Amylosucrase mutant for improving output of turanose
An amylosucrase and mutant technology, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems of reducing the concentration of sucrose and affecting the final yield of turanose, and achieves the effect of improving the conversion rate.
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Embodiment 1
[0038] Embodiment 1: Construction of recombinant bacteria
[0039] According to the amino acid sequence of amylosucrase dgas on NCBI (PDB ID: 3UER), the dgas gene encoding amylosucrase was synthesized by chemical synthesis. The plasmid used to construct recombinant E. coli was pET24a(+) with T7 promoter. The pET24a(+) plasmid and dgas gene were double-digested with Nde I and Hind III, respectively, and the digested products were recovered by tapping the rubber, then ligated with T4 ligase, and the ligated products were transformed into E.coli JM109 competent cells to obtain recombinant cells. The recombinant cells were cultured at 37°C for 8 hours, and the transformants were picked and cultured with shaking in LB liquid medium (containing 30 mg / L kanamycin), the plasmid was extracted, and the expression plasmid dgas / pET24a(+) was obtained after enzyme digestion verification.
[0040] Transform the plasmid dgas / pET24a(+) into E.coli BL21(DE3) host bacteria, spread on LB plates...
Embodiment 2
[0041] Embodiment 2: the preparation of amylosucrase mutant
[0042] According to the dgas gene sequence of the amylosucrase synthesized by the chemical synthesis method in Example 1, the primers for introducing the D447V mutation were designed and synthesized, the dgas gene of the amylosucrase was subjected to site-directed mutation, the DNA sequence after the mutation was determined, and the original code 447 was identified The codon for Asp at bit 1 is changed to the codon encoding Val. The expression vector carrying the mutated gene encoding amylosucrase is introduced into Bacillus subtilis, Escherichia coli or Bacillus pumilus for expression to obtain the single mutant amylosucrase.
[0043]Site-directed mutation of the single mutation D447V; rapid PCR technology was used to use the expression vector dgas / pET24a(+) carrying the wild-type amylosucrase gene as a template. The primers for introducing the D447V mutation are:
[0044] The nucleotide sequence is the forward p...
Embodiment 3
[0052] Example 3: Fermentation of amylosucrase mutants
[0053] Pick the recombinant strain E.coli J BL21(DE3) / dgas / pET24a(+)-D447V and grow it in LB liquid medium (containing 30μg / mL kanamycin) for 8-10h. Received into TB medium (containing 30 μg / mL kanamycin), cultured OD in a shaker at 37°C 600 After reaching 0.2, add 0.4mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for induction, ferment at 25°C for 24h, centrifuge the fermentation broth at 4°C, 8000rpm for 10min and discard clear, collect the bacteria to 30OD, and reconstitute with pH 7.4 PBS buffer, then perform high-pressure homogenization to break the wall, centrifuge at 8000rpm for 10min, and collect the supernatant to obtain the mutant crude enzyme solution.
[0054] Using the same method, the broken enzyme solution obtained by fermenting the recombinant strain E.coli J BL21(DE3) / dgas / pET24a(+) in Example 1 was used as the wild enzyme crude enzyme solution.
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