A double fructoanhydrolase mutant c387a with enhanced enzyme activity
A technology of C387A and double fructose, which is applied in the field of genetic engineering, can solve the problems of no levan, less research on levan synthesis, restrictions on the mass production of inulin and related property research, and achieve the effect of improving catalytic efficiency
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Embodiment 1
[0020] Embodiment 1: Preparation and identification of AcDFA-IIIase
[0021] (1) Preparation of AcDFA-IIIase
[0022] The gene encoding double fructoanhydrolase derived from Arthrobacter chlorophenolicus A6 was molecularly cloned and expressed in Escherichia coli engineering bacteria. The genome number of this strain in GenBank is CP001341, and the number of the enzyme gene is Achl_2895, the full length of the gene is 1338 nucleotides, see SEQ ID No: 1 in the sequence list. The protein number compiled by the gene is ACL40859.1, with a total of 445 amino acids, as shown in SEQ ID No: 2 in the sequence listing.
[0023] The plasmid vector used for cloning was pET-22b(+), and the constructed recombinant expression plasmid was named pET-22b(+)-AcDFA-IIIase. Restriction sites Nde I and Xho I were added to the two segments of the gene fragment respectively, and 6 histidine tags were added to the C-terminus for separation and purification experiments by nickel ion affinity chromato...
Embodiment 2
[0026] Embodiment 2: Preparation and expression purification of AcDFA-IIIase enzyme mutant
[0027] (1) The preparation method of the mutant C387A of AcDFA-IIIase enzyme
[0028] ① Determine the mutation site on the basis of the AcDFA-IIIase enzyme simulation structure of Arthrobacter chlorophenolicus A6; the mutation site is located in the lid region of the structure, and the lid is the region of the amino acid sequence at positions 378-402.
[0029] ②Design primers for site-directed mutagenesis: use the vector pET-22b(+)-AcDFA-IIIase carrying the AcDFA-IIIase gene as a template for site-directed mutagenesis to construct the mutant plasmid pET-22b(+)-C387A; the mutation primers are as follows, underlined is the mutation point:
[0030]The forward mutation primer is shown in SEQ ID NO:5, wherein GG CGG AA, the underline is the mutant base;
[0031] The reverse mutation primer is shown in SEQ ID NO: 6, wherein CC GCC TT, the underline is the mutated base.
[0032] ③PCR r...
Embodiment 3
[0042] Embodiment 3: Determination of AcDFA-IIIase and its mutant C387A enzyme activity
[0043] This embodiment compares the change of enzyme activity before and after the mutation, the enzyme activity assay method: 1mL reaction system, including the final concentration of 10gL -1 Substrate bisfructose anhydride, pH 6.5 phosphate buffer at a final concentration of 50mM and 100nmL -1 For pure enzyme, react at 55°C for 10 minutes, and stop the reaction in a boiling water bath for 10 minutes. After centrifugation at 18000×g for 20min at 4°C, the reaction solution was filtered through a 0.22 μm filter membrane into a liquid phase vial, and the high-performance liquid phase was equipped with a sugar column Sugar-Pak I (4.6mm×250mm, Waters, MA, USA) and a differential refraction display. for the detection of inulin. 1U of enzyme activity is defined as the amount of enzyme required to produce 1 μmol of inulin per minute at pH 6.5 and 55°C.
[0044] Enzyme activity comparison: the...
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