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Agarase and preparation method thereof

An agarase and nucleotide technology, which is applied in the fields of medicinal chemistry and biochemical industry, can solve the problems of low activity, low yield, and has not been widely used, and achieves a simple purification and preparation method, simple culture conditions and short fermentation time. Effect

Active Publication Date: 2020-01-24
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although agarase has many advantages in preparing oligosaccharides, it has not been widely used in industry. The preparation of high-efficiency and low-cost agarase preparations has become a key factor limiting the high-value utilization of red algae industry.
At present, the market for industrial-grade agarase preparations is still blank, and commercial-grade agarase is expensive and only for research use
According to the survey of reported data, most agarase-producing strains have low activity and low yield, which hinders the in-depth development of the red algae industry

Method used

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  • Agarase and preparation method thereof
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  • Agarase and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 2

[0038] Example 2. Construction of recombinant vector derived from Pseudoalteromonas sp. Q30F CCTCC No: M 2017622 agarase

[0039] After determining the restriction enzyme cutting site analysis in the sequence, and based on this, the restriction enzyme cutting site analysis in the sequence, the front and rear primer pairs containing the restriction enzyme cutting site contain 1281bp of the agarase gene (SEQ.ID.No.8) and 1338bp (SEQ.ID.No.4) sequences were amplified by PCR, and the PCR products were purified by the Cycle-Pure Kit (OMEGA Bio-TekCo.) purification kit, respectively, with the vector pProEX- HTa and pHT43 were subjected to double enzyme digestion, the enzyme digestion conditions: 37°C, 5h. The digested product was subjected to agarose electrophoresis, electrophoresis conditions: 70V, 1h, gel extraction kit (OMEGA Bio-Tek Co.) was used for gel extraction, and T4 ligase was used for 1281bp and 1338bp containing agarase gene The sequence fragments were respectively con...

Embodiment 3

[0040] Example 3. Cloning and expression host bacteria construction containing agarase gene recombinant

[0041] Escherichia coli strain DH5α (Invitrogen, genotype: F-φ80 lac ZΔM15 Δ(lacZYA-argF) U169 endA1 recA1 hsdR17(rk-,mk+) supE44λ- thi -1 gyrA96 relA1 phoA was selected. After competent cell preparation, heat shock transformation (42°C, 60s), incubate (37°C, 160rpm, 45min), on a solid plate containing 100μg / mL ampicillin sodium (LB solid medium: tryptone: 10g, yeast extract: 5g, NaCl: 10g, distilled water: 1000mL, pH7.0), cultured at 37°C for 16h, colony PCR detection, obtained positive clone strains AgaraseNS-HTa-DH5α, AgaraseE-HTa-DH5α, AgaraseNS-pHT43-DH5α, Agarase-pHT43-DH5α, four positive strains Inoculated in LB liquid medium respectively, cultured at 37°C for 12 hours, and extracted plasmids with Plasmid Mini Kit (OMEGA Bio-Tek Co.) to obtain four recombinant plasmids: AgaraseNS-HTa, Agarase-HTa, AgaraseNS-pHT43, Agarase-pHT43 .

[0042] Select Escherichia coli e...

Embodiment 4

[0044] Example 4. Induced expression of genetically engineered expression strains containing agarase

[0045]Inoculate positive clones AgaraseNS-HTa-BL21 and Agarase-HTa-BL21 in LB liquid medium (100 μg / mL ampicillin sodium), culture at 37°C until OD600 is 0.6, add IPTG to 0.5mM concentration, and induce at 24°C After 24 hours, the activity of intracellular and extracellular agarase was measured by the calibration reducing sugar method. After measurement, the final extracellular agarase activity data of the Agarase-HTa-BL21 expression strain reached 265.6 U / L, and the intracellular agarase Live data up to 105.2 U / L. The final extracellular agarase activity data of the AgaraseNS-HTa-BL21 expression strain reached 134.5 U / mL, and the intracellular protease activity data reached 244.5 U / L.

[0046] Inoculate the positive clone strains Agarase-pHT43-WB800N and AgaraseNS-pHT43-WB800N in sterile LB liquid medium containing 5 μg / mL chloramphenicol, cultivate at 37°C until the OD600 ...

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Abstract

The invention belongs to the field of medicinal chemistry and biochemical engineering, and relates to pseudoalteromonas, agarase and a preparation method thereof. Specifically, the invention relates to pseudoalteromonas sp. Q30F derived from sludge in an intertidal zone, and the strain has the characteristics of simple nutritional conditions and easiness in culture. The invention also discloses the agarase, an amino acid sequence and the gene coding thereof. The invention also relates to a coding gene of the agarase, and an expression vector and a cell line thereof.

Description

technical field [0001] The invention belongs to the fields of medicinal chemistry and biochemical industry. Background technique [0002] Agar (Agar) is mainly produced from edible red algae such as Gracilaria and Laver. Together with alginate and carrageenan, it is the three major marine polysaccharides with the largest output and widest application. It is widely used in food, medicine and chemical industries. Agarose (Agarose) is one of the main components of agarose, which is a neutral polysaccharide. Its molecular backbone consists of disaccharide units β-D-galactose and 3,6-inner ether-α-L-galactose with α- 1,3 and β-1,4 glycosidic linkages are alternately connected. Agar oligosaccharides are low molecular weight oligosaccharides obtained after hydrolysis of agar gel. There are two types of agar oligosaccharides, agar oligosaccharides and new agar oligosaccharides. Agar oligosaccharides have anti-inflammatory, anti-allergic, anti-tumor, immune regulation, whitening, ...

Claims

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Application Information

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IPC IPC(8): C12N9/24C12N15/56C12N15/70C12N15/75C12N1/21C12P19/14C12R1/19C12R1/125
CPCC12N9/2402C12N15/70C12N15/75C12P19/14
Inventor 江晓路朱常亮崔欣
Owner OCEAN UNIV OF CHINA
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