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Enzyme preparation for preparing uridylic acid and method for preparing uridylic acid through enzyme catalysis

A technology of uridylic acid enzyme and uridine acid, which is applied in the field of compound biotechnology production, can solve the problems of short conversion cycle, high price, high regeneration cost, etc., and achieve the effects of easy cultivation, reduced production cost and superior yield

Active Publication Date: 2019-01-08
TIANJIN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

This method has a shorter conversion cycle and a higher yield of uridine acid, but the uridine kinase used is highly dependent on GTP as a phosphate donor, and GTP is expensive and has high regeneration costs

Method used

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  • Enzyme preparation for preparing uridylic acid and method for preparing uridylic acid through enzyme catalysis
  • Enzyme preparation for preparing uridylic acid and method for preparing uridylic acid through enzyme catalysis
  • Enzyme preparation for preparing uridylic acid and method for preparing uridylic acid through enzyme catalysis

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preparation example Construction

[0035] Preferably, the preparation method of above-mentioned uridine acid, concrete steps are as follows:

[0036] (1) Heterologously express the gene encoding uridine kinase in E. coli E. coli BL21 (ACCC11171), and construct a strain E. coli UDK with uridine kinase activity;

[0037] (2) Heterologous expression of polyphosphokinase coding gene in Escherichia coli E.coli BL21 (ACCC11171) to construct a strain E.coli PPK with polyphosphokinase activity;

[0038] (3) Culture the recombinant strains E.coli UDK and E.coli PPK constructed above in culture medium to produce uridine kinase and polyphosphate kinase with high enzymatic activity;

[0039] (4) Breaking the Escherichia coli cells (E.coli UDK and E.coli PPK cells) with uridine kinase and polyphosphokinase activities obtained by culturing in step (3) to obtain a crude enzyme solution;

[0040] (5) Mix the crude enzyme liquid obtained in step (4) with 50-150mM uridine solution or uridine fermentation broth, 50-150mM sodium ...

Embodiment 1

[0049] Construction of strain E.coli UDK with uridine kinase activity

[0050] ①According to the nucleotide sequence of the uridine kinase encoding gene udk 1 of Thermus thermophiles (ATCC27634) on Genbank, design a point mutation on the 93rd amino acid sequence to mutate the tyrosine residue to histidine acid residues, and codon-optimize them with common codon optimization methods in Escherichia coli, and send the optimized sequence plus the enzyme cutting sites BamH I and EcoR I (see Appendix ) to Jinweizhi Company for synthesis .

[0051] ②According to the nucleotide sequence of the uridine kinase sequence encoding gene udk2 of Bacillus sp. (NCBI: txid1960589) on Genbank, the codon optimization method was used to optimize the codon in Escherichia coli, and the optimized sequence Plus restriction sites BamH I and EcoR I (see appendix ) were sent to Jinweizhi Company for synthesis.

[0052] ③ Use Takara restriction endonuclease BamH I and EcoR I to double digest the target ...

Embodiment 2

[0056] Construction of strain E.coli PPK with polyphosphokinase activity

[0057] ①Using PCR technology to use Rhodobacter sphaeroides (ATCC17023) genome as a template, design a pair of gene amplification primers (see appendix 7>) to amplify and obtain the target gene fragment. The pair of primers respectively comprise enzyme cutting sites EcoR I and Hind III.

[0058] ② Use PCR technology to design a pair of gene amplification primers ( See Appendix ) to amplify and obtain target gene fragments. The pair of primers respectively comprise enzyme cutting sites EcoR I and HindIII.

[0059] ③Use Takara restriction endonuclease EcoR I and Hind III to obtain the target fragment and pET-28a vector plasmid (see Appendix ) in steps ① and ② to obtain the target gene with the same cohesive end and linearize it Plasmid fragments.

[0060] ④ Ligate the gene fragments obtained in step ③ using Takara T4 DNA ligase to obtain two recombinant expression vectors pET-28a-ppk 1 and pET-28a-pp...

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Abstract

The invention relates to an enzyme preparation for preparing uridylic acid and a method for preparing the uridylic acid through enzyme catalysis. The method comprises the following steps: taking a material liquid containing uridine as a substrate, adding an escherichia coli cell crushing liquid with activity of uridine kinase and poly-phosphokinase, sodium hexametaphosphate, magnesium sulfate anda small amount of ATP (Adenosintriphosphat), and carrying out an enzymatic reaction under conditions that the pH value is 8.0 and the temperature is 30 DEG C to synthesize the uridylic acid. In a coupling catalysis reaction system, the uridine and the ATP are catalyzed by the uridine kinase to generate the uridylic acid, and ADP (Adenosine Diphosphate) is formed along with the dephosphorylation ofthe ATP. Poly-phosphokinase is in charge of catalyzing the sodium hexametaphosphate and the ADP to generate the ATP, so that the regeneration of the ATP is achieved in reactions. The uridylic acid production method provided by the invention has the advantages of being low in raw material price, short in period, simple and convenient to operate, green and environmentally-friendly and high in yield, and has good industrial application value.

Description

technical field [0001] The invention relates to the field of compound biotechnology production, in particular to an enzyme preparation for preparing uridine acid and a method for preparing uridine acid by enzymatic catalysis. Background technique [0002] Uridine is an important nucleotide product, which can be used in different fields as food additives, drugs and drug precursors. As the second largest nucleotide in breast milk, it is a commonly used additive in infant food; it can participate in the synthesis of glucuronide, a detoxification substance in the liver, and can also be used as a precursor of membrane phospholipids to increase brain cytidine diphosphate cholesterol Alkaline and acetylcholine levels; iodine glucoside, which is its precursor, has also been widely used in clinical practice. [0003] The existing production methods of uridine acid are mainly chemical synthesis and enzymatic hydrolysis. [0004] Chemical synthesis methods include phosphorylation and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N1/21C12P19/30C12R1/19
CPCC12N9/1205C12N9/1229C12P19/305C12Y207/01048C12Y207/04001
Inventor 范晓光陈宁吴思佳张通陈珂谢希贤徐庆阳张成林
Owner TIANJIN UNIV OF SCI & TECH
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