ATC racemase and coding gene thereof, and application of recombinant expression protein thereof

A racemase and protein technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problem of few reports of L-cysteine-related enzymes, achieve good industrial value, high catalytic efficiency, The effect of convenient separation and purification

Inactive Publication Date: 2012-04-18
天津启仁医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there are few reports on enzymes related to enzymatic synthesis of L-cysteine

Method used

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  • ATC racemase and coding gene thereof, and application of recombinant expression protein thereof
  • ATC racemase and coding gene thereof, and application of recombinant expression protein thereof
  • ATC racemase and coding gene thereof, and application of recombinant expression protein thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Cloning and Primary Structure Characterization of ATC Racemase

[0027] The present inventor extracted genomic DNA from Pseudomonas sp.QR-101, then digested genomic DNA with Hind III, and recovered 2-9kb Hind III enzymes from 1.2% agarose gel with a gel recovery kit Cut the fragments, connect the recovered enzyme-digested fragments to the pUC18 vector that has been treated with the same restriction enzymes, transfer them into E.coli JM109, and perform blue-white primary screening on a plate coated with X-gal and IPTG.

[0028] Recombinant white single colonies containing insert fragments were picked into each well containing 50 μL of LB medium, and after overnight culture at 37°C, 100 μL of 0.6% D was added, L-ATC was used as a substrate, and L-ATC hydrolase (the For the gene sequence, see SEQ ID No.3 in the sequence table) and 0.5 U each of L-NCC amidohydrolase (see the sequence table, SEQ ID No.4 for the gene sequence), shake at 35°C for 30 min, and add 150 μL of acid...

Embodiment 2

[0031] Construction of Genetic Engineering Bacteria of Escherichia coli and Expression of Recombinant ATC Racemase

[0032] According to the sequencing results, primers P1 and P2 were designed according to the two-end sequences of the protein-coding gene, wherein the upstream P1: 5'-CCGGAA TTC ATG AAG CAT CAT CAG ACG GGC AT-3' contains the EcoR I restriction site; the downstream primer P2 : 5'-CCC AAGCTT CTA GCC CAA CAG TTT TCC CAGGC-3' contains a HindIII restriction site.

[0033] Using the genome of strain Pseudomonas sp.QR-101 as a template, DNA amplification was performed according to the following PCR procedure:

[0034] Denaturation at 94°C for 1 min, renaturation at 66°C for 1 min, extension at 72°C for 1 min, and 30 cycles of amplification reaction.

[0035] The PCR amplified product was double digested with EcoR I and Hind III, and connected to the vector pET-21a(+) after the same digestion to construct the recombinant pET21-a(+) / atcA.

[0036] The obtained recombin...

Embodiment 3

[0038] Example 3 Verification of Recombinant ATC Racemase Activity

[0039] Take the enzyme source cell suspension in Example 2, adjust the final concentration to 20g / L, and add 3mL of 0.5% substrate DL-ATC and 1.5mL of mixed enzyme solution in sequence to a 5mL reaction tube, wherein, in each group The composition of the mixed enzyme solution is shown in Table 1.

[0040] Table 1 Composition of Mixed Enzyme Solution

[0041]

[0042] In a water bath at 35°C for 2 hours, each reaction solution was freeze-dried, then dissolved in 300 μL of isopropanol, and the contents of D-ATC and L-ATC were determined by high-performance liquid chromatography.

[0043] High performance liquid chromatography adopts CHIRALPAK IC column (0.46cm I.D.×25cm L) (Daicel chiral technologies CO., LTD., Shanghai, CHINA), the liquid phase conditions are: ultraviolet detector SPD-10A, detection wavelength is 220nm, flow The phase was n-hexane-isopropanol (85:15), containing 0.2% trifluoroacetic acid ...

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Abstract

The invention discloses ATC racemase and a coding gene thereof, and a preparation method and an application of a recombinant expression protein thereof. The provided ATC racemase is derived from pseudomonas with a collection number of CGMCC No. 5315. The ATC racemase is a protein with an amino acid residue sequence represented by SEQ ID No.1 or a protein derived from that with an amino acid residue sequence represented by SEQ ID No.1, wherein the derived protein has a same activity with that of SEQ ID No.1, and the derived protein is derived from the protein with the amino acid residue sequence represented by SEQ ID No.1 through substitution, deletion and addition of one or more amino acid residues. The sequence of the coding gene is a nucleotide sequence represented by SEQ ID No.2 in the sequence list. According to the invention, an ATC racemase gene derived from pseudomonas is cloned and used for expressing and preparing recombinase. The recombinase can be used for catalytically separating DL-ATC into L-ATC with a biological method; the produced L-ATC can be used as a substrate in enzymatic production of L-cysteine.

Description

【Technical field】: [0001] The invention belongs to the field of biotechnology, and relates to a coding gene of an ATC racemase derived from pseudomonas, and expression and application of recombinant protein. 【Background technique】: [0002] ATC racemase (ATC racemase) is a catalytic split DL-ATC (DL-2-amino-Δ2-thiazoline-4-carboxylic acid, DL-2-amino-Δ2-thiazoline-4-carboxylic acid) to generate L - ATC (L-2-amino-Δ2-thiazoline-4-carboxylic acid, L-2-amino-Δ2-thiazoline-4-carboxylic acid) enzyme. L-ATC can be used as a substrate, through S-carbamyl-L-cysteine ​​(S-carbamyl-L-cysteine, L-SCC) or N-carbamyl-L-cysteine ​​(N -carbamyl-L-cysteine, L-NCC) is the transformation pathway of the intermediate product to generate the final product L-cysteine, so ATC racemase is also an important factor involved in the enzymatic conversion of DL-ATC to produce L-cysteine One of the members, the specific process can be found in the appendix figure 1 . [0003] L-cysteine ​​(L-cysteine)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12N15/61C12N15/63C12N15/70C12N1/21C12P41/00C12P13/12C12R1/38C12R1/19
Inventor 高智慧张奇段静静刘磊姚瑞娟王文芳
Owner 天津启仁医药科技有限公司
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