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Recombinant ribose-5-phosphate isomerase and application thereof

A phosphate isomerase and ribose technology, applied in the field of bioengineering, can solve the problems of excessive generation of by-products, non-reusable substrates, complex purification steps, etc., and achieves the effect of good heat resistance

Inactive Publication Date: 2012-12-26
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The production of D-allose by chemical method requires complex purification steps, which has the disadvantages of more by-products, lower yield and non-reusable substrates.

Method used

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  • Recombinant ribose-5-phosphate isomerase and application thereof
  • Recombinant ribose-5-phosphate isomerase and application thereof
  • Recombinant ribose-5-phosphate isomerase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of recombinant expression vector pET22-tlRpib and acquisition of genetic engineering strain BL21(DE3) / pET22-tlRpib

[0036] Synthetic with NdeI / XhoI restriction siteThermotoga lettingae The ribose-5-phosphate isomerase gene 435bp was ligated into pET-22b (+) to obtain the pET22-tlRpib plasmid.

[0037] Add 2 μL pET22-tlRpib plasmid to 100 μL BL21 competent cell suspension, and mix gently.

[0038] Let it stand in an ice bath for 30 minutes, evenly spread the LB plate containing 100 μg / mL ampicillin, and culture it upside down at 37°C for 12-15 hours to obtain a positive clone, named recombinant Escherichia coli BL21(DE3) / pET22-tlRpib.

[0039] Sequencing of the recombinant plasmid pET22-tlRpib showed that the inserted fragment was a 435bp DNA fragment encoding a protein consisting of 145 amino acids.

Embodiment 2

[0040] The acquisition of embodiment 2 recombinant tl-Rpib enzyme

[0041] The single clone of the genetically engineered strain BL21(DE3) / pET22-tlRpib was picked to the LB seed medium containing 50 μg / mL of ampicillin, and cultured with shaking at 200 rpm at 37°C for 12 hours.

[0042] The seed culture solution was added to the fermentation LB medium at a ratio of 1:50 and cultured at 37°C until OD600=0.8-2.0, and IPTG was added to a final concentration of 0.1mM.

[0043] Incubate at 100 rpm at 28°C for 6 hours, and collect the bacterial liquid. The cells were collected by centrifugation at 10,000 rpm at 4°C for 10 min, the supernatant was discarded, and washed 3 times with normal saline.

[0044] According to the ratio of 1g bacteria: 20mL equilibration buffer, add buffer to sonicate cells, supernatant is centrifuged and passed through Chelating Sepharose Fast Flow affinity medium chromatography column, low imidazole elution buffer elutes impurity protein, high imidazole el...

Embodiment 3

[0050] Example 3 The influence of pH on the enzyme activity of recombinant tl-Rpib

[0051] The substrate D-psicose concentration was 50mM, the reaction temperature was 65°C, and the reaction time was 15min. Three kinds of buffer systems, citrate buffer (50mM, pH 5-5.5), phosphate buffer (50 mM, pH 6.0 -7.5) and Tris hydrochloric acid buffer (50 mM, pH 8.0-9.0) were reacted. The optimal pH value of the recombinant tl-Rpib enzyme is 8, and 80% of the maximum enzyme activity can be retained between pH 6-9, see image 3 .

[0052] Dissolve the recombinant tl-Rpib enzyme powder in phosphate buffer solution with pH values ​​of 6, 7, and 8 respectively, and place it at room temperature for a certain period of time, adjust the pH of the enzyme solution to 8, and adjust the volume to detect its enzyme activity. Figure 4 The results showed that the recombinant tl-Rpib enzyme had good stability at pH 8, and kept 60% of the enzyme activity after 24 hours at room temperature.

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Abstract

The invention relates to recombinant ribose-5-phosphate isomerase and application of the ribose-5-phosphate isomerase, belonging to the technical field of biological engineering. A recombinant expression carrier pET22-tlRpib is constructed, and a gene-engineered strain BL21(DE3) / pET22-tlRpib is obtained; and the recombinant Thermotogalettingae ribose-5-phosphate isomerase is obtained by fermentation, and recombinase tl-Rpib with purity more than 95% is obtained by separation and purification of ni<2+> affinity column. The recombinase can be used for catalyzing ketoaldehyde isomerization reaction between D-allulose and D-allose to produce D-allose, and the conversion rate can reach 31% after the reaction reaches the equilibrium. The enzyme has excellent heat resistance, does not need metal ions to participate in catalytic reaction, is low in separation and purification difficulty of the product, can keep more than 80% maximal enzyme activity within wider pH range (6-9), and can be applied to large-scale industrial production of rare sugar D-allose.

Description

technical field [0001] The present invention relates to a Thermotoga lettingae Soluble expression of ribose-5-phosphate isomerase gene (EC 5.3.1.6, ribose- 5-phosphate isomerase, RPI) from bacteria in E. coli BL21(DE3), and the isolation and purification of the expression product recombinant enzyme The application in the production of sugar D-allose (D-allose) belongs to the technical field of bioengineering. Background technique [0002] D-psicose is an epimer at the C-3 position of D-glucose and an aldose isomer of D-psicose. D-allose, a rare sugar that is non-toxic, calorie-free, and has a wide range of physiological functions, is becoming a research hotspot of functional rare sugars in recent years. The physiological functions of D-allose include: inhibiting the occurrence of cancer caused by oxidation, and inhibiting cancer cell lines from different sources; acting as an antioxidant to inhibit the oxidative damage caused by reactive oxygen species; inhibiting high sod...

Claims

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Application Information

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IPC IPC(8): C12N15/61C12N9/90C12N15/70C12P19/24C12P19/02C12R1/01
Inventor 江波沐万孟冯再平张涛
Owner JIANGNAN UNIV
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