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Cell for expressing phosphotransferase and non-ribosomal peptide synthetase and application thereof

A technology of phosphotransferase and peptide synthetase, applied in the direction of transferase, enzyme, bacteria, etc., can solve the problems of decarboxylated carnosine with many by-products and large pollution

Pending Publication Date: 2022-05-06
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the defects such as many by-products of decarboxylated carnosine prepared by the chemical synthesis method of the prior art and relatively large pollution, the present invention provides a cell expressing phosphotransferase and non-ribosomal peptide synthetase and its application

Method used

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  • Cell for expressing phosphotransferase and non-ribosomal peptide synthetase and application thereof
  • Cell for expressing phosphotransferase and non-ribosomal peptide synthetase and application thereof
  • Cell for expressing phosphotransferase and non-ribosomal peptide synthetase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of recombinant expression plasmids

[0051] 1. Primers

[0052] The sfp, ebony gene, p15A replicon and pET28a, pACYC, pTrc99A, pTrc-p15A vectors were amplified in vitro as follows:

[0053] (1) construct the pACYC-sfp plasmid; the PCR primer sequences used in this embodiment are listed in Table 5, and the Bsusfp gene is amplified in vitro (the nucleic acid sequence is as shown in SEQ ID NO.1, derived from the Bacillus subtilis genome, and the primer pair Bsusfp-F, Bsusfp-R), linearize the pACYC plasmid (pACYCDuet-1 plasmid, the primer pair is pACYC-F, pACYC-R) by inverse PCR at the MCS site. The PCR-amplified products of the above plasmids were digested with endonuclease Dpn I at 37°C for 1 h, the template DNA was removed, purified with a purification kit, and then purified using the ClonExpress II One Step Cloning Kit (Novizan Biotechnology Co., Ltd., Cat. No.: C112-01 ) One-step cloning of Bsusfp gene and linearized pACYC fragment (one-step c...

Embodiment 2

[0073] Embodiment 2: Construction of recombinant genetically engineered bacteria

[0074] The transformant that embodiment 1 obtains is carried out as follows:

[0075] 1. Sequencing verification: use a sterilized pipette tip to select 5 transformants on the plate for colony PCR (the primers used are shown in Table 6). The correct bands were inoculated in LB liquid medium containing the corresponding resistance (see Table 7), cultured at 37°C with shaking at 200r / min for 12 hours, sequenced and preserved. The constructed strains are shown in Table 7.

[0076] Transformant required colony PCR primers in table 6 embodiment 1

[0077]

[0078] The bacterial strain constructed in the embodiment 2 of table 7

[0079]

[0080]

[0081] 2. Induced expression of recombinant plasmids: under sterile conditions, take 1 mL of seed liquid from the LB liquid medium in step 1, and transfer to 50 mL of LB medium (containing Kana at a final concentration of 100 μg / mL; containing Kan...

Embodiment 3

[0082] Embodiment 3: Preparation reaction and detection of decarboxylated carnosine

[0083] 1. Centrifuge the thalline containing the induced recombinant genetically engineered bacteria at 12000rpm for 15min, discard the supernatant and collect the thalline; the collected thalline is resuspended using 20mL pH 8, 50mM trisaminomethane (Tris-HCl) buffer , 8000rpm, high-speed centrifugation for 15min, discard the supernatant to collect the bacteria. Take 0.05g of collected bacteria, 0.0017g of β-alanine, 0.0019g of magnesium chloride, and 0.0044g of ATP and dissolve them in 2mL of pH 7 sodium phosphate buffer solution, and react in a constant temperature oscillator at 25°C, and the oscillation speed is 600~ 800r / min, add 0.0044g of histamine after the system reacts for 30min, then react for 48h.

[0084] 2. Take the reaction sample from step 1, centrifuge at 12,000 rpm for 2 minutes, take 300 μL of the supernatant, add 40 μL of NaOH (0.1M) and 600 μL of DNS (dansyl chloride, 10...

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Abstract

The invention provides a cell for expressing phosphotransferase and non-ribosomal peptide synthetase and application thereof. The cell for expressing phosphotransferase and non-ribosomal peptide synthetase is obtained by transferring an expression plasmid containing a phosphotransferase gene and an expression plasmid containing a non-ribosomal peptide synthetase gene into a host cell. The decarboxylated carnosine is prepared by constructing the recombinant genetically engineered bacteria and using a biological enzyme catalysis method, and the method is more environment-friendly than an existing chemical synthesis method. The method for preparing the decarboxylated carnosine through a whole-cell catalysis method is reported for the first time for preparing the decarboxylated carnosine through a biological method.

Description

(1) Technical field [0001] The invention relates to a method for preparing decarboxylated carnosine, in particular to a cell expressing phosphotransferase and non-ribosomal peptide synthetase and application thereof. (2) Background technology [0002] Carcinine, scientific name β-alanyl-L-histamine (β-alanyl histamine), is an imidazole dipeptide composed of β-alanine and histamine. Decarboxylated carnosine was first discovered in crustaceans in 1975, and later in the hearts of some mammals. Compared with carnosine, vitamin C and tea polyphenols, decarboxylated carnosine has a stronger antioxidant effect, and it also has the functions of anti-glycation, UV protection, anti-aging of skin, improvement of skin wrinkles and post-sun repair. In addition, it also has a significant "reversal" function, which can effectively reverse the glycation and oxidation of the skin. Moreover, the metabolism is slow, the structure is stable, and it can function more effectively. Relevant exp...

Claims

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Application Information

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IPC IPC(8): C12N1/21
CPCC12N15/70C12N9/12C12N9/00C12P21/02
Inventor 赵嫚成浩刘薇柳志强郑裕国
Owner ZHEJIANG UNIV OF TECH
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