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Isolated polypeptide, nucleic acid and application thereof

A nucleic acid and nucleotide sequence technology, applied in the field of biochemistry, can solve the problems of non-conformity with green chemistry, high energy consumption, harsh reaction conditions, etc.

Pending Publication Date: 2021-09-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The process of chemical synthesis is complicated, many toxic substances will be produced, and the reaction conditions are harsh, the energy consumption is large, and it does not meet the requirements of green chemistry

Method used

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  • Isolated polypeptide, nucleic acid and application thereof
  • Isolated polypeptide, nucleic acid and application thereof
  • Isolated polypeptide, nucleic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] 1. Construction of recombinant plasmids

[0105] 1. For the E. coli system, optimize the codons of the β-aminopeptidase sequence gene found in the deep-sea metagenomic library, and synthesize the related gene (SEQ ID NO: 4).

[0106] 2. Take the gene synthesized in step 1, perform double digestion with restriction endonucleases NcoI and XhoI, and recover the digested product.

[0107] 3. Take the pET-26b plasmid, perform double digestion with restriction endonucleases NcoI and XhoI, and recover the vector skeleton.

[0108] 4. Ligate the digested product of step 2 with the vector backbone of step 3 to obtain the recombinant plasmid pET26b-WTCar. According to the sequencing results, the structure of the recombinant plasmid pET26b-WTCar is described as follows: a DNA molecule shown in SEQ ID NO:4 is inserted between the NcoI and XhoI restriction sites of the pET-26b plasmid. The DNA molecule shown in SEQ ID NO:4 encodes the wild-type protein shown in SEQ ID NO:1.

[01...

Embodiment 2

[0163] Example 2. Whole-cell catalysis and enzyme activity detection of wild-type protein and each mutant protein

[0164] 1. Protein expression

[0165] Each of the recombinant bacteria prepared in Example 1 was used to prepare each protein with a His6 tag fused at the N-terminal, and the specific steps were as follows

[0166] 1. Inoculate the recombinant bacteria into a liquid LB medium containing 40 μg / mL kanamycin, and culture it with shaking at 37° C. and 200 rpm until the OD600nm value = 0.6-0.8.

[0167] 2. After completing step 1, add isopropyl-β-D-thiogalactopyranoside (IPTG) to the system at a concentration of 0.02mM, shake and culture at 16°C and 200rpm for 8 hours.

[0168] 3. After completing step 2, collect the cells by centrifugation.

[0169] 4. Take the cells obtained in step 3, suspend them with binding buffer, then perform ultrasonic disruption (10% power, break for 3 seconds and stop for 5 seconds, total time 10 minutes), then centrifuge at 12000g for 20...

Embodiment 3

[0187] Embodiment 3, the impact of G310A mutant whole cell catalytic synthesis of L-carnosine

[0188] In order to further improve the enzyme activity of G310A and simplify the process flow, L-carnosine was catalyzed and synthesized by whole cells of G310A mutant, and the reaction conditions were optimized.

[0189] 1. Preparation of whole cell extracts

[0190] The G310A prepared in Example 1 was used to prepare the whole cell extract, and the specific steps were as follows:

[0191] 1. Inoculate the recombinant bacteria into a liquid LB medium containing 40 μg / mL kanamycin, and culture it with shaking at 37° C. and 200 rpm until the OD600nm value = 0.6-0.8.

[0192]2. After completing step 1, add isopropyl-β-D-thiogalactopyranoside (IPTG) to the system at a concentration of 0.02mM, shake and culture at 16°C and 200rpm for 8 hours.

[0193] 3. After completing step 2, collect the cells by centrifugation.

[0194] 4. Take the cells obtained in step 3, wash twice with Na2CO3...

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Abstract

The invention provides a separated polypeptide. Compared with a wild carnosine synthetase SEQ ID NO: 1, the polypeptide has 310 amino acid mutations. The polypeptide provided by the invention can be used for efficiently preparing carnosine, is green and environment-friendly, and is suitable for large-scale production.

Description

technical field [0001] The present invention relates to the field of biochemistry, specifically, to isolated polypeptides, nucleic acids and applications thereof, more specifically, to isolated polypeptides, nucleic acids, constructs, recombinant cells, methods for improving the activity of carnosine synthase, and methods for preparing carnosine. Background technique [0002] L-carnosine (β-alanyl-L-histidine) is a dipeptide composed of β-alanine and histidine, which is found in high concentrations in vertebrate skeletal muscle and central nervous system (Boldyrev, 2013). L-carnosine has many important physiological functions in the body, including anti-oxidation, anti-glycation, intercellular buffering and elimination of free radicals. Therefore, L-carnosine has attracted much attention as a biologically active compound and is widely used in medicine, cosmetics, food additives and other fields (Yin et al.2019). The research on the production of L-carnosine by chemical syn...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N1/21C12N15/70C12P17/10C12R1/19
CPCC12N9/93C12Y603/02011C12N15/70C12P17/10
Inventor 陈晶瑜黄灿于波张臻峰
Owner CHINA AGRI UNIV
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