Cubilose acid aldolase mutant as well as coding gene and application thereof
An acid aldolase and mutant technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of high production cost, low conversion rate, low catalytic activity of bird's nest acid aldolase, etc., and reduce production costs. , the effect of improving market competitiveness
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[0027] The general process of the preparation of the bird's nest acid aldolase mutant is as follows: first construct the carrier plasmid containing the parent bird's nest acid aldolase gene, then set the site for site-directed mutation and the amino acid species after mutation, and then synthesize appropriate primers , using the carrier plasmid containing the parent bird's nest acid aldolase gene as a template, PCR amplifies the DNA fragment, assembles the amplified DNA fragment and PCR amplifies the full-length mutant gene. Then the full-length mutated gene is cloned into an appropriate vector and transformed into an appropriate host cell, and a positive clone with bird's nest acid aldolase activity is selected through culturing. Finally, plasmid DNA is extracted from the positive clones, and DNA sequence analysis is performed to determine the introduced mutation. After confirming that the target fragment is inserted into the vector, it can be screened by LB medium to obtain b...
Embodiment 1
[0036] Amplification and cloning of parent bird's nest acid aldolase gene:
[0037] Primers EC-F and EC-R were designed according to the gene sequence of GenBank (GenBank NC_012971). The gene encoding bird's nest acid aldolase was amplified from Escherichia coli BL21(DE3) with primer pair EC-F and EC-R.
[0038] Amplification conditions were: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F, 400 nM primer EC-R, 1.0 U Pfu DNA polymerase (Promega, USA), Pick a little Escherichia coli BL21 (DE3) cells with an inoculation loop, and adjust the reaction volume to 50 ml with sterile water.
[0039] The PCR amplification reaction program was: 95°C for 3 minutes, 40 cycles: 95°C for 50 seconds, 50°C for 30 seconds and 72°C for 1 minute, and finally 72°C for 10 minutes. The amplified product was digested with restriction endonucleases NdeI and AscI and then ligated with ve...
Embodiment 2
[0043] Site-directed mutagenesis of site 25 of bird's nest acid aldolase
[0044] In order to obtain the mutant A25E by mutating Ala(A) at the 25th position in the parental amino acid sequence to Glu(E), the plasmid pRSET-EC in Example 1 was used as a template to design primer pairs 25EF and 25ER (as shown in Table 1 Show).
[0045] The primer pair EC-F and 25ER was used to amplify the F-ER fragment, and the primer pair 25EF and EC-R was used to amplify the EF-R fragment. The specific sequences of primers EC-F and EC-R are shown in Table 1.
[0046] The above amplification reaction conditions are: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 400 nM primer EC-F and 400 nM primer 25ER, or 400 nM primer 25EF and 400 nM primer EC-R, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng pRSET-EC, the reaction volume was adjusted to 50 microliters with sterile water.
[0047] The PCR ampli...
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