Nucleotide sequence of encoded fucoidin glucoside hydrolase and application thereof

A fucoidan glycoside and nucleotide sequence technology, applied in the fields of genetic engineering, molecular biology, and sugar chemistry

Active Publication Date: 2016-08-03
SHANDONG JIEJING GROUP CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, biodegradation requires the use of highly efficient and specific glycoside hydrolases
At present, there are relatively few reports on the study of fucoidan hydrolase, and there are no fucoidan oligosaccharides prepared by biological methods on the market

Method used

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  • Nucleotide sequence of encoded fucoidin glucoside hydrolase and application thereof
  • Nucleotide sequence of encoded fucoidin glucoside hydrolase and application thereof
  • Nucleotide sequence of encoded fucoidin glucoside hydrolase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Expression of Fucoidan Hydrolase in Recombinant Strains

[0020] (1) Prepare LB medium containing Kan resistance: 5g / L yeast extract, 10g / L tryptone, 10g / L sodium chloride, sterilize at 120°C for 20min, cool to room temperature and add Kan to make the final concentration 50ug / L ml.

[0021] (2) Inoculate the recombinant strain obtained above on solid LB medium containing Kan resistance, culture overnight at 37°C, pick a single colony and inoculate it into 5ml of liquid LB culture containing Kan resistance, 37°C, 200rmp shaker After culturing for 24 hours, inoculate the above bacterial liquid into 500ml liquid LB culture containing Kan resistance, and culture it to OD at 37°C and 200rmp bed 600 When nm=0.6, add 0.5mMIPTG, induce expression at 16°C, 200rmp for 24 hours, centrifuge at 5000rmp, and collect the bacteria.

Embodiment 2

[0022] Example 2 Detection of expressed fucoidan hydrolase

[0023] The bacterial cells collected in Example 1 were suspended into BufferA (50mM Tri-HCl+500mMNaCl, pH7.9) buffer solution for sonication, and the supernatant was collected by centrifugation at 12000rmp, and SDS-Page detection was carried out, as shown in figure 2 shown. The predicted protein molecular weight is 58.8kDa, and then WesternBlot detection, such as image 3 shown.

Embodiment 3

[0024] Example 3 Purification of expressed fucoidan hydrolase

[0025] The above proteins were purified using a nickel column:

[0026] (1) Equilibrate the column with BufferA (50mMTri-HCl+500mMNaCl, pH7.9), flow rate 1ml / min;

[0027] (2) Load the sample with BufferA at a flow rate of 1ml / min, and collect the breakthrough.

[0028] (3) Elution with BufferA, flow rate 1ml / min, elution 30ml;

[0029] (4) Use Buffer A containing 20mM, 60mM, 100mM, 160mM, and 500mM imidazole in order to elute at a flow rate of 1ml / min, and collect one tube every 5min;

[0030] (5) G250 detects the protein content in each collected fraction;

[0031] (6) Select the group with high eluted protein content at each imidazole concentration, original solution, sample breakthrough, 1×BindingBuffer elution, and perform SDS-PAGE detection, such as figure 2 shown.

[0032] Purified fucoidan glycoside hydrolase protein was obtained according to the above steps, and concentrated using an 8000Da protein ...

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Abstract

The invention relates to a nucleotide sequence of encoded fucoidin glucoside hydrolase and an application thereof. The nucleotide sequence is SEQ ID NO.1, encoded protein contains 529 amino acids, the nucleotide sequence is SEQ ID NO.2, and the predicted molecular mass of protein is 58.8 kDa. According to the nucleotide sequence and the application thereof, the gene GenBank No:AJ877239 of the encoded fucoidin hydrolase is subjected to codon optimization, 1599bp basic groups exist after optimization, and are loaded into a cloning vector pET28a, genes of the optimized fucoidin hydrolase can be expressed in escherichia coli BL21, glucoside hydrolase capable of efficiently and exclusively hydrolyzing fucoidin is obtained, and a biological degrading enzyme is provided for fucoidin hydrolysis.

Description

technical field [0001] The invention relates to a nucleotide sequence encoding fucoidan glycoside hydrolase and its application, belonging to the fields of molecular biology, genetic engineering and sugar chemistry. Background technique [0002] Fucoidan is a unique water-soluble polysaccharide combined with sulfate groups, mostly present in brown algae and marine invertebrates, composed of sulfate-based fucose, galactose, glucose, xylose, rhamnose, Monosaccharides such as acids. Its polysaccharide structure mainly includes two main chain types: I is composed of (1→3)-linked α-L-fucose residues; II is composed of alternating (1→3), (1→4)-linked α-L-fucose residue composition. [0003] Fucoidan, as a biological polysaccharide widely found in brown algae, has attracted more and more attention from researchers on its biological activity, and various biological activities have been discovered and reported one after another. Studies have found that fucoidan has anticoagulant, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/24
CPCC12N9/24
Inventor 林成彬李建军任立世王斌惠锋基
Owner SHANDONG JIEJING GROUP CORP
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