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Beta-agarase and applications thereof

An agarase and agar technology, which is applied in the directions of application, enzyme, introduction of foreign genetic material using a carrier, etc., can solve problems such as not being widely used, and achieve the effect of good industrial application prospects.

Inactive Publication Date: 2013-07-10
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although the preparation of oligosaccharides by agarase has many advantages, it is not widely used in industry at present

Method used

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  • Beta-agarase and applications thereof
  • Beta-agarase and applications thereof
  • Beta-agarase and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Isolation of β-agarase gene YM01-3

[0015] Fifteen agarase genes and their gene sequences were obtained by sequencing the entire genome of Streptococcus agarophilus YM01, including 13 β-agarase genes and 2 α-agarase genes, among which YM01-3 A β-agarase gene. The upstream primer (5′-CCGGAATTCATGTATGCAGCAGACTGGGAT-3′) and the downstream primer (5′-CCGCTCGAGTTGGAACTTCCATTGCTGG-3′) were designed using the biological software Primer5.0. Genomic DNA was used as a template for PCR reaction. The composition of the PCR reaction was as follows (25 μl reaction system) :ddH 2 O 10.5 μl, upstream and downstream primers 0.5 μl each, DNA template 1 μl, 2×MasterMix 12.5 μl. The reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 1.5 min, and final extension at 72°C for 10 min, a total of 30 cycles. After the reaction, the PCR product was recovered to obtain β-agarase gene YM01-3...

Embodiment 2

[0016] Embodiment 2: the construction of Escherichia coli cloning vector PUCm-T—YM01-3

[0017] The β-agarase gene YM01-3 was connected to the carrier PUCm-T using DNA Ligation Kit. The connection system (10 μl) was as follows: Solution Ⅰ 5 μl, DNA fragment 4 μl, PUCm-T carrier 1 μl. The connection solution obtained by connecting at 16°C for 16 hours can be used to obtain the E. coli cloning vector PUCm-T—YM01-3, which is used to transform E. coli JM109.

Embodiment 3

[0018] Embodiment 3: Construction of Escherichia coli recombinant strain JM109-PUCm-T-YM01-3

[0019] Add 200 μl of thawed competent E.coli JM109 and 10 μl of the connection solution obtained in Example 2 to a 2 ml Eppendorf tube, ice-bath for 30 minutes, 42°C for 90 seconds, ice-bath for 2 minutes, add 800 μl of LB medium, and shake at 37°C for 1 hour. The bacterial solution was mixed with 4 μl IPTG and 40 μl X-gal, spread on the LB plate containing 100 μg / ml ampicillin, and incubated at 37°C for 12-14h. Pick white colonies for PCR and double enzyme digestion detection, the enzyme digestion system (20μl) is as follows: ddH 2 O 8 μl, PUCm-T—YM01-3 plasmid DNA 8 μl, EcoRI 1 μl, XholⅠ 1 μl, 10×H Buffer 2 μl, those with a specific band of 1260 bp in agarose gel electrophoresis were positive transformation clones, that is, those containing PUCm-T—YM01-3 Escherichia coli JM109-PUCm-T-YM01-3. Send 1ml of the positive clone bacteria liquid for testing, and determine the nucleotide ...

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Abstract

The invention relates to a beta-agarase, the amino acid sequence of the beta-agarase is SEQ ID NO:1, and the nucleotide sequence of encoding gene is SEQ ID NO:2. The beta-agarase has the high temperature resistance characteristic, and can specifically degrade agar to produce neoagarool igosaccharode with the polymerization of 4-10, and the final product is neoagarotetraose and neoagarohexaose, and the beta-agarase has good industrial application prospects.

Description

technical field [0001] The invention belongs to the technical field of gene screening application, and specifically relates to a β-agarase and its application, that is, a β-agarase derived from Catenivulum agarivorans gen. nov. sp. nov. YM01 Gluease gene YM01-3 and its application. Background technique [0002] In the natural environment, agarase is widely distributed, and many microorganisms and some marine molluscs can produce agarase. At present, most of the agarases isolated and studied are from microorganisms, and marine bacteria are the group that produces the most agarases. Agar-degrading bacteria are widely distributed in marine ecosystems, and are distributed in marine plants, animals, seawater and marine sediments. According to the similarity of amino acid sequence, agarase is classified as GH-16, GH-50 and GH-86 in the glycoside hydrolase family (Glycoside hydrolase family). According to the different ways of degrading agarose by agarase, agarase can be divided...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/70C12P19/14C12R1/19
Inventor 史晓翀张晓华董素洁崔方元
Owner OCEAN UNIV OF CHINA
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