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Preparation method of 4-phosphoric acid erythrose

A technology of red fresh sugar phosphate and fructose phosphate is applied in the application field of biotechnology, which can solve the problems of high requirements for reaction equipment, complex product components, low yield and the like, and achieves low requirements for reaction equipment, high product yield and high yield. high effect

Active Publication Date: 2013-07-10
ANHUI BBCA FERMENTATION TECH ENG RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above methods all need to be carried out under high temperature and high pressure conditions, which require high reaction equipment, complex product components, and low yield

Method used

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  • Preparation method of 4-phosphoric acid erythrose
  • Preparation method of 4-phosphoric acid erythrose
  • Preparation method of 4-phosphoric acid erythrose

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0024] Example 1 Cloning of the Histoplasma dermatitidis (ATCC 18188) transketolase gene (tktA)

[0025] With 0.5 μg of Histoplasma dermatitidis genomic DNA as PCR reaction template, according to the sequence of transketolase (as shown in SEQ ID NO.1) design forward primer tktA-F is 5'-TTCGGGATCCTACCCCTCGATGTGACTGCA-3', reverse Primer tktA-R is 5′-TTGCCTCGAGAATCTACTAAACGACCTTCCGC-3′, in which the parts in italics are restriction enzyme cutting sites BamHI and XhoI respectively. The design of the 3' primer ensures that the expression product contains 6 histidine tags. The PCR reaction was carried out in a total volume of 50 μL, and the reaction conditions were 30 cycles of denaturation at 94°C for 5 min, followed by denaturation at 94°C for 50 s, annealing at 56°C for 1 min, and extension at 72°C for 2 min. Take 5 μL of PCR amplification products for agarose gel electrophoresis verification ( figure 1 ). Take 100 μL of the PCR product for agarose gel electrophoresis, and rec...

Embodiment 2

[0026] Example 2 Construction of tktA expression vector and its expression in E.coli JM109

[0027] 1. Construction of tktA gene: Gel recovery of the PCR product in Example 1. The purified PCR product and the pET-22b(+) vector were digested with BamH I and XhoI respectively, and recovered by the gel extraction kit, then ligated (16°C, 16h), transformed into E. coli DH5a competent cells, and selected Positive clones were subjected to enzyme digestion verification analysis with BamHI and XhoI after plasmid extraction ( figure 2 ), and then carry out DNA sequencing identification after verification. Construct pET-22b(+)-tktA expression vector.

[0028] 2. Expression of tktA gene: Transform Escherichia coli JM109 competent cells with the pET-22b(+)-tktA expression plasmid verified by sequencing; pick positive clones, inoculate in LB medium containing kanamycin, shake at 37°C Culture OD 600 When the temperature reaches 0.6-0.8, add IPTG with a final concentration of 1 mmol / L, ...

Embodiment 3

[0029] Example 3 Separation and purification of TK and detection of enzyme activity

[0030] 1. the purification of Histoplasma dermatitidis transketolase: fermented liquid in embodiment 2 is collected thalline through centrifugation, with 50mmol / L, pH 8.0Tris-HCl (containing 1mmol / L) buffer suspension (wet thallus The ratio of the solution to the buffer solution is 1 g: 5 mL), the cells were disrupted by ultrasonic waves in an ice bath, and the supernatant was collected after centrifugation. Pack the column with NTA medium, wash with 2 times the column volume of deionized water, add NiSO 4 The solution was added to the NTA medium to saturate the binding degree, and the column was washed with 5 times column volume of NAT-1 buffer (20mM Tris-HCl pH7.9, 0.5M NaCl). Add the supernatant obtained above to Ni 2+ -In the NTA resin column, after repeated addition 3 to 5 times, wash the medium with 5 times the column volume of NAT-2 buffer (NAT-0 buffer contains 80mmol / L imidazole) t...

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Abstract

The invention relates to a preparation method of 4-phosphoric acid erythrose, comprising the following steps: immobilizing heat-resistant transketolase at the temperature of 55-65 DEG C; and then taking 6-fructose phosphate and 3-glyceraldehyde phosphate as raw materials to prepare the 4-phosphoric acid erythrose by enzymatic reaction. In the preparation method, the heat-resistant and specifically transformed transketolase of the 4-phosphoric acid erythrose is prepared by adopting a molecular biology technology, and the 6-fructose phosphate and the 3-glyceraldehyde phosphate are taken as the raw materials to efficiently prepare the 4-phosphoric acid erythrose by adopting an immobilized enzyme technology. The preparation method is simple and practical.

Description

technical field [0001] The invention relates to the application field of biotechnology, in particular to a method for preparing erythrose 4-phosphate by using molecular biology technology. Background technique [0002] One way in which glucose is oxidatively broken down is the pentose phosphate (ppp) metabolic pathway. Since this pathway is initiated by glucose-6-phosphate (G-6-P), it is also known as the hexose phosphate bypass. This pathway is carried out in the cytoplasm of the cell. The entire metabolic pathway goes through a series of enzymatic reactions in the oxidation stage and the non-oxidation stage, and at the same time produces NADPH+H + , providing restoring power. Specifically, the first stage starts with the dehydrogenation of G-6-P to generate 6-phosphogluconolactone, then hydrolyzes to generate 6-phosphogluconic acid, and then dehydrogenates and decarboxylates to generate 5-phosphate ribulose; the second stage The stage is that ribulose 5-phosphate underg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/02
Inventor 李荣杰薛培俭尚海涛徐斌段绪果薛亮胡长浩
Owner ANHUI BBCA FERMENTATION TECH ENG RES
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