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Recombinant mannuronic acid C-5 epimerase as well as coding gene, preparation method and application thereof

A technology of mannuronic acid and epimerase, applied in the field of genetic engineering, can solve the problems of low enzyme activity and the like

Pending Publication Date: 2022-06-03
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on this kind of enzymes, and due to the low activity of the enzymes, they are still in the laboratory stage, and the actual application of them to the regulation of the G content of fucoidan is still blank.

Method used

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  • Recombinant mannuronic acid C-5 epimerase as well as coding gene, preparation method and application thereof
  • Recombinant mannuronic acid C-5 epimerase as well as coding gene, preparation method and application thereof
  • Recombinant mannuronic acid C-5 epimerase as well as coding gene, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Mannuronic acid C-5 epimerase PmC5A mutant full-length gene cloning

[0043] The genomic DNA of Pseudomonas mendoza was extracted by referring to the procedure of the genomic DNA purification kit (Thermo, LOT 00105781). After the alignment and analysis of the mannuronic acid C-5 epimerase PmC5A gene sequence, two pairs of primers were designed as follows:

[0044] SEQ ID NO. 5:

[0045] 238N-S-F: 5'-ACGTGGTGATCAAGGGCAGCACCTACCGCGACAACATCGTCTACGGC-3'; SEQ ID NO. 6:

[0046] 238N-S-R: 5'-GCCGTAGACGATGTTGTCGCGGTAGGTGCTGCCCTTGATCACCACGT-3'; SEQ ID NO. 7:

[0047] 348S-N-F: 5'-ACGGCATTCGCATCCGCAACAGCGAGAACATTCGCCTCTACGACAAC-3'; SEQ ID NO. 8:

[0048] 348S-N-R: 5'-GTTGTCGTAGAGGCGAATGTTCTCGCTGTTGCGGATGCGAATGCCGT-3'. Using the extracted genomic DNA of Pseudomonas mendoza as a template, the gene sequence encoding the mature protein of the bifunctional enzyme (excluding the signal peptide gene) was amplified. PCR reaction conditions were: 94°C for 2 min, 1 cycle; 9...

Embodiment 2

[0049] Example 2 Gene sequence analysis of mannuronic acid C-5 epimerase mutants

[0050] The sequencing results were analyzed by the Basic Local Alignment Search Tool (BLAST) in the GenBank database, DNAMAN software was used for multiple sequence alignment, and Vector NTI was used to analyze the sequence information.

[0051] The obtained two PmC5A mutant genes (named PmC5A 238N-S and PmC5A 348S-N ) coding region length 1428bp, PmC5A 238N-S and PmC5A 348S-N The nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.3, respectively. The PmC5A mutant encodes 467 amino acids and a stop codon, PmC5A 238N-S and PmC5A 348S-N The amino acid sequences of are shown in SEQ ID NO.2 and SEQ ID NO.4, respectively.

Embodiment 3

[0052] Example 3 Recombinant expression and purification of PmC5A mutant gene in Escherichia coli

[0053] In order to facilitate the recombinant expression of the gene, NdeI and XhoI restriction sites were introduced into the designed upstream and downstream primers respectively. The PCR product PmC5A mutant and the expression vector pET21a were double digested with NdeI and XhoI, respectively. 4 DNA ligase ligation (ligation system: (5 μL 4 DNA Ligase 0.5μL, 10×T 4 DNA LigaseBuffer 0.5μL, pET21a 2μL, PCR product 2μL), ligation conditions: overnight ligation at room temperature. ). 5 μL of the ligation product was transformed into E. coli TOP10 competent cells, spread on solid Luria-Bertani medium containing 100 μg / mL ampicillin, and cultured at 37°C for 12-16 h. Pick a single clone, use degenerate primers for colony PCR verification, insert the correctly amplified single clone into the liquid Luria-Bertani medium containing 100 μg / mL ampicillin, and extract the plasmid; ...

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Abstract

The invention discloses recombinant mannuronic acid C-5 epimerase as well as a coding gene, a preparation method and application thereof, and belongs to the field of gene engineering. In particular discloses two mutants of mannuronic acid C-5 epimerase PmC5A genes derived from Pseudomonas mendocina as well as a preparation method and application of enzymes of the two mutants, namely the genes of the mutated enzymes are cloned to an escherichia coli expression vector by utilizing a genetic engineering technical method, so that an escherichia coli recombinant strain capable of heterologously expressing the enzymes is obtained, and the two mutants can be used for preparing the two mutants of the mannuronic acid C-5 epimerase PmC5A genes of the Pseudomonas mendocina and the enzymes of the two mutants of the two mutants of the mannuronic acid C-5 epimerase PmC5A genes of the Pseudomonas mendocina. The enzyme prepared by heterologous expression of the strain can efficiently convert beta-D-mannuronic acid (M) in fucoidan into alpha-L-guluronic acid (G), and the degradation effect is basically avoided. The mannuronic acid C-5 epimerase provided by the invention can be widely applied to the fields of food, medicine and the like.

Description

technical field [0001] The invention relates to a recombinant mannuronic acid C-5 epimerase and its encoding gene, as well as a preparation method and application, and belongs to the field of genetic engineering. Background technique [0002] Fucoidan is a linear polymerization composed of two monomers, β-D-mannuronic acid (Mannuronate, M) and its C-5 epimer, α-L-guluronic acid (Guluronate, G). thing. There are three forms of M and G in fucoidan: poly-mannuronate (PM), poly-guluronate (PG) and mannuro-guro Uronic acid mixed block (MG block). It is shown in the following structural formula: [0003] [0004] Different sources of fucoidan not only have different intermolecular M / G ratios, but also different order of M and G. The differences in structure lead to great differences in the physicochemical properties of fucoidan such as gel strength and water holding capacity. The study found that the larger the M / G value of the fucoidan, the smaller the gel strength and the...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12N15/61C12P19/24C12P19/00
CPCC12N9/90C12Y501/00C12P19/24C12P19/00
Inventor 尹恒孙明王文霞
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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