Method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis

A technology of algin oligosaccharides and algin, which is applied in botany equipment and methods, biochemical equipment and methods, enzymes, etc., can solve the problems of low efficiency and inability to prepare algin oligosaccharides in a targeted manner, and achieves wide application prospects, Improve immunity, the effect of simple process

Active Publication Date: 2017-06-23
OCEAN UNIV OF CHINA
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  • Abstract
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  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for the directional preparation of alginic oligosaccharides by enzymatic hydrolysis. The modified enzyme can efficiently and directionally prepare the target alginic oligosaccharides, and solve the problems of low efficiency and inability to directional preparation of brown algae in the prior art. Disadvantages of gum oligosaccharides

Method used

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  • Method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis
  • Method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis
  • Method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: Preparation of algin oligosaccharides using alginase lyase before modification

[0023] Dissolve alginate in water with pH=7 adjusted by NaOH, prepare 200 mL of alginate solution with a concentration of 5%, add 2 mL of alginate lyase (amino acid sequence such as: SEQ ID NO: 1, nucleotide sequence is SEQ ID NO: 2), stir and enzymolyze in a warm bath at 45°C for 2 hours, then add 2 mL of alginate lyase, continue to stir and enzymolyze in a warm bath at 45°C for 2 hours. The enzymolysis solution was centrifuged at 8000rpm for 10min to remove the residue, and the supernatant was the enzymolysis product. The enzymatic hydrolysis product was subjected to 4-fold alcohol precipitation to obtain the product, and the product was subjected to rotary evaporation and freeze-drying to obtain a crude alginate oligosaccharide with a yield of 43.9%. The product is detected by mass spectrometry, and the results show that the product includes a degree of polymerization of 3-10...

Embodiment 2

[0028] Example 2 Transformation of alginate lyase gene and construction of recombinant vector

[0029] In view of the low efficiency of enzymatic hydrolysis of alginate lyase, the wide distribution of enzymatic hydrolysis products, and the inability to directional produce target products, the applicant designed upstream primers based on the complete sequence of alginate lyase gene on the basis of further analysis of alginate lyase enzyme F1 and downstream primer R1 were amplified by PCR to obtain the complete sequence of the gene. The PCR conditions were: pre-denaturation at 94°C for 3 minutes, followed by 30 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 2 minutes, and finally extension at 72°C for 5 minutes. Primers F1, R2 and F2, R1 were used to amplify the first 54 bases and the last 768 bases of the complete gene sequence respectively. The two PCR products were digested with AvrⅡ and ligated with T4 ligase to form a genetically modified sequence. The genetically modi...

Embodiment 3

[0031] Example 3 Production of Recombinant Alginate Lyase Using Escherichia coli Recombinant Strains

[0032] Pick the recombinant strain of Escherichia coli, inoculate it into 5 mL liquid LB medium containing 100 ug / mL ampicillin, and culture it with shaking at 170 r / min at 37°C for 12-16 hours. Inoculate 50mL of liquid LB medium containing 100ug / mL ampicillin according to 2% inoculum amount, shake culture at 37°C at 170r / min until OD600 is 0.5-0.8, add IPTG with a final concentration of 0.9mM, continue at 23°C at 150r / min Cultivate to 24h. Centrifuge at 1000 rpm for 10 min to obtain supernatant extracellular enzymes.

[0033] The enzyme activity was measured by the DNS method, and the results showed that the enzyme activity of the recombinant alginate lyase after genetic modification was 29.7 U / mL, which was 5.2 times that of the enzyme activity before genetic modification.

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Abstract

The invention provides a method for directionally preparing alginate oligosaccharides by enzymatic hydrolysis. An amino acid sequence of alginate lyase used in the method is SEQ ID NO:3. According to the method provided by the invention, the alginate oligosaccharides are directionally prepared by utilizing the genetically-modified recombinant alginate lyase; compared with an existing method for preparing the alginate oligosaccharides by acid hydrolysis, the method provided by the invention overcomes the defects of large destructiveness and low efficiency; compared with an existing method for preparing the alginate oligosaccharides by enzymatic hydrolysis, the method provided by the invention overcomes the defects that the enzymolysis time is long, the cost is high, and the alginate oligosaccharides with specific degree of polymerization cannot be directionally prepared by applying the improved recombinant alginate lyase. The method provided by the invention is simple in process, low in cost and suitable for industrialized production, and can reduce the alginate viscosity within a short time; the total yield of the prepared oligosaccharide is 90 percent or above. The prepared alginate oligosaccharides has the efficacies of resisting tumor, resisting inflammation, reducing blood fat, improving the immunity and the like, can be used in the field of food and health products, and has wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of algin oligosaccharide preparation, and in particular relates to a method for directional preparation of algin oligosaccharide by enzymatic hydrolysis. Background technique [0002] Alginate oligosaccharides (alginate oligosaccharides) are low molecular weight fragments obtained by hydrolyzing alginate with a small degree of polymerization and good water solubility. Because alginate oligosaccharide has various physiological activities such as hypolipidemic, antiviral, antitumor and antioxidative, it can be widely used in the fields of food, medicine and cosmetics, and has great application and development value. The latest research shows that the poly-M segment oligosaccharide drug "971" prepared from algin can inhibit the aggregation and cytotoxicity of β-amyloid cells, and is being used in the second phase clinical research of anti-Alzheimer's disease; poly-G oligosaccharide can Synergistically inhibits...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/00C12N9/88C12N15/60
CPCC12N9/88C12P19/00C12Y402/02011
Inventor 牟海津杨敏李丽
Owner OCEAN UNIV OF CHINA
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