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Novel D-allulose 3-epimerase and application thereof

A technology of epimerase and psicose, applied in the field of genetic engineering, can solve the problems of unfavorable cost control and low thermal stability of D-psicose, and achieve low requirements for production conditions, high thermal stability, The effect of high conversion rate

Active Publication Date: 2019-02-05
JILIN COFCO BIOCHEM +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Wherein, the recombinant escherichia coli containing the dpe gene of Clostridium cellulolyticum and Agrobacterium tumefaciens has higher conversion rate of D-fructose and D-psicose, about 33%, but the latter is under the optimal reaction condition ( 50℃) half-life is only 63.5min
[0005] In addition, the current epimerase has the problem of low thermal stability under the optimal reaction conditions, which is not conducive to the cost control of industrial large-scale production of D-psicose

Method used

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  • Novel D-allulose 3-epimerase and application thereof
  • Novel D-allulose 3-epimerase and application thereof
  • Novel D-allulose 3-epimerase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0031] Example 1D-Expression of psicose 3-epimerase (MesDPE)

[0032]In the database, a potential thermostable sugar isomerase was searched and screened again, and a sugar phosphate isomerase from Mesoaciditoga lauensis was found. Its protein sequence is shown in SEQ ID NO: 1, and it is speculated that it has Ketose 3-epimerase activity. For high-efficiency expression in Escherichia coli, the coding sequence was optimized and screened through experiments to obtain the coding sequence shown in SEQ ID NO.2.

[0033] The construction of the Escherichia coli expression strain comprising SEQ ID NO.2 is as follows:

[0034] The coding sequence shown in SEQ ID NO.2 was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd., and a BamH I restriction site was added at its 5' end, and a histidine tag was added before the stop codon, and at the 3' end Add Pst I and Not I restriction sites. The sequence was ligated to pET 30a (+), pTac28 and pTST vectors (preserved in our laboratory) ...

Embodiment 2

[0038] The enzyme activity assay of embodiment 2 recombinant MesDPE

[0039] 1. The standard reaction system is as follows (1 mL): 143 μL purified enzyme MesDPE (final concentration: 0.02 mg / mL); 100 μL D-fructose solution (final concentration: 50 mg / mL); 10 μL Co 2+ (final concentration is 0.1mM CoCl 2 ); 690 μL Tris-HCl buffer (pH 8.0). The reaction conditions are: react in a water bath at 55°C for 20 minutes, and then treat in a boiling water bath for 5 minutes to inactivate the activity of the enzyme.

[0040] 2. The reaction system was filtered with a 0.45 μm microporous membrane, and the filtrate was used for high performance liquid phase analysis. The high performance liquid chromatography conditions are as follows: Agilent 1260HPLC and G1362A RID detector; analytical column: Waters Sugar-Pak I, 6.5×300mmcolumn; mobile phase: water; flow rate: 0.4mL / min; column temperature: 80°C; detector: RID , the detector temperature was 55°C; the sample volume was 20 μL.

[0041...

Embodiment 3

[0043] Embodiment 3 Identification of recombinant MesDPE enzymatic properties

[0044] 1. Determination of metal ion dependence: The reaction system was configured according to the final concentration of substrate fructose of 50 mg / mL, the final concentration of metal ions of 1 mM, and the final concentration of enzyme of 0.02 mg / mL; carried out according to the reaction conditions in Example 2. like Figure 5 As shown, the activity of MesDPE mainly depends on Co 2+ , followed by Mn 2+ .

[0045] 2. The influence of pH on enzyme activity: according to the standard reaction among the embodiment 2, take 50mg / mL fructose as substrate, Co 2+ The final concentration is 0.1mM, and the following buffers are used respectively: 50mM sodium dihydrogen phosphate-sodium hydroxide buffer solution of pH5-7; 50mM Tris-HCl buffer solution of pH7.5-9.5; according to the reaction conditions in Example 2 . The results show( Image 6 ), MesDPE had the highest activity at pH 6.0; as the pH v...

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Abstract

The invention belongs to the technical field of genetic engineering and in particular relates to novel D-allulose 3-epimerase, DNA (Deoxyribonucleic Acid) for encoding the novel D-allulose 3-epimerase, an expression vector and a transformant containing the DNA and application of the enzyme to production of D-allulose. The D-allulose 3-epimerase is derived from thermoacidophile mesoaciditoga lauensis of a deep-sea spring and has relatively high conversion rate and thermal stability when being used for converting D-fructose into the D-allulose.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a D-psicose 3-epimerase derived from Mesoaciditoga lauensis and an application thereof. Background technique [0002] With the acceleration of urbanization, increasing environmental pollution, changes in people's lifestyles, and population aging, the incidence of chronic metabolic diseases such as obesity and diabetes has risen sharply in my country and the world. Excessive consumption of carbohydrates is an important cause of obesity and diabetes. How to maintain the level of sweetness that people are accustomed to, while reducing the absorption of sugar in the intestines and reducing energy intake is one of the current research hotspots in the field of nutrition and medicine, and it is also an important issue that the food industry needs to solve urgently. [0003] D-psicose (D-psicose) is the epimer of D-fructose (D-fructose) C-3, its sweetness is equiv...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12N15/70C12N1/21C12P19/24C12P19/02C12R1/19
CPCC12N9/90C12N15/70C12P19/02C12P19/24C12Y501/03
Inventor 佟毅沈雪梅王靖李义王小艳陈博丁子元李浩然张媛
Owner JILIN COFCO BIOCHEM
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