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A kind of endo-algin lyase, its encoding gene and application

A technology of endo-algin cutting and cutting algin, which is applied in the field of bioengineering, can solve the problems of industrial application limitations, interference with alginate oligosaccharide correlation analysis, poor acid-base and metal ion tolerance, etc.

Active Publication Date: 2022-08-05
THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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  • 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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  • A kind of endo-algin lyase, its encoding gene and application
  • A kind of endo-algin lyase, its encoding gene and application
  • A kind of endo-algin lyase, its encoding gene and application

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Embodiment 1

[0034] The preparation of embodiment 1 endo-alginate lyase

[0035] Proceed as follows:

[0036] Experimental Materials

[0037] Vibrio sp.MCCC 1A13243 (preserved by the China Marine Microorganism Culture Collection and Management Center, the preservation number is 1A13243, which can be obtained by purchasing), Escherichia coli E.coli TOP10, Escherichia coli E.coil BL21 (DE3) (purchased from ThermoFisher company), expression vector pET-22b (purchased from ThermoFisher company); Substance bacterial genome DNA extraction kit (purchased from Xiamen Jingju company); DNA polymerase (purchased from Quanjinjin company); restriction endonuclease Nde I and Xho I (purchased from Quanjinjin); T4 ligase (purchased from Takara); LB medium (10 g peptone per liter, 5 g yeast extract, 10 g NaCl); binding buffer (1× PBS buffer: 10 mM phosphate, pH 7.2 to 7.4); rinse buffer (500 mM NaCl, 10 to 20 mM imidazole, 20 mM phosphate, pH 7.4); elution buffer (500 mM NaCl, 50 to 500 mM imidazole, 20 m...

Embodiment 2

[0049] Enzymatic properties of endo-algin lyase

[0050] (1) Determination method of alginate lyase activity

[0051] The reducing sugar content was determined by DNS method. Mix 50 μL of the diluted enzyme solution and 200 μL of 0.3% sodium alginate substrate, and react at 35° C. for 30 min. After the reaction was completed, 500 μL of DNS reagent was added, boiled for 5 min, immediately placed in ice water to cool, and the supernatant was taken after a brief centrifugation, and the OD was measured. 540 value (with the 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 measurement conditions, the amount of enzyme required to generate 1 μmol of reducing sugar by cleaving sodium alginate per minute is defined as one enzyme activity unit (U).

[0052] (2) The temperature of enzyme action

[0053] The enzyme activity of the diluted enzym...

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Abstract

The invention discloses an endo-algin lyase, its encoding gene and application. The present invention takes the genomic DNA of Vibrio sp.MCCC 1A13243 as a template, designs and synthesizes primers, amplifies the endo-alginate lyase gene by PCR, and clones the gene into the pET-22b expression vector. Induced expression in BL21 and purified by Ni-Sepharose affinity chromatography to obtain a higher-purity recombinant endo-algin lyase protein. The optimum temperature of the enzyme is 35℃, the optimum pH is 8.5, and the stability is good in the range of 0~65℃ and pH 4.5~10.5; Co 2+ , Cu 2+ , Mn 2+ and Fe 3+ It can obviously promote the activity of the enzyme; the enzyme has the strongest degradation effect on sodium alginate, and can also degrade polyguluronic acid and polymannuronic acid, and the main product of degrading these three substrates is disaccharide. The enzyme has advantages in the production of fucoidan oligosaccharides, especially fucoidobiose.

Description

technical field [0001] The invention relates to obtaining an endo-alginate lyase by means of genetic engineering and a preparation method thereof, and belongs to the technical field of bioengineering. Background technique [0002] Algin is a kind of water-soluble acidic polysaccharide mainly extracted from the cell wall of brown algae. It is randomly arranged by two monomers, β-D-mannuronic acid (M) and α-L-guluronic acid (G). composed of polymers, the 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) fragment, polyα-L -Guluronic acid (Polyguluronic acid, PolyG) fragment and both hybrid fragments (PolyMG). Because of its physiological activities such as anti-tumor and lowering blood pressure, algin is widely used in food, medical treatment, agriculture, energy and so on. [0003] At present, biological enzymatic method is an important method for degrading algin. C...

Claims

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Application Information

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Patent Type & Authority Patents(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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