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A kind of agarase with uniform degradation product and application thereof

A degradation product, agarase technology, applied in the field of enzyme engineering, can solve problems such as complex components, and achieve the effect of good industrial application prospects

Active Publication Date: 2022-02-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, currently reported β-agarase hydrolysis products have complex components, usually containing neoagarose, tetraose and hexaose

Method used

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  • A kind of agarase with uniform degradation product and application thereof
  • A kind of agarase with uniform degradation product and application thereof
  • A kind of agarase with uniform degradation product and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the construction of genetically engineered bacteria

[0037] According to the complete genome sequence of Paenibacillus odorifer, specific primers (Table 1) were designed, and PCR amplification was performed using the Paenibacillus odorifer genome as a template and primers F1 and R1. After obtaining the target product, it was sent to Tianlin Biotechnology (Wuxi) Co., Ltd. for sequencing to obtain the agarase AgaP1 gene sequence whose nucleotide sequence is shown in SEQ ID NO.2 (amino acid sequence is shown in SEQ ID NO.1). The PCR product and plasmid vector pColdII were double digested with restriction endonucleases KpnI and XbaI, and ligated to obtain a recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21(DE3) competent cells to obtain the recombinant strain E.coli BL21-pColdII-AgaP1.

[0038] Table 1 Primer Sequence

[0039]

Embodiment 2

[0040] Embodiment 2: expression and purification of agarase (AgaP1)

[0041] LB medium (g / L): sodium chloride 10, tryptone 10, yeast extract 5, pH 7.

[0042] Taking the original strain E.coli BL21(DE3) and the empty strain E.coli BL21(DE3) into the pClodII plasmid) as a control, the recombinant E. coli BL21-pColdII-AgaP1 was inoculated in the In LB liquid medium, culture at 37°C, 200rmp for 12h, then inoculate 500μL of the above seed solution into 50mL LB medium containing 50μL ampicillin, and culture at 37°C for 2.5h, until OD 600 to 0.5, lower the temperature of the shaker to 15°C and let it stand for 30 minutes. 20 μL of IPTG with a final concentration of 0.2 mM was added to each bottle as an inducer, and no inducer was used as a control group, and cultured at 15° C. and 200 rpm for 24 hours.

[0043] Collect the bacterial liquid, centrifuge at 4°C, 8000rmp for 10 minutes to obtain the bacterial cells, add 5 mL of phosphate buffer (0.02mol / L, pH 7.0) to resuspend the bac...

Embodiment 3

[0044] Embodiment 3: the enzyme activity assay of agarase (AgaP1)

[0045]In disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH 7), with agarose as substrate, in the range of 20-60 ° C, every 10 ° C, the activity of agarase AgaP1, it can be seen that the activity of agarase AgaP1 The optimum temperature is 40°C (see figure 1 ). Under the optimum reaction temperature of 40°C, within the range of pH 4.0-9.0, measure the enzyme activity at intervals of 1 unit, and determine that the optimal reaction pH is 7.0 (see figure 2 ).

[0046] In disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7.0), under the condition of 40 DEG C, the enzyme activity and The specific enzyme activity of the purified agarase AgaP1, wherein the enzyme activities of AgaP1 in the crude enzyme solution are 5716.68U / L and 4116.96U / L respectively, and the specific enzyme activities of the purified agarase AgaP1 are 694.61U / mg and 490.11U / mg.

[0047] Temper...

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Abstract

The invention discloses agarase with uniform degradation products and its application, belonging to the field of enzyme engineering. In the present invention, agarase AgaP1 derived from Paenibacillus odorifer (the amino acid sequence of which is shown in SEQ ID NO. 1) is heterologously expressed in Escherichia coli, wherein agarose and agar powder in the crude enzyme solution are substrates The enzymatic activities were 5716.68U / L and 4116.96U / L, respectively. The purified agarase AgaP1 pure enzyme solution can degrade agarose to obtain a single degradation product, new agarobiose, and is an agarase with industrial application prospects.

Description

technical field [0001] The invention relates to an agarase with uniform degradation product and application thereof, belonging to the field of enzyme engineering. Background technique [0002] Agar gum, also known as agar, is a polysaccharide extracted from red algae such as Geliflower and Gracilaria, and belongs to one of the three major alginates. Its main components are agarose and agar gel. Agarose is β-D-galactose (G) linked by 1,3 glycosidic bonds and 3,6-inner ether-α-glucose linked by 1,4 glycosidic bonds. L-galactose (LA) residues are repeatedly and alternately linked into linear polysaccharide molecules, non-ionic polysaccharides that do not contain sulfate (salt), and are the components that form gels. The structure of agar gel is that the cross-linked galactose sugar chain often contains D-galactose, 3,6-galactoside, galacturonic acid and charge groups such as sulfuric acid, carboxylic acid, pyruvic acid, etc. Inorganic elements such as calcium and magnesium. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/24C12N15/56C12P19/14C12P19/12C12N15/70C12N1/21C12R1/19
CPCC12N9/2402C12P19/14C12P19/12
Inventor 廖祥儒李静杨邵岚黄琳蔡宇杰管政兵
Owner JIANGNAN UNIV
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