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Novel tagatose 6-phosphate 4-locus epimerase and application thereof

A technology of tagatose phosphate and epimerase, applied in the direction of racemase/epimerase, isomerase, application, etc., can solve the problem of unsuitable large-scale production of tagatose and poor thermal stability advanced questions

Active Publication Date: 2019-05-14
TIANJIN YEAHE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low thermal stability of the enzyme, it is not suitable for large-scale production of tagatose
In addition, when Wichelecki et al. were studying the metabolism of altritol and galactitol in Agrobacterium tumefaciens, they found that an enzyme encoded by a gene Atu3167 could catalyze the conversion of tagatose 6 phosphate into fructose 6 phosphate (J Biol Chem 2015; 290:28963-28976.), but the thermostability of the enzyme is also not high, not suitable for large-scale production of tagatose

Method used

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  • Novel tagatose 6-phosphate 4-locus epimerase and application thereof
  • Novel tagatose 6-phosphate 4-locus epimerase and application thereof
  • Novel tagatose 6-phosphate 4-locus epimerase and application thereof

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Effect test

Embodiment 1

[0051] The cloning of embodiment 1TiT4E gene

[0052] We obtained the amino acid sequence of the polypeptide 6-phosphate tagatose 4-position epimerase from NCBI (https: / / www.ncbi.nlm.nih.gov / ), and the NCBI Reference Sequence of the sequence is WP_019907213.1 (SEQ.No.1), we named the polypeptide TiT4E, and TiT4E was annotated as a hypothetical protein on NCBI, and was classified as a 6-phosphate tagatose kinase, which is to convert tagatose under the action of ATP Enzyme that phosphorylates tagatose 6-phosphate. We then entrusted Wuxi Qinglan Biotech Co., Ltd. (http: / / qinglanbiotech.com / ) to design the polynucleotide (DNA) sequence encoding the polypeptide based on the amino acid sequence, and optimized the codons in the E. coli expression system. After optimization, The polynucleotide sequence is shown in SEQ.NO.2.

[0053] Wuxi Qinglan cloned the codon-optimized sequence into pMV vector to form pMV-TiT4E plasmid. We then used the forward primer GAA CATATG AACACCGAACATCC...

Embodiment 2

[0054] Expression and purification of embodiment 2TiT4E

[0055] Transform pET20b-TiT4E into Escherichia coli BL21(DE3), pick a single clone into 3ml LB medium containing 100μg / ml ampicillin, and culture overnight at 37°C and 220rpm. Take 1ml of overnight bacteria into 200ml of LB medium containing 100μg / ml ampicillin, 37°C, 220rpm until the OD600 value reaches about 0.8, add Isopropylβ-D-1-thiogalactopyranoside (IPTG) with a final concentration of 100μM, respectively at 37 °C and 18°C ​​induced protein expression. Induce for 4 hours at 37°C and 20 hours at 18°C. After the induction, the bacteria were collected by centrifugation, resuspended with 30mM phosphate buffer (pH 7.0), and ultrasonically disrupted to obtain the cell disruption liquid, and the expression level of the enzyme was detected by SDS-PAGE, as shown in image 3 In Lane 1 and Lane 4 of A, perform high-speed centrifugation (12000rpm, 10min) on the cell lysate, and also detect the supernatant by SDS-PAGE, such ...

Embodiment 3

[0056] The enzyme activity assay of embodiment 3TiT4E

[0057]The method for measuring the enzyme activity of TiT4E is as follows: a reaction system contains fructose-6-phosphate, tagatose-6-phosphate phosphatase, buffer solution, and magnesium ions, and the growth amount of inorganic phosphorus in the reaction system is measured after the reaction is completed. Specifically, in a reaction system containing 100mM HEPES buffer, 10mM fructose-6-phosphate, 10U / ml tagatose-6-phosphate phosphatase (from Archaeoglobus fulgidus, the number of the gene on KEGG is AF_0444, the gene also Heterologous expression in Escherichia coli, purified by Ni-NTA column to obtain a large amount of enzyme), 5mM magnesium sulfate, 0.005 or 0.02g / L TiT4E, reacted at 60°C for 8 minutes. After the reaction was over, the reaction was terminated in an ice bath. The released inorganic phosphorus ions were determined by the method for determining inorganic phosphorus provided by literature (Anal.Chem.1956, ...

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Abstract

The invention discloses novel tagatose 6-phosphate 4-locus epimerase. The epimerase can achieve conversion between fructose 6-phosphate and tagatose 6-phosphate. The invention also discloses application of the enzyme in tagatose production.

Description

technical field [0001] The present invention relates to the fields of genetic engineering and biocatalysis, and provides a novel 4-position epimerase of tagatose 6-phosphate, which has the ability to make the 4-position carbon of 6-phosphate fructose (fructose 6-phosphate) To isomerization, conversion to tagatose 6-phosphate (tagatose 6-phosphate) and conversion of tagatose 6-phosphate to fructose 6-phosphate. The invention also provides the application of the enzyme in the production of tagatose. Background technique [0002] The 4-position epimerization of 6-carbon sugars plays an important role in the production of rare sugars and their derivatives. For example, a Korean patent (application number: WO2015016544A1) reported that 6-phosphate fructose can be converted to 4-position epimerization into tagatose 6 phosphate, followed by dephosphorylation of tagatose 6 phosphate to tagatose. The 6-phosphate tagatose 4-position epimerase reported in this patent is 1,6-diphospha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12P19/24C12P19/02
CPCC12N9/90C12N15/63C12P19/02C12P19/24C12Y503/01C12N15/52C12N9/1051C12Y204/01001C12Y504/02002C12Y503/01009C12Y207/01101C12N9/1205C12Y501/03C12Y207/01144
Inventor 马延和孙媛霞李运杰杨建刚
Owner TIANJIN YEAHE BIOTECHNOLOGY CO LTD
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