BBa_B0034, BBa_K5520008, BBa_K731721.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 149
Illegal NheI site found at 897 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 182
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 641
Usage and Biology
Plasmid construction
Test of CsnB-K260Y protein
1. SDS-PAGE
Recombinant mutant strain with PET-28a-CsnB-K260Y was successfully expressed in E. coli BL21(DE3) following IPTG induction. Purification of CsnB-K260Y 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 2 was observed in the unpurified enzyme sample within the molecular weight range of 30 kDa, which corresponds to the expected theoretical value. After purification, mutant lane 2 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 K260Y mutant shows the lowest enzyme activity, with a 69.7% decrease compared to the wild type.
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. However, the final productions of the K260Y mutant was primarily composed of (GlcN)2, with minimal (GlcN)3.
4. Analysis of Enzyme Activity and Product Change Mechanisms
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.
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