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Beta-agarase capable of degrading agarose to produce neoagarotetraose

A new technology of agartetraose and agarase, applied in the directions of hydrolase, glycosylase, enzyme, etc., can solve the problems of enzyme activity and stability that do not meet industrialization requirements, poor specificity, severe reaction conditions, etc. The effect of good industrial application prospects

Active Publication Date: 2014-06-25
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional method of preparing agar oligosaccharides uses chemical methods, the reaction conditions are relatively severe, and the specificity is poor; while the enzymatic preparation has the advantages of mild reaction conditions, high catalytic efficiency, and easy control of the process.
However, the research on the preparation of agar oligosaccharides by enzymatic methods is mostly in the laboratory stage. On the one hand, the indicators such as enzyme activity and stability have not met the requirements of industrialization; on the other hand, the key problem is that it is difficult to obtain high-specificity agarase.

Method used

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  • Beta-agarase capable of degrading agarose to produce neoagarotetraose
  • Beta-agarase capable of degrading agarose to produce neoagarotetraose
  • Beta-agarase capable of degrading agarose to produce neoagarotetraose

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Embodiment 1

[0019] Cloning of embodiment 1β-agarase gene agWH50B

[0020] The β-agarase gene agWH50B in Agarophage agarophora WH0801 (NRRL B-59247(T) or CGMCC1.10131(T)) was cloned by degenerate primer PCR and nested PCR. Specific operation: Cultivate Agarophore WH0801 in 2216E seawater medium until the end of the logarithm, and extract the DNA genome. Using degenerate primers (5′-HTRCCNAAYCAYHTRTAYHTRGGN-3′; 5′-VACRAARCCVACRTTRTARTTTTC-3′), using the genome as a template, the conditions are: 94°C for 5 minutes, then 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 3 minutes Perform 30 cycles in 3 steps, and perform PCR to obtain a fragment of the β-agarase gene, then design primers based on the end sequence of the fragment, and perform three rounds of nested PCR to recover PCR products with a size of 500Kb-2000Kb Fragments (using the chromosome walking kit from Takara Company). The PCR products in each step were ligated to the T-A cloning vector and then transformed into DH5α and...

Embodiment 2

[0021] Example 2 Construction of expression plasmid pETB containing β-agarase gene agWH50B

[0022] According to the obtained full-length sequence of the β-agarase gene, the upstream and downstream primers of the gene were designed (see Example 1), and the genome of Agarophore chrysogenum WH0801 was used as a template to obtain β-agarase by PCR amplification The full-length sequence of the gluease gene. Conditions were 94°C for 2 minutes, followed by 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 3 minutes, 30 cycles and finally 72°C for 10 minutes. Agarose gel electrophoresis showed that there was an obvious single band at the position of 2.8Kb, and the single band was cut and recovered, and the sequencing results showed that the sequence obtained by splicing in Example 1 was correct. The recovered band was double-digested with EcoRI and SacI, and the expression vector pET21a was also double-digested with EcoRI and SacI, and then both the PCR product and the digested vec...

Embodiment 3

[0023] Example 3 Construction of engineering bacteria pETB / BL21 highly expressing β-agarase gene agWH50B

[0024] The expression vector plasmid pETB was transformed into Escherichia coli BL21 (DE3) according to the standard calcium chloride heat shock transformation, and positive transformants with ampicillin resistance were screened. The plasmid was extracted by standard alkaline lysis method, and the plasmid was double digested with EcoRI and SacI. The gel electrophoresis bands showed two clear bands of 2.9Kb and 5.3Kb, corresponding to the full length of the β-agarase gene and the length of the plasmid. It was proved that the expression vector plasmid pETB containing β-agarase gene had been transformed into Escherichia coli BL21 (DE3).

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Abstract

The invention provides novel beta-agarase, namely, beta-agarase capable of specifically degrading agarose to produce neoagarotetraose, application of the novel beta-agarase, and a gene for encoding beta-agarase. The novel beta-agarase is characterized in that the amino acid sequence is as shown in SEQ ID No.: 1; and the nucleotide sequence of the gene is shown as SEQ ID No.: 2. The beta-agarase is used for degrading agarose to produce neoagarotetraose and can specifically degrade agarose to produce the unique product, namely, neoagarotetraose; and therefore, the beta-agarase can be used for preparing high-purity neoagarotetraose; the conversion rate of the beta-agarase to agarose within 48 hours is more than 90%, and the purity of neoagarotetraose is higher than 95%; and therefore, the beta-agarase has a good industrial application prospect.

Description

technical field [0001] The invention belongs to the technical field of functional gene screening, and in particular relates to a β-agarase for degrading agarose to generate new agarose and its application. Background technique [0002] Agar gum is mainly a kind of polysaccharide extracted from red algae such as agarose, river fence, and chicken feather, and is composed of agarose (agarose) and agaropectin (agaropectin). Agarose can be hydrolyzed by agarase to generate agarose oligosaccharides, which have good functions of scavenging free radicals, inhibiting inducible NO synthase and promoting the growth of probiotics, and are widely used in the food and pharmaceutical industries. Moreover, the functional activity of agar oligosaccharides is closely related to its degree of polymerization. Agarose oligosaccharides with a degree of polymerization of two to four can inhibit the production of tumor necrosis factor, excess NO free radicals and prostaglandin E2 (PGE2); six to ei...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12N1/21C12P19/14
CPCC12N9/2468C12N15/70C12P19/14C12Y302/01081
Inventor 毛相朝刘楠杨孟薛长湖魏东芝
Owner OCEAN UNIV OF CHINA
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