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Gene of novel alginate endolyase, engineering bacterium and application

A kind of alginate lyase, endotype technology, applied in the field of the gene sequence of novel endotype algin lyase AlgM, can solve the problems such as low substrate concentration, less than 50% effective components, can not meet the requirements of production, Achieving the effect of low production cost

Active Publication Date: 2015-12-16
BINZHOU MEDICAL COLLEGE
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0003] In recent years, there have been more and more researches on oligosaccharides, and a variety of functional oligosaccharide products have been put into the market, but most of the oligosaccharide products are ordinary oligosaccharides, and the active ingredients are less than 50%
Using alginate lyase to enzymatically hydrolyze alginate to produce alginate oligosaccharides has a good market prospect. In general production, the activity of general alginate lyase is poor, the hydrolysis efficiency is low, and the substrate concentration is low, so it cannot be adapted to production. requirements

Method used

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  • Gene of novel alginate endolyase, engineering bacterium and application
  • Gene of novel alginate endolyase, engineering bacterium and application
  • Gene of novel alginate endolyase, engineering bacterium and application

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Experimental program
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Effect test

Embodiment 1

[0064] 1. The first step of the inventive method is to screen the alginate lyase AlgM gene:

[0065] According to the newly isolated strain Vibriosp.BZM-1 from alginate lyase AlgM, the genome library of the bacteria was established. First, the genomic DNA of the bacteria was extracted, partially digested with the restriction endonuclease Sau3AI, and recovered from the agarose gel. For 10kb fragments, add dGTP to these fragments, recover 3-7kb fragments by re-gelation, and linearize the vector pBZM201 with restriction endonuclease SapI, and recover this fragment as a carrier, and make up 4-10kb fragments Ligate with the pBZM201 vector linearized by SapI digestion at 16°C for 24 hours, and then transform Escherichia coli Xl10-gold competent cells to obtain the genome library of the bacteria (see figure 1 ). Cultivate on the LB medium plate containing sodium alginate and ampicillin for 20 hours, and finally immerse it in 5% cetylpyridine solution for about 10 minutes, extract p...

Embodiment 2

[0071] Inoculate Pichia pastoris strains with three-copy, four-copy, five-copy, six-copy and seven-copy expression cassette structures in 100ml BMGY medium respectively, and culture them at 28°C-30°C, 260rpm for 40-50h until OD 600 20-30, centrifuge at room temperature at 5000rpm for 5min, and collect the bacteria; transfer the bacteria to 100ml BMMY medium, culture at 28°C-30°C, 260rpm, and add methanol every 12h (addition amount is 1.0% of the volume of the medium) %), after induced expression, the supernatants at the time of the highest expression were taken for SDS-PAGE electrophoresis, and the protein expression of Pichia strains with different copy expression cassette structures were analyzed, and three-copy, four-copy and five-copy expression cassettes were taken The expression time of Pichia pastoris strains of the structure is the supernatant of 96h, 120h and 144h. Take 8 microliters of the supernatant from each sample for SDS-PAGE electrophoresis. It is found that at ...

Embodiment 3

[0073] Using the recombinant alginate lyase crude enzyme liquid obtained in the shake flask in Example 2, hydrolyzing sodium alginate with different concentrations to prepare alginate oligosaccharides.

[0074] Raw material: sodium alginate

[0075] Prepare the concentration of the hydrolyzed substrate as 10%, 15%, 20%, 25%, and 30% respectively; use 100ml of alkaline water with a temperature of 30-40°C and a pH between 8 and 9 as a solvent, and add an appropriate amount of sodium alginate, Stir and prepare 100ml of reaction solutions with substrate concentrations of 10%, 15%, 20%, 25%, and 30%, respectively, and then add 1000ul, 1500ul, 2000ul, 2500ul, 3000ul of crude enzyme solution, adjust the pH to 8-9. The hydrolysis time was 16, 14, 12, 10, 8 hours respectively. The reaction solution is centrifuged to remove residues, the supernatant is filtered, ultrafiltered, and vacuum spray-dried to obtain the finished product of fucoidan oligosaccharide. The calculation yield is ...

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Abstract

The invention discloses recombinant alginate lyase, an engineering bacterium thereof and a method for preparing alginate oligosaccharide through hydrolysis. The method is characterized by screening an alginate lyase gene AlgM according to a strain Vibrio sp.BZM-1 newly isolated from seaweeds; constructing an expression vector pHBM905BDM-AlgM containing the gene, and converting pichia sp GS115 competent cells to obtain a single copy gene engineering bacterium; and constructing, culturing and verifying the multi-copy gene engineering bacterium to prepare recombinant alginate lyase through high-efficiency expression, and producing alginate oligosaccharide by hydrolyzing sodium alginate with alginate lyase. The recombinant alginate lyase gene engineering bacterium GS115 / AlgM4 (CCTCC No:M2015500) containing four copies has a higher expression level. The activity of recombinant alginate lyase is as high as 31000U / mL.

Description

technical field [0001] The invention relates to a gene sequence of a novel endo-type alginate lyase AlgM. The invention also relates to the construction of the Pichia pastoris genetically engineered strain of the recombinant alginate lyase and the method for expressing the alginate lyase by using the Pichia pastoris. The invention also relates to a method for preparing alginate oligosaccharides by using the recombinant alginate lyase to hydrolyze higher concentration sodium alginate. Background technique [0002] Fucoidan oligosaccharides are the latest generation of functional oligosaccharides. Fucoidan oligosaccharides with a polymerization degree of less than 20 obtained by the international leading separation and degradation technology are composed of β-D-mannuronic acid and α-L-ancient Linear low polymer composed of glucuronic acid. Seaweed oligosaccharides have low molecular weight, strong water solubility, and high stability. Experiments and clinical studies have fo...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N15/81C12N1/19C12P19/00C12R1/63C12R1/84
Inventor 武玉永姚庆收谭秀华马朋秦加阳孙业盈
Owner BINZHOU MEDICAL COLLEGE
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