Cyclodextrin glycosyltransferase mutant under weakened inhibition of beta-cyclodextrin
A glucose-based and mutant technology is applied in the fields of genetic engineering and enzyme engineering, which can solve the problems of low starch conversion rate, decreased CGT enzyme cyclization activity, and limited application of cyclodextrin, and achieves the advantages of improving yield and reducing inhibitory effect. Effect
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Embodiment 1
[0026] Example 1 Determination of Mutation Sites
[0027] The substrate binding model of CGTase in the cyclization reaction process is: the first step is amylose binding to maltosyl binding site 1 (MBS1), then through maltosyl binding site 2 (MBS2), and with the help of The amylose chain is positioned on the subsite through the substrate-binding groove. After the amylose chain is severed, the cyclization is completed with the help of amino acid residues near the subsite, and the resulting cyclodextrin is quickly separated from the enzyme protein. However, during the enzyme-catalyzed reaction, cyclodextrin can combine with the enzyme at MBS1, MBS2 and the active center by hydrogen bonding or hydrophobic interaction, thereby causing product inhibition.
[0028] According to the analysis of the PDB crystal structure (PDB code 1D3C) of the cyclodextrin glucosyltransferase derived from B.circulansstrain251, it can be seen that the CGTase with linear mixed inhibition has multiple am...
Embodiment 2
[0029] The preparation of embodiment 2 mutants L600Y, L600E and L600R
[0030] (1) Site-directed mutation
[0031] Using rapid PCR technology, site-directed mutagenesis was carried out with the expression vector pST / cgt containing the wild CGTase gene as a template.
[0032] Primers for introducing the Leu600Tyr mutation:
[0033] Forward primer: 5'-CGACGACGGCC TAT GGGCAAAAT-3', the underline is the mutant base,
[0034] Reverse primer: 5'-ATTTTGCCC ATA GGCCGTCGTCG-3', the underline is the mutant base;
[0035] Primers for introducing the Leu600Glu mutation:
[0036] Forward primer: 5'-CGACGACGGCC GAA GGGCAAAAT-3', the underline is the mutant base,
[0037] Reverse primer: 5'-ATTTTGCCC TTC GGCCGTCGTCG-3', the underline is the mutant base;
[0038] Primers for introducing the Leu600Arg mutation:
[0039] Forward primer: 5'-CGACGACGGCC CGT GGGCAAAAT-3', the underline is the mutant base,
[0040] Reverse primer: 5'-ATTTTGCCC ACG GGCCGTCGTCG-3', the underline is ...
Embodiment 3
[0046] Embodiment 3 Enzyme assay analysis
[0047] (1) Determination of enzyme activity
[0048] Determination of β-cyclization activity: Take 0.1 mL of appropriately diluted enzyme solution, add 0.9 mL of 1% (w / v) maltodextrin (DE=5) prepared in advance with 50 mM phosphate buffer (pH 6.0) In the test tube of the solution, after reacting at 50°C for 10 min, add 3.5 mL of 30 mM NaOH and 0.5 mL of 5 mM NaOH 2 CO 3 The solution was reacted with 0.02% (w / v) phenolphthalein solution, kept at room temperature for 15 minutes, and the absorbance was measured at 550 nm. The inactivated enzyme was used as a blank. One enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol β-cyclodextrin per minute under the above conditions.
[0049] (2) Enzyme product inhibition comparison
[0050] Under certain reaction temperature and pH conditions, add a quantitative enzyme solution, use maltodextrin (DE=5) solution as the reaction substrate, select the reaction sub...
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