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Alginate lyase Alga and coding gene and application thereof

A technology of alginate lyase and sodium alginate, which is applied in the fields of lyase, application, and genetic engineering, and can solve problems such as time-consuming, difficult to control degradation conditions, and complicated operation

Pending Publication Date: 2016-06-29
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

[0003] Among them, the degradation conditions of acid hydrolysis and oxidative degradation methods are difficult to control, and the operation is relatively complicated and time-consuming.

Method used

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  • Alginate lyase Alga and coding gene and application thereof
  • Alginate lyase Alga and coding gene and application thereof
  • Alginate lyase Alga and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cultivation of Vibrioalginolyticus40B and Extraction of Strain Genomic DNA

[0031] The strain used was Vibrio W13, and the monoclonal colony of Vibriosp.W13 strain was picked and inoculated into 50ml liquid medium, and then placed in a shaker with a temperature of 30°C and a rotation speed of 220rpmin for 24 hours, and then cultured The cells were collected by centrifugation and the supernatant was discarded.

[0032] The liquid medium formula used is (g / L): beef extract 5g, glucose 15g, yeast extract powder 1.0g, NaCl5.0g, MgSO4 7H2O0.5g, CaCl20.2g, KH2PO41.0g, FeSO47H2O0.02g, The pH value is 7.0.

[0033] Take 4mL of the cultured bacterial solution, put it in a 2mL tube, centrifuge at 5000g for 10min. The supernatant was discarded, and the genome was extracted using a bacterial genome extraction kit (Thermal Scientific, USA). The operation was as follows: resuspend the bacteria with 180 μL of digestion solution (Digestion Solution), then add 20 μL of proteinase K, mi...

Embodiment 2

[0034] Example 2 Cloning and identification of the gene encoding alginate lyase Alga

[0035] The following amplification primers were designed according to the possible alginate lyase gene in the Vibrioalginolyticus40B genome: upstream primer (5'-CGCATGAAGCATATTTTCTTCAA-3') and downstream primer (5'-CCGGCCTTGGTACTTACCAAAAG-3'). Using the extracted strain genome as a template, PCR amplification was performed to obtain the full-length sequence of alginate lyase Alga 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 between 1.0kb and 2.0kb, which was excised from the agarose gel and purified using a DNA gel recovery kit.

[0036] The purified DNA fragment was connected to the cloning vector pMD19-T, transformed into Escherichia coli DH5α competent cells, and cultured in LB solid medium ...

Embodiment 3

[0045] Construction of recombinant expression vector of alginate lyase Alga

[0046] According to the complete sequence of the alginate lyase gene (signal peptide removed), the upstream primer (5'-CGCGGATCCGTGGGCTGTAATAGTACGGC-3') and the downstream primer (5'-CCGCTCGAGGCCTTGGTACTTACCAAAAG-3') were designed, and PCR amplification was carried out to obtain the complete gene of the alginate lyase Alga long sequence. 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 PCR product was digested with BamHI and XhoI and recovered; the Escherichia coli expression vector pET21a(+) was also digested with BamHI and XhoI and recovered, connected with the above-mentioned target gene and transformed into E. coli DH5α, and screened for the presence of ampicillin Penicillin-resistant transformants. The positive transformant plasmid was extracted with a ...

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Abstract

The present invention discloses an alginate lyase Alga genetic sequence and a preparation method and application thereof. Alginate lyase Alga is derived from Vibrio alginolyticus 40B. The present invention provides a process for preparing the novel alginate lyase Alga, a genetic engineering method is used, an alginate lyase Alga coding gene is cloned into an E. coli expression vector to obtain a recombinant E. coli strain capable of heterologous expression of the alginate lyase Alga, and the alginate lyase Alga prepared by heterologous expression of the recombinant E. coli strain has the function of degrading sodium alginate to prepare sodium alginate oligosaccharide. The alginate lyase Alga can be widely used in chemical industry, agriculture, food, feed additives, pharmaceuticals and algae genetic engineering and other fields.

Description

technical field [0001] The invention designs a alga gum lyase gene alga. The invention also provides the production method and application of the vector containing the gene, the Escherichia coli recombinant strain and the expression product thereof. Background technique [0002] Alginate is a linear acidic polysaccharide composed of α-L-guluronic acid (G) and β-D-mannuronic acid (M) two kinds of monouronic acid, widely present in kelp, wakame, etc. In the cell walls of macroalgae. With the gradual deepening of research on glycobiology and glycochemistry, many marine oligosaccharides, especially the degradation products of alginate-algin oligosaccharides, have various biological activities. For example, alginate oligosaccharides with a low degree of polymerization have biological activities such as hypolipidemic, antiviral, antitumor, and antioxidative, and some have been developed into medicines. There are many methods to prepare alginate oligosaccharides from alginate: a...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12P19/00
Inventor 尹恒朱本伟王文霞谭海东李曙光
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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