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Mannuronan C-5 epimerase/alginate lyase encoding gene, and mannuronan C-5 epimerase/alginate lyase enzyme and preparation and application thereof

A technology of alginate lyase and mannuronic acid, which is applied in the field of genetic engineering, can solve the problems of difficulty in finding, large molecular weight of sodium alginate, and poor solubility

Active Publication Date: 2020-10-30
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

[0011] Sodium alginate has a wide range of applications due to different M / G ratios, but natural sodium alginate has a large molecular weight and poor solubility, so the degradation of sodium alginate has once become a research hotspot
In the study, it was found that alginate lyase, as a biological enzyme with mild reaction conditions and high efficiency, has attracted everyone's attention. However, during the cleavage process, the ratio of M / G will change uncontrollably. Therefore, it is necessary to find a specific ratio Or the sodium alginate oligosaccharides arranged in sequence become a big problem
There are no reports of mannuronate C-5 epimerase from Pseudomonas sp. having these two functional properties

Method used

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  • Mannuronan C-5 epimerase/alginate lyase encoding gene, and mannuronan C-5 epimerase/alginate lyase enzyme and preparation and application thereof
  • Mannuronan C-5 epimerase/alginate lyase encoding gene, and mannuronan C-5 epimerase/alginate lyase enzyme and preparation and application thereof
  • Mannuronan C-5 epimerase/alginate lyase encoding gene, and mannuronan C-5 epimerase/alginate lyase enzyme and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1 bifunctional enzyme full-length gene cloning

[0046] Genomic DNA of Pseudomonas mendoza was extracted according to the operation steps of Genomic DNA Purification Kit (Thermo, LOT 00105781). After multiple sequence alignment analysis of bifunctional enzyme gene sequences in The National Center for Biotechnology Information (NCBI) database, degenerate primers PmC5A-F: 5'-ATACATATGAACCCGMHGSARSACCAGTTC-3'; PmC5A-R: 5'-TATCTCGAGRTTGGTCAKYKKCTCGG- 3', using the extracted genomic DNA of Pseudomonas mendoza as a template to amplify the gene sequence (excluding the signal peptide gene) encoding the mature protein of the bifunctional enzyme. The PCR reaction conditions were: 98°C for 2 min, 1 cycle; 98°C for 10 s, 62°C for 30 s, 72°C for 2 min, 30 cycles; 72°C for 5 min, 1 cycle. PCR products were analyzed by agarose gel electrophoresis (see figure 1 ), the target gene was recovered by cutting the gel, connected to the prokaryotic expression vector pET21a by dou...

Embodiment 2

[0047] Embodiment 2 bifunctional enzyme gene sequence analysis

[0048] The sequencing results were analyzed using 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 sequence information.

[0049] The coding region of the obtained alginate lyase gene (named PmC5A) is 1428 bp long, and its nucleotide sequence is shown in SEQ ID NO 1. PmC5A encodes 467 amino acids and a stop codon, its amino acid sequence is shown in SEQID NO 2, the theoretical molecular weight of the protein is 52.39kDa, and the predicted isoelectric point is 5.61.

Embodiment 3

[0050] Example 3 Recombinant expression and purification of PmC5A gene in Escherichia coli

[0051] In order to facilitate the recombinant expression of the gene, NdeI and XhoI restriction sites were respectively introduced into the designed upstream and downstream primers. The PCR cleaning product PmC5A and the expression vector pET21a were double-digested with NdeI and XhoI respectively. 4 DNA ligase connection (ligation system: (5μLT 4 DNA Ligase 0.5μL, 10×T 4 DNA LigaseBuffer 0.5 μL, pET21a 2 μL, PCR product 2 μL), ligation conditions: overnight at room temperature. ). Take 5 μL of the ligation product to transform E.coli TOP10 competent cells, spread on solid Luria-Bertani medium containing 100 μg / mL ampicillin, and culture at 37°C for 12-16h. Pick a single clone, use degenerate primers for colony PCR verification, insert the correctly amplified single clone into liquid Luria-Bertani medium containing 100 μg / mL ampicillin, and extract the plasmid; use endonucleases Nd...

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Abstract

The invention discloses a mannuronan C-5 epimerase / alginate lyase encoding gene, and a mannuronan C-5 epimerase / alginate lyase enzyme and preparation and application thereof, belonging to the field ofgene engineering. The invention particularly discloses an enzyme gene which is derived from pseudomonas mendocina and has two functions of epimerizing mannuronan C-5 and cracking alginate, and a preparation method and application of the enzyme of the enzyme gene. According to the invention, a gene of the enzyme is cloned to an escherichia coli expression vector by utilizing a technical method ofgenetic engineering to obtain a recombinant escherichia coli strain capable of heterologously expressing the enzyme, and the enzyme prepared by heterologously expressing the strain can efficiently convert beta-D-mannuronan (M) into alpha-L-guluronate (G) and crack sodium alginate into oligosaccharide. The bifunctional enzyme provided by the invention can be widely applied to the fields of agriculture, food, feed additives, medicines and the like.

Description

technical field [0001] The invention relates to a mannuronic acid C-5 epimerase / alginate lyase coding gene, enzyme, preparation and application, and belongs to the field of genetic engineering. Background technique [0002] Brown algae is an economical seaweed plant that is abundant in the ocean. It contains various polysaccharides such as algin, fucoidan and kelp starch. Among them, the content of alginate is relatively high, accounting for 20%-40% of the dry weight of the algal body. In algae, alginate mainly exists in the form of alginic acid, alkali metal salts (sodium salt, potassium salt) and divalent salts (calcium salt, magnesium salt). Alginate acts as a filling material for the cell wall of brown algae, providing hardness and toughness to the algal body, and its physiological characteristics are similar to cellulose in land plants. [0003] Alginate has the common characteristics of polysaccharides, such as water retention and variable solution viscosity, but as ...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N9/90C12N15/60C12N15/61C12P19/04C12P19/02C12P19/00C12P19/12C12P19/24
CPCC12N9/90C12Y501/00C12N9/88C12P19/02C12P19/04C12P19/12C12P19/00C12P19/24
Inventor 尹恒孙明
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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