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Mutant of D-psicose 3-epimerase and application of mutant

A technology of epimerase and psicose, applied in the field of enzyme protein engineering, can solve the problems of limited large-scale industrial application and poor thermal stability of wild enzymes

Active Publication Date: 2021-09-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most suitable substrate for DPEase is usually D-psicose, which can catalyze the epimerization of the hydroxyl group on the C3 position of D-fructose to convert it into D-psicose, which is the key to the production of D-psicose Commonly used enzymes, but the thermal stability of DPEase wild enzyme is poor, which is limited to large-scale industrial applications, so how to effectively improve the thermal stability of DPEase is of great significance

Method used

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  • Mutant of D-psicose 3-epimerase and application of mutant
  • Mutant of D-psicose 3-epimerase and application of mutant
  • Mutant of D-psicose 3-epimerase and application of mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: the establishment of DPEase mutant library

[0025] With the plasmid pET-24a(+)-dpe (synthesized from Gene Synthesis Company, both ends enzyme The cleavage sites are NdeI and HindIII) as templates, F1 and R1 as primers to amplify the dpe gene, and connect it to the plasmid pET-20b(+)-His6 by Megaprimer PCR of Whole Plasmid (MEGAWHOP) to construct recombinant Expression vector pET-20b(+)-dpe-His6.

[0026] MEGAWHOP specific steps:

[0027] Reaction system: 2×Super Pfx Master Mix 25μL, 50ng Template, 500ng megaprimer, ddH 2 O to make up to 50 μL.

[0028] Reaction program: 5 min at 72°C; 2 min at 98°C; 25 cycles (30s at 98°C, 30s at 55.5°C, 5 min at 72°C); extension at 72°C for 10 min, and incubation at 8°C.

[0029] Use pET-20b(+)-dpe-His6 as a template, and use F1 and R1 as primers to perform error-prone PCR on the dpe gene. The product is verified by 1% agarose gel electrophoresis. After the verification is correct, the error-prone PCR product The ge...

Embodiment 2

[0036] Example 2: 96-well plate expression of DPEase mutants

[0037] Seed culture: select a single clone from the LB ampicillin resistance plate in Example 1 and inoculate it into a 96-well flat-bottom shallow well plate containing 160 μL LB liquid medium in each well (Amp, 30 μg·mL -1 ), placed in an incubator at 37°C, 750r·min -1 Under the cultivation of 12h.

[0038] 96-well plate expression: transfer the 96-well plate seed solution into a 96-well cone-bottomed shallow well plate containing 160 μL LB medium in each well (Amp, 100 μg·mL -1 ), first at 37°C, 750r·min -1 Cultivate in a modular incubator for 4 hours, add final concentration of 0.4mMIPTG and transfer to 25℃, 750r min -1 Continue to express for 24h.

Embodiment 3

[0039] Example 3: Shake flask expression of KpRD

[0040] The KpRD protein expressed in the shake flask will be used in the high-throughput screening of Example 4.

[0041] The recombinant plasmid pET-24a(+)-kpRD (ribitol 2-dehydrogenase, ChemBioChem, 2015, 16(4), 592–601) was transformed into E.coli BL21(DE3) competent cells (see Stratagene for specific transformation steps Company BL21 (DE3) specification), coated on LB resistance (Kan, 30μg·mL -1 ) plate, cultivated at 37°C for 10-16h, and waited for a single colony to grow; pick a single colony from the plate and put it into 10mL of LB liquid medium (Kan, 30μg·mL -1 ), at 37℃, 200r·min -1 Cultivate for 8-10 hours to obtain seed solution; take the seed solution and transfer it to 200mL TB medium to adjust the initial OD of TB medium 600 0.1; at 37°C, 200r·min -1 cultured to OD 600 To reach 0.5-0.6, add the final concentration of 1mmol L -1 After IPTG, transfer to 25°C, 200r·min -1 Expressed in a shaker for 24h. The ...

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Abstract

The invention relates to a mutant of D-psicose 3-epimerase and application of the mutant. A series of mutants with improved fermentation enzyme activity are obtained by performing directed evolution on wild type DPEase from Clostridium cellulyticum. The fermentation enzyme activity of the DPEase mutant H56Q is 1.61 times that of the wild type; and the half-life period of the mutant H56Q at 55 DEG C is prolonged by 87.5% compared with that of the wild type, the thermal stability is improved, and the good industrial application potential is achieved.

Description

technical field [0001] The invention relates to a mutant of D-psicose 3-epimerase and application thereof, belonging to the technical field of enzyme protein engineering. Background technique [0002] D-psicose is the C-3 epimer of D-fructose. D-psicose was initially isolated from the antibiotic psicose adenosine. D-Psicose is a white tasteless crystal, easily soluble in water, its taste is similar to sucrose, its sweetness is 70% of the traditional sweetener sucrose, but it provides almost no calories, and it is evaluated by the US Food Navigator as the most Potential sucrose substitute. D-Allulose has been identified as food safety grade GRAS by the US Food and Drug Administration (FDA). D-Psicose can enhance insulin resistance, inhibit postprandial blood sugar rise, prevent diabetes; reduce fat accumulation, scavenge active oxygen free radicals, increase intracellular glutathione levels and play a role in neuroprotection and related diseases It plays an important role...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12P19/24C12P19/02
CPCC12N9/90C12Y501/03001C12P19/24C12P19/02
Inventor 吴敬刘展志龙乃云刘姝含
Owner JIANGNAN UNIV
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