L-carnosine synthetase ATPGD derived from novel shellfish and application of L-carnosine synthetase ATPGD

A technology for synthesizing enzymes and carnosine, applied in the field of L-carnosine synthetase ATPGD, which can solve problems such as environmental pollution and complex routes

Pending Publication Date: 2022-03-18
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the production of carnosine mainly relies on chemical synthesis, but this route is relatively complicated and easy to cause environmental pollution. Therefore, the mild and environmentally friendly biological enzyme catalysis method has become one of the exploration directions for the synthesis of this dipeptide in recent years.

Method used

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  • L-carnosine synthetase ATPGD derived from novel shellfish and application of L-carnosine synthetase ATPGD
  • L-carnosine synthetase ATPGD derived from novel shellfish and application of L-carnosine synthetase ATPGD
  • L-carnosine synthetase ATPGD derived from novel shellfish and application of L-carnosine synthetase ATPGD

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Experimental program
Comparison scheme
Effect test

Embodiment 1A

[0023] The acquisition of embodiment 1 ATPGD full-length gene

[0024] 1. RNA extraction

[0025] Total RNA was extracted using Trizol (Invitorgen), and the specific steps were as follows: (1) Put 50 mg of Tridacna gill tissue into a centrifuge tube with 1 ml of Trizol solution, grind thoroughly, and place at room temperature for 10 min; (2) Add 200 μL of chloroform, vigorously Shake for 40 seconds, and place at room temperature for 5 minutes; (3) Centrifuge at 12,000×g for 10 minutes at 4°C, and transfer the supernatant to a new tube; (4) Add 0.5ml of isopropanol, mix well, and let stand at room temperature for 10 minutes; (5) 4 Centrifuge at 12,000×g for 10 minutes at ℃, discard the supernatant; (6) wash the precipitate twice with 1ml 75% ethanol, and centrifuge at 12,000×g for 10 minutes at 4℃; RNA was stored at -80°C for later use. All containers and pipette tips in the above steps have been treated without RNAase.

[0026] 2. Reverse transcription and ATPGD full-length...

Embodiment 2

[0029] Prokaryotic expression of embodiment 2 recombinant protein

[0030] 1. Prokaryotic expression vector construction

[0031] (1) Design a pair of primers covering the ATPGD ORF of giant clam (F: 5'-ATGACGAGTTTCCGTGAAAGGTTCG-3'; R: 5'-CTAATCAGTCGTATGAGATGAAGAAGGCTC-3'); (2) Use this pair of primers for PCR amplification (reaction system : 0.5 μl of 10-fold diluted gill tissue cDNA, 1 μl of each primer (10 mM), 12.5 μl of Premix TaKaRaEx Taq (TaKaRa), 10 μl of ddH2O. Reaction program: 95°C for 3min; 95°C for 15s, 60°C for 15s, 72°C for 3min; ℃ 5min). After the PCR product was detected to meet the expected size by 1.5% agarose gel, it was recovered and purified using HiPure Gel Pure DNA Mini Kit (Magen), and the product was stored at -20°C after sequencing verification. (3) Using the PCR product as a template, use a primer pair (5'-CCGCGTGGATCCCCGGAATTCATGACGAGTTTCCGTGAAAGGT-3'; 5'-GTCACGATGCGGCCGCTCGAGCTAATCAGTCGTATGAGATGAAGAAGG-3') to amplify again and add a recombinatio...

Embodiment 3

[0036] The synthesis of embodiment 3L-carnosine

[0037]Using the 0.2ml solution (PBS containing 20mMGSH as a solvent) containing about 15μg Tridacna maxima ATPGD recombinant protein obtained in Example 2 and 1ml containing 0.5mM β-alanine, 0.5mM L-histidine and 0.5mM The PBS substrate solution of MgATP was incubated at 37°C for 60min and 120min; the control group was 0.2ml solution containing 15μg BSA protein (PBS containing 20mM GSH as solvent) and 1ml containing 0.5mM β-alanine, 0.5 A PBS substrate solution of mM L-histidine and 0.5 mM MgATP was mixed. Three biological repetitions were set up for each group, and then 200 μl of 30% (v / v) HCl was added to terminate the reaction, and macromolecules such as proteins were removed after filtration with a 3 kDa ultrafiltration membrane. Use HPLC to measure the concentration of the filtrate containing carnosine, and find that the reaction system containing the ATPGD recombinant protein of giant clam can produce 76.8 μ M and 167.2 ...

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Abstract

The invention discloses L-carnosine synthetase ATPGD derived from novel shellfish and application of the L-carnosine synthetase ATPGD, and belongs to the technical field of marine organisms. The protein ATPGD of the ATPgrasp structural domain is found from tridacna squamosa for the first time, the amino acid sequence of the protein ATPGD is shown as SEQ ID NO.2, beta-alanine and L-histidine can be catalytically synthesized into the L-carnosine, and a brand new enzyme preparation is provided for biosynthesis of the L-carnosine. The invention further discloses a method for efficiently synthesizing the L-carnosine by using the tridacna squamosa ATPGD.

Description

technical field [0001] The invention belongs to the technical field of marine organisms, and in particular relates to a novel L-carnosine synthase ATPGD derived from shellfish and its application. Background technique [0002] L-carnosine (β-alanyl-L-histidine) and its analogs (homocarnosine, anserine) are natural active dipeptides widely present in the brain, muscle and other important tissues of mammals. The dipeptide has good antioxidant activity and has a significant effect in scavenging free radicals and reducing lipid peroxidation, so it is widely used in cosmetics, food preservation, and cell oxidation-related diseases such as hypertension, heart disease, cataracts, tumors, etc. adjuvant therapy, etc. At present, the production of carnosine mainly relies on chemical synthesis, but this route is relatively complicated and easy to cause environmental pollution, so the mild and environmentally friendly biological enzyme catalysis method has become one of the exploration...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/70C12N1/21C12P17/10C12R1/19
CPCC12N9/93C12N15/70C12P17/10C12Y603/02011
Inventor 张扬杨倬喻子牛
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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