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A kind of preparation method of homogeneous fucoidan

A technology of fucoidan and alginate, which is applied in the direction of fermentation, etc., can solve the problems of low degradation efficiency of a single enzyme, poor product uniformity, and restrictions on wide application

Active Publication Date: 2022-03-04
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the currently used alginate lyase still has the problems of low single-enzyme degradation efficiency and poor product uniformity, which limits the wide application of homogeneous alginate oligosaccharide preparation technology based on enzymatic methods and becomes a bottleneck restricting industrial development.

Method used

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  • A kind of preparation method of homogeneous fucoidan
  • A kind of preparation method of homogeneous fucoidan
  • A kind of preparation method of homogeneous fucoidan

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Gene Cloning of AlyV and AlyF

[0022] 1 gene clone

[0023] 1.1 Gene cloning of AlyV

[0024] First, genome sequencing and homologous protein sequence analysis of Vibrio pelagius with alginate degradation activity were carried out, and the gene sequence information of AlyV was obtained (its amino acid sequence is SEQ ID NO: 1; the sequence of the coding gene is SEQ ID NO: 2). Sequence analysis showed that AlyV is a new uncharacterized alginate lyase, which belongs to the polysaccharide lyase 7 family (PL-7). According to the CAZy database, among the characterized members of the PL-7 family, the sequences of the three proteins A9mT, AlxM and AlyVOA are the most similar to AlyV, with homology of 47.1%, 43.4% and 42.8%, respectively. The amino acid sequence alignment is as follows: figure 1 shown. These three homologous alginate lyases of AlyV all have the substrate specificity of polyM, A9mT and AlyVOA are derived from Vibrio, and AlxM is derived from Phot...

Embodiment 2

[0073] Embodiment 2: Combined degradation of alginate

[0074] 1. Synergistic research on enzyme activity

[0075] The reaction conditions of the present embodiment are as follows:

[0076] The substrate was 2 mg / ml AL, the buffer was 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, and reacted at 30° C. for 1 h.

[0077] AlyV and AlyF have polyM and polyG-specific alginate lyase activities, respectively, but when they act alone, they cannot completely crack alginate (AL) into fucoidan oligosaccharides, but the joint reaction of AlyV and AlyF can completely cleavage AL degradation, such as Figure 4 As shown, the "10:0" and "0:10" lanes in the figure represent the degradation of AL by AlyV and AlyF respectively.

[0078] Set the final concentration of the "AlyV+AlyF" group to 10nM AlyV+10nM AlyF, the final concentration of the "AlyV" group to 20nMAlyV, and the final concentration of the "AlyF" group to 20nM AlyF.

[0079] Take the ΔOD of the "AlyV+AlyF" group 235 The value is 100%, ...

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Abstract

The invention provides a method for degrading alginate combined with alginate lyase, which is to use alginate lyase AlyV and AlyF to degrade alginate to prepare fucoidan; wherein the amino acid sequence of alginate AlyV is SEQ ID NO: 1, The sequence of its coding gene is SEQ ID NO: 2; the amino acid sequence of alginate lyase AlyF is SEQ ID NO: 3, and the sequence of its coding gene is SEQ ID NO: 4. Wherein the molar ratio of alginate lyase AlyV and AlyF is 1:10-10:1, and the most preferred molar ratio is 1:1. The invention adopts the enzymatic preparation technology, which is green and environment-friendly; the combined use of double enzymes with different substrate binding specificities improves the conversion efficiency of the substrate; at the same time, the combined use of double enzymes greatly improves the uniformity of the product.

Description

technical field [0001] The invention belongs to the technical field of oligosaccharide preparation, and in particular relates to a method for preparing uniform fucoidan. Background technique [0002] Alginate is a non-homopolymeric linear molecule composed of α-L-guluronic acid (G) and β-D-mannuronic acid (M) through 1,4-glycosidic bonds in different combinations. Alginate lyase catalyzes the 1,4-glycosidic bond between monomers through the β-elimination reaction, and forms an unsaturated double bond between C4 and C5 at the non-reducing end. The double bond has a maximum absorbance at 235nm. In the determination of enzyme activity, mainly by OD 235 The generation of unsaturated double bonds was detected by ultraviolet absorption method and the homogeneity of degradation products was determined by thin layer chromatography (TLC). [0003] Studies have found that the degradation product of alginate, fucoidan oligosaccharides, has a variety of different biological activities...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/00
CPCC12P19/00
Inventor 刘伟治李智剑律倩倩张晓华何新新
Owner OCEAN UNIV OF CHINA