D-xylulose 4-epimerase, mutant and application thereof

A technology of epimerase and xylulose, applied in the field of D-xylulose 4-epimerase, can solve the problems of high production cost and complex production process of L-pentose

Pending Publication Date: 2021-07-16
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] Based on the defects of high production cost and complex production process of L-pentose in the prior art, it is necessary to provide a new method for producing L-pentose

Method used

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  • D-xylulose 4-epimerase, mutant and application thereof
  • D-xylulose 4-epimerase, mutant and application thereof
  • D-xylulose 4-epimerase, mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0332] Example 1. Separation and detection of pentose sugars

[0333] D-xylose, D-xylulose, D-ribulose and L-arabinose are separated by using any of the methods described in (1)-(4) below:

[0334] (1) Bio-Rad (Bio-Rad) Aminex HPLC HPX-87H liquid-phase ion exchange column was used for separation, and the separation conditions were as follows: the column temperature was 60° C., the mobile phase was 5 mM sulfuric acid, and the flow rate was 0.6 mL / min.

[0335] (2) Bole Aminex HPX-87P lead ion exchange column, the column temperature is 80℃, the mobile phase is deionized water, and the flow rate is 0.6mL / min;

[0336] (3) Waters Sugar Pak I calcium ion exchange column, the column temperature is 80°C, the mobile phase is deionized water, and the flow rate is 0.5mL / min; and

[0337] (4) Shodex Sugar KS-801 sodium ion exchange column, the column temperature is 70°C, the mobile phase is deionized water, and the flow rate is 0.5 mL / min.

[0338] For D-xylose, D-xylulose, D-ribulose,...

Embodiment 2

[0340] Example 2. Mining of enzymes with Xu4E function from L-ribulose-5-phosphate 4-epimerase

[0341] Considering the similarity of the substrate structure and the possible enzymatic catalytic mechanism, we selected from the L-ribulose-5-phosphate 4-epimerase family (RP4E, EC 5.1. Conversion of xylulose to L-ribulose functional Xu4E enzyme. We cloned four RP4Es from Bacillus subtilis 168, Geobacillus stearothermophilus, Escherichia coli, and T. maritima, respectively, and cloned them to the pET plasmid. E. coliBL21 (DE3) carrying the expression plasmid was grown and the recombinant protein was expressed.

[0342] After the protein with His-tag was purified by affinity adsorption, the protein with His-tag was purified by the method described in this disclosure for " Judgment more Determination of whether a peptide / enzyme has Xu4E enzymatic activity”, and tested the obtained recombinant protein. From the test results, the three RP4Es derived from Thermus marinus, Bacillu...

Embodiment 3

[0345] Example 3. Mining of new enzymes with Xu4E function from tagatonic acid 3-epimerase

[0346] Considering the similarity of the substrate structure and the possible enzymatic catalysis mechanism, we adopted a method similar to Example 2 to select a possible enzyme from tagatonic acid 3-epimerase (UxaE, EC 5.1.2.7). A new enzyme capable of converting D-xylulose to L-ribulose. We cloned multiple uxae genes from different microorganisms and cloned them into pET plasmids. E. coli BL21 (DE3) carrying the expression plasmid was grown and the recombinant protein was expressed.

[0347] After the protein with His-tag was purified by affinity adsorption, the protein with His-tag was purified by the method described in this disclosure for " Judgment more Determination of whether a peptide / enzyme has Xu4E enzymatic activity ”, to detect the obtained recombinant protein. From the detection results, the tagatonic acid 3-epimerase (Tm0440) from Thermus marinus has no optimized re...

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Abstract

The present invention discloses a D-xylulose 4-epimerase, a mutant and application thereof. Specifically, the invention relates to a polypeptide with the activity of the D-xylulose 4-epimerase, a preparation method of the polypeptide, and an application of the polypeptide in preparing L-pentose by taking D-xylose or D-xylulose as a raw material. Compared with a traditional production method in the prior art, the novel method for preparing the L-pentose disclosed by the invention has the advantages that the production process is simpler, and the production cost of the L-pentose is reduced.

Description

technical field [0001] The present disclosure belongs to the fields of biocatalysis and synthetic biology. The present disclosure relates to novel polypeptides (enzymes) capable of reversibly catalyzing the interconversion between D-xylulose and L-ribulose and their mutants, named D-xylulose 4-epimerase (Xu4E); And by constructing multiple artificial enzyme pathways, using this enzyme, L-pentose (i.e. L-arabinose, L-ribose, L-ribulose, L-xylose) will be produced from the most abundant pentose D-xylose in nature. ketose, L-xylose and L-lyxose). Background technique [0002] Pentose (or pentose monosaccharide), chemical formula C 5 H 10 O 5 , is a five-carbon monosaccharide. They can be divided into two broad categories: aldopentose and ketopentose; there are eight aldopentoses and four ketopentose, each of which corresponds to two aldopentose. Twelve pentose sugars can also be divided into L-type sugars and D-type sugars, and each type of sugar has four aldopentose and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12P19/24C12P19/02
CPCC12N9/90C12P19/24C12P19/02C12Y501/03004C12Y501/03005
Inventor 张以恒周伟
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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