68th and 109th double mutant enzyme of D-psicose 3-epimerase and application thereof
A technology of epimerase and psicose, which is applied in the field of genetic engineering of enzymes, can solve problems such as limiting the scope of application, and achieve high catalytic activity and improved functions
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Embodiment 1
[0028] Example 1: DPE enzyme site-directed mutation analysis and mutant preparation method
[0029] Through the analysis of the 3D spatial structure of DPE enzyme, it was found that G109 is located in the β-turn region of the active site, and the stability of the enzyme molecule can be changed by changing the hydrophobicity of the 109-position amino acid. Therefore, the weaker hydrophobic glycine Gly was replaced by the stronger hydrophobic proline Pro to increase its thermal stability. Y68 is located in the active pocket. By changing the amino acid at position 68, the shape of the hydrophobic pocket can be changed, thereby changing the enzyme activity. The study found that replacing the 68-position tyrosine Tyr with isoleucine Ile can increase its catalytic activity.
[0030] Construction of pet22b(+)-Y68I / G109P mutant plasmid by rapid PCR site-directed mutagenesis;
[0031] The construction of the pet22b(+)-Y68I / G109P mutant plasmid uses the pet22b(+)-cb-dpe plasmid as a t...
Embodiment 2
[0050] Embodiment 2: Clostridia ( Clostridium boltae ) Expression and purification method of DPE enzyme mutant.
[0051] The mutant plasmid pet22b(+)-Y68I / G109P verified by sequencing was transformed into E. coli BL21(DE3) cells, and the positive transformants were picked and cultured in LB medium at 37°C and 200rpm shaking overnight, and then inserted into LB medium at 37°C Cultivate for 3-4 hours until the OD value is 0.6-0.8, cool down to 25°C, and add IPTG at a final concentration of 0.8mM to induce for 8 hours.
[0052] The fermentation broth was centrifuged at 4°C and 10000rpm for 20min to collect the bacterial cells. Add 15 mL Binding Buffer (50 mM Na 2 HPO 4 , 50 mM NaH 2 PO 4 , 500 mM NaCl, adjust the pH to 7.4) to fully resuspend the bacteria, then place the centrifuge tube in an ice bath and put it into an ultrasonic cell disruptor. The conditions for ultrasonic disruption are: working time 1 s, stop time 2 s, total 20 min. Centrifuge the broken solution at l...
Embodiment 3
[0054] Example 3: Thermostability of DPE enzymes at 55°C
[0055] 1. DPE enzyme activity assay method: Add 700 μL of 100 g / L D-fructose to 1 mL of reaction system, 200 μL of diluted enzyme solution, and 100 μL of Co at a final concentration of 1 mM 2+ ion. Incubate at 55°C for 5 min, then boil for 10 min to terminate the enzyme reaction.
[0056] 2. Place the purified DPE enzyme solution in a 55°C water bath to keep it warm, and regularly measure the residual enzyme activity. Draw a curve of residual enzyme activity versus time (e.g. figure 2 ), according to the curve to get the half-life of DPE enzyme at this temperature.
[0057] Table 1. DPE enzyme thermostability at 55°C
[0058] t 1 / 2 (55℃) (min) t 1 / 2 (55℃) increase multiple wild-type DPE 42.9 1 Y68I / G109P 78.3 1.8
[0059] Through measurement, it was found that the half-life of the mutant enzyme Y68I / G109P at 55°C was greatly improved, from 42.9 min to 78.3 min, which was 1.8 tim...
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