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Method for producing neoagarobiose

A new technology of agarose and new agarose, which is applied in the field of enzyme engineering and bioengineering, can solve the problem of inability to determine the effective or main active components, large differences in the composition of agar degradation products, and the degree of polymerization of agar oligosaccharides. Great impact and other problems, to achieve the effect of improving uniformity, simple operation, and improving degradation ability

Active Publication Date: 2019-01-11
AQUABRAIN BIOTECH XIAMEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As for the substrate, most of α-agarase and β-agarase in the prior art can only use agarose as a substrate, and it is difficult to directly use crude agarose as a substrate
For the product, the agar oligosaccharide produced by the biodegradation method is usually a complex substance composed of oligosaccharides of different molecular sizes, and the degree of polymerization of the agar oligosaccharide is not uniform. The enzymes of sugars, octasaccharides, and decasaccharides only contain relatively high agar oligosaccharides in the product under a specific production process, and the degree of polymerization of agar oligosaccharides in the product is greatly affected by the process, resulting in different Production batches of agar degradation products vary widely in composition
[0004] In addition, more and more studies have shown that agar oligosaccharides with different degrees of polymerization have great differences in antioxidant and other biological activities. or main active ingredient

Method used

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  • Method for producing neoagarobiose
  • Method for producing neoagarobiose
  • Method for producing neoagarobiose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Source and Enzyme Activity Analysis of Agarase Agaz306 and Agaz308

[0052] Our research group isolated a marine strain Flammeovirgapacifica WPAGA1 that can degrade agarose from deep sea sediments. Genome sequencing analysis obtained 13 β-agarases, and PCR technology was used to amplify the related agarase genes. The related agarase gene obtained was recovered by agarose gel electrophoresis, and then cloned into the expression vector pEASY-Blunt E2, and transferred into E.coli DH5α, and then sequenced and analyzed to verify the gene sequence. The correct positive clone was selected, cultured overnight at 37°C in LB medium containing 50 μg / mL ampicillin, the plasmid was extracted and transformed into the expression host E.coli BL21(DE3). The positive clones containing related recombinant plasmids were cultured in 50 mL SOC medium containing 50 μg / mL ampicillin at 37 °C until OD 600 When it is about 0.5, add 1 mM IPTG and induce at 16°C for 12 hours. After c...

Embodiment 2

[0055] Embodiment two, the preparation of recombinant plasmid

[0056] 1. Construction of recombinant plasmid pET-Sul1

[0057] (1) First, using primers Sulz308F and Sulz308R, the sulfatase Sulz308 was amplified from the genome of the strain Flammeovirga pacifica WPAGA1;

[0058] (2) Synthesize the signal peptide oligonucleotide chain PelB-Sulz308, the 18 bases at the end of the sequence are the sequence of sulfatase Sulz308;

[0059] (3) Using the above-mentioned Sulz308 gene sequence and signal peptide PelB-Sulz308 as a template, using primers PelB-Sulz308F and Sulz308RC to carry out PCR amplification to obtain the sulfatase Sulz308 sequence fused with the signal peptide PelB at the 5' end;

[0060] The PCR amplification system is as follows:

[0061]

[0062] The PCR reaction conditions are as follows:

[0063]

[0064] The second step to the fourth step reaction were repeated for 35 cycles.

[0065] (4) After recovering the PCR fragment, digest it with restrictio...

Embodiment 3

[0101] Embodiment 3, preparation and growth curve determination of engineering bacteria E.coli BL21 (pET-Sul1, pACY-NAB1)

[0102] (1) Prepare the E.coli BL21 strain containing the recombinant plasmid pACY-NAB1 into competent cells according to the method in the Molecular Cloning Experiment Guide.

[0103] (2) Transfer the recombinant plasmid pET-Sul1 into E.coli BL21 containing the recombinant plasmid pACY-NAB1 to obtain expression strains containing two recombinant plasmids pET-Sul1 and pACY-NAB1.

[0104] (3) Inoculate the recombinant E.coli BL21 containing two recombinant plasmids pET-Sul1 and pACY-NAB1 into 50 mL of SOCB medium containing 25 μg / mL chloramphenicol and kanamycin respectively; E.coli BL21 was inoculated in 50mL SOCA medium containing 25μg / mL chloramphenicol, cultured on a shaker at 37°C and 200rpm, until OD 600 When the temperature is 0.6, add IPTG with a final concentration of 0.1 mM, and continue to culture on a shaker at 24°C.

[0105] (4) E.coli BL21 c...

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Abstract

The invention relates to a method for producing neoagarobiose. According to the method, agarase Agaz306, agarase Agaz308 and selectable sulfatase are combined for use, and crude agar-agar and / or agarose can converted into neoagarobiose. The invention further provides a method for producing neoagarobiose through fermentation of recombinant escherichia coli for co-expressing the above three enzymes.The purity of neoagarobiose produced by virtue of the method is high, the active oxygen content of fibroblasts can be reduced, the content of Aquaporin 3 of keratinocytes can be increased, and the method has wide application prospects in drugs, foods or cosmetics.

Description

technical field [0001] The invention relates to the technical fields of enzyme engineering and bioengineering, in particular to the preparation of agar oligosaccharides by degrading agar with enzymes or recombinant bacteria, and in particular to a method for producing new agarose by using agar degrading enzymes in combination with recombinant bacteria. Background technique [0002] Agarose is an important polysaccharide component in marine algae - red algae, which has important application value in food, medical, biological applications and other fields. Agar oligosaccharide is a new type of marine functional oligosaccharide with a degree of polymerization of 2-20 after hydrolysis of agar polysaccharide. It is mainly composed of repeating units of agarose, including agaroligo saccharides and new agarose Two series of oligosaccharides (neoagarooligo saccharides), agar oligosaccharides with 3,6-inner ether-α-L-galactose residues as reducing ends, and neoagarooligo oligosacchar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/12C12P19/14C12N1/21C12R1/19
CPCC12N9/2468C12P19/12C12P19/14C12Y302/01081
Inventor 产竹华易志伟陈兴麟高波良曾润颖
Owner AQUABRAIN BIOTECH XIAMEN CO LTD
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