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A new type of endo-alginate lyase gene, engineering bacteria and application

A technology of alginate lyase and engineering bacteria, applied in the field of gene sequences of novel endo-type alginate lyase AlgM, can solve problems such as low substrate concentration, inability to meet production requirements, low hydrolysis efficiency, etc., and achieve production cost low effect

Active Publication Date: 2018-03-23
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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  • A new type of endo-alginate lyase gene, engineering bacteria and application
  • A new type of endo-alginate lyase gene, engineering bacteria and application
  • A new type of endo-alginate lyase gene, engineering bacteria 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 Vibrio sp.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 Sau3A I, and recovered from the agarose gel. For 4-10kb fragments, these fragments were supplemented with dGTP, and the 3-7kb fragments were recovered by regelation. At the same time, the vector pBZM201 was linearized by restriction endonuclease Sap I, and the fragments were recovered as vectors, and the complementary base 4- The 10kb fragment was ligated with the pBZM201 vector linearized by Sap I digestion at 16°C for 24h, and then transformed into Escherichia coli Xl10-gold competent cells to obtain the genome library of the bacterium (see figure 1 ). Cultivate on the LB medium plate containing sodium alginate and ampicillin for 20 hours...

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 the medium volume 1.0%), after induced expression, the supernatant at the time of the highest expression was taken for SDS-PAGE electrophoresis to analyze the protein expression of Pichia strains with different copy expression cassette structures, three copies, four copies and five copies of expression The expression time of Pichia pastoris strains with cassette structure was the supernatant of 96h, 120h and 144h. Take 8 microliters of supernatant from each sample for SDS-PAGE electrophoresis. It was found that at 144h, four copies of Pichia pastor...

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] The concentrations of the hydrolyzed substrates were respectively prepared as 10%, 15%, 20%, 25%, and 30%; 100ml of alkaline water with a temperature of 30-40°C and a pH between 8 and 9 was used as a solvent, and an appropriate amount of sodium alginate was added. 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 ca...

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Abstract

The invention discloses a recombinant alginate lyase, its engineering bacteria and a method for preparing alginate oligosaccharides by hydrolysis. According to the newly isolated strain Vibrio sp.BZM‑1 from seaweed, the alginate gene-AlgM is screened; The expression vector of the gene, pHBM905BDM‑AlgM, is transformed into Pichia pastoris GS115 competent cells to obtain single-copy genetically engineered bacteria; construct, culture and verify multi-copy genetically engineered bacteria, efficiently express recombinant alginate lyase, and use this enzyme to hydrolyze alginic acid Sodium production of fucoidan oligosaccharides. GS115 / AlgM4 (CCTCC No: M2015500) with four copies of recombinant alginate lyase genetically engineered strain had a higher expression level. The activity of the recombinant alginate lyase of the present invention 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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Patent Type & Authority Patents(China)
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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