Alginate lyase mutant and application thereof
An algin lyase and mutant technology, which is applied to algin lyase mutants and their application fields, can solve the problems that the algin lyase cannot meet the needs of industrialized production, etc. The effect of purification costs
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Embodiment 1
[0040] Example 1: Construction of alginate lyase mutants
[0041]Mutate the alginate lyase gene Aly01 derived from V.natriegens SK42.001, its nucleotide sequence is shown in SEQ ID NO.1, the full length is 1566bp, encoding 521 amino acids, and the encoded amino acid sequence is shown in SEQ ID NO .2 shown. Amino acids 29, 203, 293, 348, 463, 466 and 481 were mutated respectively to construct the mutants shown in Table 1.
[0042] Design primers containing mutation sites, use the recombinant plasmid pET-28a(+)-Aly01 as a template, and use PCR technology to circularly amplify the gene fragment of the recombinant plasmid with the target mutation, and transform it into E.coli BL21(DE3) to obtain the mutation Recombinant bacteria were spread on LB solid plates containing Kanna resistance, and after overnight culture, single colonies were picked and sequenced, and the recombinant strains were selected for correct sequencing. The primers for site-directed mutagenesis are listed in ...
Embodiment 2
[0049] Example 2: Induced expression and purification of original enzyme Aly01 and mutants
[0050] Extract recombinant plasmids pET28a(+)-Aly01, pET28a(+)-Aly01D29Q, pET28a(+)-Aly01E203A, pET28a(+)-Aly01D293G, pET28a(+)-Aly01E348A, pET28a(+)-Aly01D463N, pET28a(+)- Aly01D466N and pET28a(+)-Aly01E481V were respectively transferred into E.coli BL21(DE3) competent cells, the specific operation was the same as in Example 1, and a single colony was picked and placed in 5mL LB (Kan) liquid medium at 37°C , 200rpm for 12h. Inoculate 1 mL of culture solution into 100 mL of LB (Kan) liquid medium, culture at 37 °C and 200 rpm until OD600 is between 0.6 and 0.8, add IPTG (final concentration is 1 mM), and transfer it to 18 °C, Under the culture condition of 200rpm, induction was carried out for 72h, and the culture medium was centrifuged at 6000rpm for 10min, and the supernatant was taken to measure the activity of alginate lyase, and the original enzyme Aly01 was used as the control. ...
Embodiment 3
[0053] Example 3: Enzymatic properties of original enzyme and mutant D29Q
[0054] Determination of the optimum reaction pH: the original enzyme and the mutant D29Q were added to different pH buffers (sodium citrate buffer for pH 4.0-6.0; phosphate buffer (sodium phosphate buffer) for pH 6.0-8.0; pH7.5-8.5 is Tris-hydrochloric acid buffer solution; pH8.5-10.0 is glycine-NaOH buffer solution) react in sodium alginate substrate with a concentration of 0.5%, the buffer solution concentration is 50mM, and the reaction temperature is 35°C , the reaction time is 10min. Use the above-mentioned improved DNS method to measure the enzyme activity, take the highest enzyme activity as 100%, calculate the relative enzyme activity under different pH conditions, and determine the optimum reaction pH. Such as Figure 4 As shown, the optimal pH of the original enzyme and the mutant were both 8.0.
[0055] Determination of the optimum reaction temperature: Under the above optimum reaction pH...
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