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Truncated algin lyase Aly7B-CDII gene and application thereof

A alginate lyase and gene technology, applied in the field of genetic engineering technology and protein expression, can solve the problems of complicated operation, long time-consuming, difficult to control degradation conditions, etc.

Active Publication Date: 2019-11-15
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the chemical degradation method based on acid degradation has the disadvantages that the degradation conditions are difficult to control, the operation is complicated, and it takes a long time.

Method used

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  • Truncated algin lyase Aly7B-CDII gene and application thereof
  • Truncated algin lyase Aly7B-CDII gene and application thereof
  • Truncated algin lyase Aly7B-CDII gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Cultivation and genome extraction of bacterial strain Vibrio sp.W13

[0035] The strain Vibrio sp.W13 used is kept in our laboratory. The formulation of the culture medium used is:

[0036] LB medium: tryptone 10g, yeast extract 5g, NaCl 10g, pH7.0. For solid medium add 1.5% agar. Autoclaved.

[0037] Beef extract 5g, glucose 15g, yeast extract powder 1.0g, NaCl 5.0g, MgSO 4 ·7H 2 O 0.5g, CaCl 2 0.2g, KH 2 PO 4 1.0g, FeSO 4 ·7H 2 O 0.02g, pH value is 7.0. For solid medium add 1.5% agar.

[0038] Spread the preserved strains on the solid selection culture, and cultivate the activated colonies in the liquid medium. Take 4mL of the cultured bacterial solution, put it in a 2mL tube, centrifuge at 5000g for 10min. Discard the supernatant, resuspend the bacteria with 180 μL of Digestion Solution, then add 20 μL of proteinase K, mix thoroughly, and incubate at 56°C for 30 min. Add 20 μL of RNaseA, mix thoroughly, and place at room temperature for 10 mi...

Embodiment 2

[0039] Embodiment 2: Extraction of truncating enzyme Aly7B-CDII

[0040] According to the gene sequence of the second catalytic domain in Vibrio sp.W13 genome, the following amplification primers were designed: forward primer (5'-TGGAATATTGACGATTGG-3') and reverse primer (5'-ATGAAGAGTGCTCAAAGCAC-3'). Using the extracted strain genome as a template, PCR amplification was performed to obtain the full-length sequence of the truncated Aly7B-CDII gene. The PCR conditions were: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 2 minutes, and finally extension at 72°C for 10 minutes. Agarose gel electrophoresis showed a specific band near 1.0kb, which was excised from the agarose gel and purified using a DNA gel recovery kit.

[0041] Ligate the purified DNA fragment to the cloning vector pEASY-Blunt Zero Cloning vector, transform Escherichia coli DH5α competent cells, culture in LB solid medium (containing ampicillin), pick wh...

Embodiment 3

[0042] Example 3: Construction of alginate lyase Aly7B-CDII recombinant expression vector and corresponding expression purification.

[0043] The forward primer (5'-TGGAATATTGACGATTGG-3') and the reverse primer (5'-ATGAAGAGTGCTCAAAGCAC-3') were designed according to the truncation Aly7B-CDII. PCR amplification was performed to obtain the full-length sequence of the truncated Aly7B-CDII gene. The PCR conditions were: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 2 minutes, and finally extension at 72°C for 10 minutes. The transformant with ampicillin resistance was screened, the positive transformant plasmid was extracted with a plasmid extraction kit, and the recombinant plasmid was named pET21a-Aly7B-CDII.

[0044] The recombinant expression plasmid pET21a-Aly7B-CDII was transformed into Escherichia coli strain BL21(DE3) (purchased from Novagen, USA), and then the truncated Aly7B-CDII was induced, expressed and puri...

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Abstract

The invention relates to a truncated algin lyase gene Aly7B-CDII. The nucleotide sequence of the truncated algin lyase gene Aly7B-CDII is as shown in SEQID NO.1. The algin lyase gene Aly7B-CDII is cloned and transformed to an escherichia coli expression carrier, an escherichia coli recombinant strain capable of performing heterologous expression is obtained, and the strain is used for performing heterologous expression on the truncated body. The enzymology property characterization result indicates that the truncated body Aly7B-CDII exhibits activity on algin, poly M and poly G. The truncatedbody Aly7B-CDII exhibits substrate specificity similar to that of holoenzyme, but the pH and the temperature stability of the truncated body are better than those of the holoenzyme. The truncated algin lyase gene Aly7B-CDII has enormous reference value on reconstructing and exploring of novel algin lyase, and the obtained algin lyase truncated body can be used as a potential tool for producing andpreparing alginate-derived oligosaccharide.

Description

technical field [0001] The invention belongs to the field of genetic engineering technology and protein expression, in particular to a truncated alginate lyase gene and its application. Background technique [0002] my country has extremely rich marine biological resources, especially seaweed resources. As a large algal resource, brown algae is favored by developers because its cell wall is rich in alginate, a polysaccharide with unique properties and great development value. Alginate is a straight-chain acidic polysaccharide composed of β-D-mannuronic acid (mannuronic, M) and α-L-guluronic acid (guluronic, G) through α-1,4-glycosidic bonds. into linear anionic polysaccharides. In the natural state, alginate mainly exists in water-soluble alginates such as sodium alginate and potassium alginate, and water-insoluble alginic acid. Sodium alginate (trade name: sodium alginate) or other alginates currently on the market are mainly extracted from brown algae. Studies have sho...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21C12P19/00C12P19/02C12R1/19
CPCC12N9/88C12N15/70C12P19/00C12P19/02
Inventor 倪芳朱本伟胡富姚忠孙芸李谦
Owner NANJING UNIV OF TECH
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