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

A technology of alginate lyase and alginate cutting, applied in the field of exoalginate lyase and its preparation, can solve the problems of poor acid-base and metal ion tolerance, industrial application limitations, etc.

Active Publication Date: 2022-08-05
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

The exo-alginate lyase found so far has poor tolerance to acid-base and metal ions, and its industrial application is greatly limited.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Preparation of exo-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, 20 to 50 mM imidazole, 20 mM phosphate, pH 7.4); elution buffer (500 mM NaCl, 500 mM imidazole, 20 mM phosphate, ...

Embodiment 2

[0049] Enzymatic properties of exo-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 30° 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 enzyme...

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Abstract

The present invention disclosed an outer -cut brown algae cracking enzyme, its encoding gene and application.The invention is based on the genome DNA of the vibriosp.mccc 1A13243 as a template, which is designed and synthesized. The gene is cloned on the PET‑22B expression carrier through PCR amplification.The induction expression is performed in BL21, and the Ni‑sePharose affinity is purified to obtain a higher -purity reorganized external cut brown algae cracking enzyme protein.The optimal temperature of this enzyme is 30 ° C, the most suitable pH is 8.5, and the insulation can be kept by more than 50 % of the relative enzyme vitality in the range of pH4.5 ~ 10.0; MN MN; MN 2+ And Co 2+ It has a significant promotion of this enzyme activity; the enzyme has a good substrate specificity for sodium alginate. Its degradation is mainly divided into monosaccharides. It has good application prospects in the development and utilization of brown algae resources.

Description

technical field [0001] The invention relates to an exo-alginate lyase obtained 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, the biological enzymatic method is an important method for degrading algin....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/70C12N15/66C12N1/21C12R1/19
CPCC12N9/88C12N15/70C12Y402/02
Inventor 邵宗泽周梅先陈琳
Owner THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES