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D-psicose-3-epimerase mutant with improved catalytic activity and application of mutant

A technology of epimerase and psicose, applied in racemase/epimerase, isomerase, application, etc., can solve the problem of poor catalytic activity and limit the large scale of D-psicose Industrial production, high application costs

Active Publication Date: 2018-07-03
LIVINGZONE SHANGHAI BIO CHEM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the poor catalytic activity of the unmodified DPE enzyme derived from wild strains, there are certain limitations in industrial application, resulting in a large amount of enzyme usage and high application costs, which limit the large-scale production of D-psicose Industrial production

Method used

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  • D-psicose-3-epimerase mutant with improved catalytic activity and application of mutant
  • D-psicose-3-epimerase mutant with improved catalytic activity and application of mutant
  • D-psicose-3-epimerase mutant with improved catalytic activity and application of mutant

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Experimental program
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Effect test

preparation example Construction

[0106] The preparation method of allulose

[0107] The present invention also provides a method for preparing allulose, the method comprising the steps of:

[0108] (1) contacting the mutated D-psicose-3-epimerase of the present invention with a reaction substrate to carry out a catalytic reaction, thereby generating the allulose;

[0109] (2) Optionally, isolating and purifying the psicose.

[0110] The present invention also relates to the application of the D-psicose prepared by the above method in the production of human food, animal feed, cosmetics or medicines.

[0111] The main advantages of the present invention include:

[0112] (1) The present invention provides a DPE enzyme mutant whose catalytic activity is significantly improved, and its catalytic activity (unit enzyme activity) is that of wild-type D-psicose-3-epimerase under the same conditions The catalytic activity is 9.6 times, greatly reducing the amount of enzymes used in the process of synthesizing D-ps...

Embodiment 1D

[0120] Example 1D-Establishment of a mutant library of psicose-3-epimerase

[0121] According to the BLAST comparison of the reported D-Psicose-3-epimerase (DPE) sequence, it was found that the amino acid sequence of an unknown protein derived from Paenibacillus senegalensis was consistent with other The similarity of several DPE genes is relatively large, and it is speculated that this gene has the ability to convert D-fructose into D-psicose, and it is a potential DPE gene. After the DNA sequence was optimized, the whole gene was synthesized by Changzhou Jiyu Biotechnology Co., Ltd., and recombined into the pET29a(+) vector (Novagen); after expression experiments and functional experiments, the protein can convert D-fructose into D - Allulose, which has the function of D-psicose-3-epimerase, belongs to this family, and its amino acid sequence is shown in SEQ ID NO.:1. Subsequently, protein directed evolution research was carried out on the basis of the wild-type protein.

...

Embodiment 2

[0130] Activation and induced expression of embodiment 2 mutant library

[0131] Pick a single clone from the above-mentioned cultured overnight plate to the LB liquid medium (ingredients: tryptone (Tryptone) 10g / L, yeast extract (Yeast Extract) ) 5g / L, sodium chloride 10g / L, wherein tryptone (Tryptone) and yeast extract (Yeast Extract) were purchased from Oxoid, and sodium chloride was purchased from Sinopharm Chemical Reagent Co., Ltd.) in 96 deep-well plates, in Culture overnight at 37°C with shaking at 220rpm. On the next day, draw 100 μl of the culture solution from the 96 empty plate cultured overnight, and add it to a fresh 96 deep-well plate containing LB liquid medium with a final concentration of 50 μg / ml kanamycin sulfate, at 37°C, 220rpm After 4 hours of shaking culture, IPTG with a final concentration of 1 mM was added for induction, and then culture was continued at 30° C. for 20 hours. A total of 400 single clones were picked for activity screening.

[0132] ...

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Abstract

The invention provides a D-psicose-3-epimerase mutant with improved catalytic activity and an application of the mutant and particularly provides mutational D-psicose-3-epimerase. The mutational D-psicose-3-epimerase is obtained by mutation in at least three loci selected from the following groups: the 73rd aspartic acid (D), the 111st tyrosine (Y), the 188th asparaginate (N) and the 250th glycine(G) of wild D-psicose-3-epimerase corresponding to SEQ ID NO:1. The mutational D-psicose-3-epimerase has very high catalytic activity and catalytic efficiency (reaching up to 29.6%).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mutant of D-psicose-3-epimerase with improved catalytic activity and its application. Background technique [0002] D-psicose is a new functional rare sugar with special health functions discovered in recent years. Its sweetness is equivalent to 70% of fructose, and its energy is only 0.3% of sucrose. It has low energy and improves intestinal flora. , lower blood sugar, anti-caries, prevent obesity and other physiological functions. In 2011, the FDA approved that D-psicose can be used as a food additive. Since then, D-psicose has been developed rapidly. [0003] In nature, the content of D-psicose is extremely small, and it is extremely difficult to obtain. Biological preparation of D-psicose is a research hotspot in recent years. D-psicose-3-epimerase (D-Psicose-3-Epimerase, abbreviated as DPE) belongs to the class of epimerases, which can catalyze the epimerization of various...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12P19/24C12P19/02
CPCC12N9/90C12P19/02C12P19/24C12Y501/03
Inventor 余允东祝俊
Owner LIVINGZONE SHANGHAI BIO CHEM TECH CO LTD
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