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Improved method for producing 5'-flavor nucleotide

A technology of nucleotides and nucleotide sequences, applied in the field of producing 5'-taste nucleotides, can solve the problems of low transformation rate and high usage of genetically engineered bacteria, and achieves short cycle, low usage of bacteria and low cost. low effect

Inactive Publication Date: 2012-03-21
SHANGHAI FUCHANG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has the disadvantages of high usage of genetically engineered bacteria and low conversion rate.

Method used

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  • Improved method for producing 5'-flavor nucleotide
  • Improved method for producing 5'-flavor nucleotide
  • Improved method for producing 5'-flavor nucleotide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 1. Construction of genetically engineered bacteria

[0040] Primers were designed according to the phoc gene sequence (GeneBank access: AB044338.1) of Enterobacter aerogenes provided in GeneBank as follows:

[0041] Primer 1: 5'-cg ggatcc catgaaaaagcgcgttctcgccctctg-3'(BamHI) (denoted as SEQ.ID.NO.5),

[0042] Primer2: 5'-ccg ctcgag cgatgacgttacttctgcgttttggcg-3' (XholI) (denoted as SEQ.ID.NO.6),

[0043] PCR was performed using the chromosomal DNA of Enterobacter aerogenes (Enterobacter aerogenes W8401) as a template: 95° C. for 5 min, 30× (95° C. for 30 s, 55° C. for 30 s, 72° C. for 60 s), and 72° C. for 10 min. A PCR product with a length of about 760bp was obtained, which was digested with the pBV220 vector and then ligated with T4 DNA ligase to transform E.coli DH5α competent cells. Transformants were identified by PCR with pBV220 vector general primers and restriction enzyme digestion to obtain genetically engineered bacteria DH5α / pBV220-phoc. The DNA sequ...

Embodiment 2

[0059] Embodiment 2 acid phosphatase gene phoc mutation

[0060] Eight pairs of primers were designed: 420, 421; 422, 423; 424, 425; 426, 427; 428, 429; 430, 431; 432, 433; 434, 435. They are recorded as SEQ.ID.NO.7-22 respectively, as shown in Table 1.

[0061] The first round of PCR: using universal primers primer1 and 421 and universal primers primer2 and 420 as primers and plasmid pBV220-phoc as a template, two sequences were amplified by PCR. PCR conditions are as described in Step 1 of Example 1. The two fragments were recovered from the gel and quantified by image scanning software (Smartview).

[0062] The second round of PCR: using primer1 and primer2 as primers and 50 ng of recovered fragments as a template (1:1), carry out a PCR reaction under the same conditions as step 1 in Example 1.

[0063] The target fragment was recovered, digested with BamH I and Xhol I overnight, connected into the pBV220 vector digested with BamH I and Xhol I, transformed into E.coli DH...

Embodiment 3

[0073] Embodiment 3 catalytic synthesis 5'-inosinic acid (5'-IMP)

[0074] Experiment 1:

[0075] Using DH5α / pBV220-mphoc as the enzyme source to catalyze the synthesis of 5’-IMP, the reaction system is as follows:

[0076] Buffer: 100 mM acetate buffer pH 4.0

[0077] Substrate: 50g / L Ir and 300g / L Na 4 P 2 o 7 10H 2 o

[0078] Bacteria volume: 0.5% (wet weight)

[0079] Reaction 2 hours 5 '-IMP concentration reaches 94.6g / L, conversion rate reaches 96%.

[0080] Experiment 2:

[0081] Using DH5α / pBV220-mphoc as the enzyme source to catalyze the synthesis of 5’-IMP, the reaction system is as follows:

[0082] Buffer: 100 mM acetate buffer pH 4.0

[0083] Substrate: 80g / L Ir and 300g / L Na 4 P 2 o 7 10H 2 o

[0084] Bacteria volume: 0.5% (wet weight)

[0085] Reaction 6 hours 5 '-IMP concentration reaches 144.4g / L, conversion rate reaches 92%.

[0086] Experiment 3:

[0087] Using DH5α / pBV220-mphoc as the enzyme source to catalyze the synthesis of 5’-IMP, the ...

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Abstract

The invention belongs to the technical field of biochemical engineering, relates to an improved method for producing 5'-flavor nucleotide and particularly relates to a method for constructing a gene engineering bacterium through a DNA (deoxyribonucleic acid) recombination technology, carrying out mutant screening on the gene engineering bacterium and utilizing the high-activity gene engineering bacterium to produce 5'-nucleotide. The method comprises the following steps: 1, through the DNA recombination technology, constructing an acid phosphatase expression vector, and converting into Escherichia coli to obtain a gene engineering bacterium capable of efficiently expressing acid phosphatase; 2, mutating the gene of the acid phosphatase, and screening the acid phosphatase having high affinity with nucleoside; and 3, using the acid phosphatase to catalyze the nucleoside and a phosphate group donor so as to synthesize the 5'-nucleotide. By using the method to realize the catalytic synthesis of the 5'-nucleotide, the invention has the characteristics of simple process, high efficiency, short period, less bacterium consumption, low cost and the like, conforms to environment protection requirements, and is suitable for industrial production.

Description

technical field [0001] The invention relates to a method for producing 5'-taste nucleotides, in particular to a method for producing 5'-taste nucleotides by using highly active genetically engineered bacteria, which belongs to the technical field of biochemical engineering. Background technique [0002] 5'-nucleotides are often used as food additives and drug synthesis intermediates, of which 5'-inosine-5'-monophosphate (5'-IMP for short) and Guanosine-5'-monophosphate (Guanosine-5'-monophosphate , referred to as 5'-GMP) as a taste agent can be mixed with monosodium glutamate (MSG) to produce a synergistic effect, which can increase the freshness several times to dozens of times; in addition, they have synergistic effects on sweetness and meat flavor It has inhibitory effect on salty, sour, bitter, fishy and burnt taste, so it has been paid more and more attention and welcome in food industry production. [0003] The methods for industrial production of 5'-nucleotides mainl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12N9/16C12N15/70C12N15/55C12N15/10C12R1/19C12R1/01
Inventor 梁胜华
Owner SHANGHAI FUCHANG TECH
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