Method for preparing new agarose by combined use of agarase, recombinant host cell and application of recombinant host cell and expression vector
A technology of recombining host cells and new agarobiose, which is applied in the fields of enzyme engineering and genetic engineering, can solve the problems of low substrate conversion rate and poor degradation efficiency, and achieve high enzyme activity, strong stability, and improved protein expression level Effect
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Embodiment 1
[0045] Excavation of embodiment 1 agarase AgaA and AgaB and construction of recombinant vector
[0046] According to the genome sequencing and annotation results of the marine bacterium Marinimicrobium sp.H1 that has been screened, the genes of agarase AgaA and AgaB were amplified by designing relevant primers using the genome of the bacterium as a template. Sequence analysis shows that the N-terminus of AgaA protein has a long repeat sequence containing a large number of serine residues. Experiments have proved that when this sequence exists, the protein expression level is very low, and the enzyme cannot be purified by Ni column affinity chromatography . Therefore, a domain optimization strategy was adopted to delete the repeated sequence to construct a recombinant expression vector for the truncated protein. The PCR conditions were: pre-denaturation at 95°C for 3 minutes, followed by 32 cycles of 30s at 95°C, 30s at 55°C, 2 minutes at 72°C, and extension at 72°C for 10 min...
Embodiment 2
[0052] Embodiment 2 Heterologous expression and purification of agarase AgaA and AgaB
[0053] The heterologous expression method of agarase is as follows:
[0054] (a) Induce the expression of the correct plasmid transfection strain Escherichia coli BL21 in the sequencing analysis, spread the plate and culture it in a 37°C incubator for 12 hours;
[0055] (b) Pick a single colony, inoculate it into a 30 mL test tube containing 5 mL of fermentation medium, and incubate with shaking at 37°C for about 12 hours;
[0056] (c) Inoculate 0.5% of the inoculum into a 250mL Erlenmeyer flask containing 100mL of fermentation medium, and culture for 3-4h at 37°C and 200r / min;
[0057] (d) OD of the bacteria solution 600 When it grows to 0.6-0.8, add IPTG (final concentration 0.5mmol / L) and induce at 16°C for about 20-24h.
[0058] (e) Collect the bacteria cultured in step (d), centrifuge at 4°C and 6000r / min for 30min, collect the bacteria, resuspend the bacteria with 2mL of pH 7 buffe...
Embodiment 3
[0065] The influence of embodiment 3 temperature on agarase
[0066] Dilute the purified agarase to an appropriate multiple, take 0.1mL of the diluted enzyme solution and add it to 0.9mL of 0.3% agar substrate (buffer solution 50mM Tris-HCl, pH=7.0), AgaA at 30-80 The optimum temperature was tested between ℃, and the optimum temperature of AgaB was tested between 30-70°C. The reaction solution was placed at the above different temperatures for 20 minutes to measure the enzyme activity and determine the optimum reaction temperature. The enzyme activity measured at the optimum temperature of the enzyme reaction is 100%, the ratio of the enzyme activity to the highest enzyme activity at other temperatures is the relative enzyme activity at this temperature, and the temperature-relative enzyme activity curve is drawn, and the results are as follows figure 1 As shown, the optimum reaction temperature of AgaA is 60℃, and the optimum reaction temperature of AgaB is 40℃.
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