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D-psicose 3-epimerase mutant with improved catalytic efficiency

A technology of epimerase and allulose, applied in the field of enzyme engineering, to achieve the effect of improving catalytic efficiency

Active Publication Date: 2018-05-11
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, little research has been done on the molecular modification of ketose 3-epimerase

Method used

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  • D-psicose 3-epimerase mutant with improved catalytic efficiency
  • D-psicose 3-epimerase mutant with improved catalytic efficiency
  • D-psicose 3-epimerase mutant with improved catalytic efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation method of Dorea sp.DPEase enzyme mutant

[0027] Construction of the recombinant plasmid pET22b-dore-dpe: According to Dorea sp.CAG:317 (accession number: CBGJ010000047.1), the D-psicose 3-epimerase gene fragment BN605_00564 was synthesized and linked to pET-22b (+) between the restriction sites Nde I and Xho I to obtain the recombinant plasmid pET22b-dore-dpe.

[0028] Construction of pET-22b(+)-A38E / G105A mutant plasmid: Using pET22b-dore-dpe plasmid as a template, the A38E / G105A site-directed mutation was introduced by PCR. The sequencing verification results showed that there was no randomness except the required mutation sites. Mutation, so the mutant plasmid pET-A38E / G105A was successfully constructed.

[0029] The mutation primers are as follows: (the underline is the mutation point)

[0030] A38E-F: 5’-GAAATCGGTGCT GAA CCATTACCGG-3’

[0031] G105A-F: 5’-CCATTTGATC GCC GGCGCTCTCTATG-3’

[0032] dore-R: 5’-GGTGTGTTTCCAATCCAACATATATTTC-3’

[0033] Sh...

Embodiment 2

[0041] Example 2: The expression and purification method of the mutant enzyme of Dorea sp. DPEase.

[0042] Transform the mutant plasmid pET-22b(+)-A38E / G105A verified by sequencing into E. coli BL21(DE3) cells, pick the positive transformants and shake them overnight in LB medium at 37°C and 200rpm, and then insert them into LB culture Incubate at 37°C for 3-4 hours to an OD value of 0.6-0.8, then cool to 30°C, add IPTG to a final concentration of 0.6mM and induce 6 hours.

[0043] The fermentation broth was centrifuged at 4°C and l0000 rpm for 20 min, and the bacteria were collected. Add 20mL buffer (50mM PBS, 200mMNaCl, adjust pH to 6.0) to fully resuspend the bacteria, then put the centrifuge tube in an ice bath, put it in the ultrasonic cell disruptor, the ultrasonic disruption conditions are: working time 1s, stop Time is 2s, totaling 18min. The obtained crushed liquid was centrifuged at low temperature and high speed, and centrifuged at 4°C and 10,000 rpm for 30 minutes to...

Embodiment 3

[0045] Example 3: Determination of the catalytic efficiency of the mutant enzyme of Dorea sp. DPEase.

[0046] The present invention refers to the method for measuring the activity of D-psicose 3-epimerase. The mutant pure enzyme is subjected to a catalytic reaction under standard reverse conditions, and the synthetic amount of D-psicose is detected by HPLC, and the mutant is calculated Enzyme activity. The enzyme activity of wild-type Doreasp.DPEase is defined as the relative enzyme activity 100%. It was found that the specific enzyme activity of the mutant A38E / G105A was 1113.7 U / mg, which was an increase of 38.6% compared to the original enzyme specific enzyme activity of 803.5 U / mg.

[0047] Table 3 Comparison of relative enzyme activity between wild enzyme and mutant enzyme under optimal reaction conditions

[0048]

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Abstract

The invention discloses a D-psicose 3-epimerase mutant with improved catalytic efficiency and belongs to the technical field of enzyme engineering. The Dorea sp. DPEase mutant enzyme A38E / G105A keepsthe optimal catalytic conditions. Under the optimal catalytic conditions, the relative enzyme activity of the enzyme for catalytic conversion of D-fructose as a substrate into D-psicose is improved by38.6%. The discovery has an important research value for the industrial production of D-psicose.

Description

Technical field [0001] The invention relates to a D-psicose 3-epimerase mutant with improved catalytic efficiency, which belongs to the technical field of enzyme engineering. Background technique [0002] In recent years, my country's nutrition and health products industry has developed rapidly, and the growth rate can be maintained at 12% to 15%. It has surpassed Japan as the second largest market for health products. With the continuous development of my country's economy, people's dietary structure will also undergo major changes, and gradually develop in a safer, more reasonable and healthier direction. Currently, chronic diseases caused by excessive intake of high-sugar foods are increasing rapidly, such as obesity, diabetes, high blood pressure, and hyperlipidemia. Therefore, low-energy rare sugars have gradually attracted the attention of the international community. [0003] D-psicose is the C-3 epimer of D-fructose. It is a rare natural monosaccharide, which is rarely fo...

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 沐万孟张文立黄嘉苇江波张涛
Owner JIANGNAN UNIV
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