Modified agarase and application thereof

A technology of agarase and new agar oligosaccharides, which is applied in the field of enzyme engineering, can solve the problems that new agar oligosaccharides with a low degree of polymerization cannot be obtained in large quantities, and achieve the effects of convenient control, low requirements, and high utilization rate

Active Publication Date: 2019-04-05
AQUABRAIN BIOTECH XIAMEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is only one agarase that can produce new agar oligosaccharides with a degree of polymerization of 8-12, but this enzyme cannot obtain new agar oligosaccharides with a low degree of polymerization in large quantities (Xu S Y, Kan J, Hu Z, et al.Quantification of Neoagaro-Oligosaccharide Production through Enzymatic Hydrolysis and Its Anti-OxidantActivities.[J].Molecules,2018,23(6):1354.)
So far, there is no enzyme that can simultaneously produce new agar-oligosaccharides with a high degree of polymerization and a new agar-oligosaccharide with a low degree of polymerization

Method used

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  • Modified agarase and application thereof
  • Modified agarase and application thereof
  • Modified agarase and application thereof

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Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: Transformation and recombinant expression of agarase gene

[0035]Bioinformatics analysis and function prediction were carried out on the existing agarase gene. The structure of the original agarase gene was predicted by NCBI-BLAST, and the results showed that there was a homologous sequence of the Porsecretion system C-terminal sorting domain in the amino acid sequence corresponding to the 5' end of the gene. By PCR technology combined with primer design, the nucleotide sequence of the region corresponding to the domain at the 3' end of the existing agarase AgaM1 gene is truncated to obtain the artificially truncated agarase gene AgaM1-01 (SEQ ID NO: 2 ). AgaM1-01 was connected into the expression vector pEASY-Blunt E1 to obtain the recombinant vector pEASY-AgaM1-01, and the recombinant vector was transformed into Escherichia coli BL21 (DE3) strain;

[0036] Insert the recombinant Escherichia coli strain into LB liquid medium, cultivate at 37°C until th...

Embodiment 2

[0037] Example 2: Optimization of production conditions for new agar oligosaccharides

[0038] Agar concentration optimization. Theoretically, the higher the agar concentration, the higher the yield of oligosaccharides. However, as the reaction proceeds, the activity of the enzyme begins to decrease, eventually resulting in part of the agar not participating in the reaction, and this phenomenon is more common in high-concentration agar. The unreacted agar solidifies into a solid as the temperature decreases after the reaction, and these colloidal solids will block the pipelines and filter membranes of the production equipment, resulting in waste of substrates, loss of equipment, and additional labor costs. Therefore, in the production process, the substrate utilization rate is the primary optimization factor considered. Enzyme activity was measured by the dinitrosalicylic acid method, and the substrate utilization ratio (substrate utilization ratio=(initial reaction system q...

Embodiment 3

[0041] Embodiment 3: Composition analysis of new agar oligosaccharide product

[0042] Degradation products and polymerization degrees of the original agarase (AgaM1) and the truncated agarase (AgaM1-01) used in this paper were determined by thin layer chromatography (TLC). As a result, it was found that the original agarase AgaM1 could only produce new agar oligosaccharides (new agarose and new agarosexose) with a low degree of polymerization ( Figure 3A ), and the truncated agarase (AgaM1-01) can obtain new agar oligosaccharides with a degree of polymerization of 4-12 under the above-mentioned optimal conditions ( Figure 3B ). The above results show that: 1) through artificial modification, the original agarase obtained new enzymatic properties after being truncated artificially; 2) the above-mentioned optimum production conditions are effective, and the Under the above optimal conditions, new agar oligosaccharides with a degree of polymerization of 4-12 can be obtained,...

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Abstract

The invention relates to a truncated modified agarase, and the truncated agarase is obtained by deleting a homologous fragment of a Por secretion system C-terminal sorting domain at the C-terminal ofwild agarase. The truncated agarase can catalyze an agar substrate to generate neoagaro-oligosaccharide 4, neoagaro-oligosaccharide 6, neoagaro-oligosaccharide 8, neoagaro-oligosaccharide 10 and neoagaro-oligosaccharide 12; however, the wild agarase only generates the neoagaro-oligosaccharide 4 and the neoagaro-oligosaccharide 6. According to the invention, the neoagaro-oligosaccharides, which aregenerated by using the modified agarase and have multiple degrees of polymerization, are final products of a reaction, and the moisture retention and resistance to oxidation of the neoagaro-oligosaccharides are obviously improved compared with existing products.

Description

technical field [0001] The present invention relates to the field of enzyme engineering, in particular to the technical field of improving enzymatic performance through transformation, in particular to improving the agarase degradation activity and product composition of agarase through genetic engineering recombination, and also to the modified agarase and the use of its products. Background technique [0002] Agarose is a kind of macromolecular polysaccharide, and the new agarose oligosaccharides derived from its degradation have important biological activities such as moisturizing, anti-oxidation, and anti-inflammation (Qing Lili, Dong Jingjing, Li Sidong. Preparation and application of agarose oligosaccharides Progress[J].Shandong Chemical Industry, 2011,40(5):28-30.). According to the degree of polymerization, new agar oligosaccharides can be divided into new agarose, new agarotetraose, new agarose hexose, new agarose octaose, new agarose decaose and so on. According ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/38C12N9/24C12N15/56C12P19/14C12P19/04
CPCC12N9/2402C12N9/2468C12P19/04C12P19/14C12Y302/01081C12Y302/01158
Inventor 曾润颖曲武产竹华陈兴麟易志伟
Owner AQUABRAIN BIOTECH XIAMEN CO LTD
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