Endo-alginate lyase and coding gene and application thereof

A technology for endo-cutting alginate and cutting alginate is applied in the field of bioengineering, and can solve the problems of narrow action temperature range, industrial application limitation, interference with algal oligosaccharide correlation analysis and the like

Active Publication Date: 2021-06-29
THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] With the development of genome sequencing technology, more and more alginate lyase genes have been annotated, and their functions have been studied by means of genetic engineering. Narrow, poor tolerance to high temperature, acid-base, and metal ions, and the degradation product is a mixture of multiple components, which interferes with the correlation analysis of the structure and function of algal oligosaccharides, which is greatly limited in industrial applications

Method used

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  • Endo-alginate lyase and coding gene and application thereof
  • Endo-alginate lyase and coding gene and application thereof
  • Endo-alginate lyase and coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Preparation of endo-alginate lyase

[0035] Proceed as follows:

[0036] Experimental Materials

[0037] Vibrio sp.MCCC 1A13243 (preserved by China Marine Microorganism Culture Collection Management Center, the preservation number is 1A13243, which can be purchased), Escherichia coli E.coli TOP10, Escherichia coli E.coil BL21 (DE3) (purchased from ThermoFisher Company), expression vector pET-22b (purchased from ThermoFisher Company); Saibaisheng bacterial genomic DNA extraction kit (purchased from Xiamen Jingju Company); DNA polymerase (purchased from Quanshijin Company); restriction endonuclease Nde I and Xho I (purchased from Quanshijin Company); T4 ligase (purchased from Takara Company); LB medium (containing 10 g of peptone per liter, 5 g of yeast extract, and 10 g of NaCl); binding buffer (1× PBS buffer: 10mM phosphate, pH7.2~7.4); washing buffer (500mM NaCl, 10~20mM imidazole, 20mM phosphate, pH 7.4); elution buffer (500mM NaCl, 50~500mM imidazole, ...

Embodiment 2

[0049] Enzymatic properties of endo-alginate lyase

[0050] (1) Alginate lyase activity assay method

[0051] The content of reducing sugar was determined by DNS method. Mix 50 μL of diluted enzyme solution with 200 μL of 0.3% sodium alginate substrate, and react at 35° C. for 30 min. After the reaction is completed, add 500 μL of DNS reagent, boil for 5 minutes, immediately cool in ice water, take the supernatant after short centrifugation, and measure the OD 540 Value (with inactivated enzyme solution as a control), the amount of reducing sugar and enzyme activity were calculated according to the standard curve. Definition of enzyme activity unit: Under the above-mentioned measurement conditions, the amount of enzyme needed to crack sodium alginate to produce 1 μmol reducing sugar per minute is defined as an enzyme activity unit (U).

[0052] (2) The action temperature of the enzyme

[0053] Measure the enzyme activity of the diluted enzyme solution in the range of 0-90°...

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Abstract

The invention discloses endo-alginate lyase and a coding gene and application thereof. A primer is designed and synthesized by taking genome DNA of a Vibriosp.MCCC 1A13243 as a template, an endo-alginate lyase gene is amplified through PCR, the gene is cloned into a pET-22b expression vector, induced expression is carried out in escherichia coli E.coli BL21, and affinity chromatography purification is carried out by adopting Ni-Sepharose to obtain the recombinant endo-alginate lyase protein with higher purity. The optimum temperature of the enzyme is 35 DEG C, the optimum pH of the enzyme is 8.5, and the enzyme has good stability in the ranges of 0-65 DEG C and pH 4.5-10.5; co<2+>, Cu<2+>, Mn<2+> and Fe<3+> can obviously promote the enzyme activity; and the enzyme has the highest degradation effect on sodium alginate and can further degrade polyguluronic acid and polymannuronic acid, and a main product for degrading the three substrates is disaccharide. The endo-alginate lyase has advantages in the aspect of producing alginate oligosaccharide, especially brown algae disaccharide.

Description

technical field [0001] The invention relates to an endo-alginate lyase obtained by means of genetic engineering and a preparation method thereof, belonging to the technical field of bioengineering. Background technique [0002] Alginate is a kind of water-soluble acidic polysaccharide mainly extracted from the cell wall of brown algae. It is composed of two monomers of β-D-mannuronic acid (M) and α-L-guluronic acid (G). These two monomers are linked by 1,4-glycosidic bonds to form three different forms of polysaccharide fragments: poly β-D-mannuronic acid (Polymannuronic acid, PolyM) fragments, poly α-L - A polyguluronic acid (PolyG) fragment and a hybrid fragment (PolyMG) of the two. Alginate is widely used in food, medical treatment, agriculture, energy and other fields because of its physiological activities such as anti-tumor and blood pressure reduction. [0003] At present, the biological enzymatic method is an important method to degrade alginate. Compared with the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N1/21C12P19/12C12R1/19
CPCC12N9/88C12N15/70C12P19/12C12Y402/02011C12Y402/02003
Inventor 邵宗泽周梅先陈琳
Owner THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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