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Alginate lyase gene algp and its preparation method and expression

An algin lyase and algin technology are applied in the field of algin lyase gene algp and its preparation, and can solve the problems of hindering the preparation of alginate oligosaccharides, hindering the in-depth progress of research and application, low yield and the like

Active Publication Date: 2016-02-03
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, at present, most of the strains producing alginate lyase have low activity and low yield, which hinders the preparation of alginate oligosaccharides and further hinders the in-depth progress of research and application.

Method used

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  • Alginate lyase gene algp and its preparation method and expression
  • Alginate lyase gene algp and its preparation method and expression
  • Alginate lyase gene algp and its preparation method and expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 4

[0096] Example 4, Construction of Escherichia coli engineering strain SW (DE3) capable of highly expressing alginate lyase

[0097] The expression vector pET28a-algp was transformed into Escherichia coli HMS174(DE3) (purchased from Shanghai Yiteng Biotechnology Co., Ltd.) according to the standard calcium chloride method, and the transformants with kanamycin sulfate resistance were screened. After culturing in LB liquid medium (containing kanamycin sulfate), PCR verification was performed using the universal primer M13. The PCR verification procedure was pre-denaturation at 95°C for 5 minutes, followed by 30 cycles of 95°C for 30s, 48°C for 30s, and 72°C for 1min. Finally, extend at 72°C for 10 min.

[0098] Agarose gel electrophoresis showed that the positive transformant had a correct band at about 1600bp, and the positive transformant was named SW(DE3).

Embodiment 5

[0099] Example 5, Production of Recombinant Alginate Lyase Using Escherichia coli Recombinant Strain SW(DE3)

[0100] Pick a single clone of Escherichia coli recombinant strain SW(DE3) and inoculate it in 5 mL of LB medium containing kanamycin sulfate, cultivate overnight at 37°C at 200 r / min, and then inoculate three 100 mL strains at a ratio of 1:100 overnight. After culturing in LB medium containing kanamycin sulfate for 5 hours, they were inoculated in a 5L fermenter at a ratio of 1:10.

[0101] The 5L fermentation tank has a liquid volume of 3L.

[0102] The components of the fermentation medium are: yeast powder 2g / L, glycerol 5g / L, foam enemy 60μl / L, Na 2 HPO 4 6g / L, KH 2 PO 4 3g / L, NH 4 Cl1g / L, NaCl0.5g / L, MgSO 4 0.5g / L, CaCl 2 Solution 2ml / L, trace elements 2ml / L.

[0103] CaCl 2 Solution: 10g anhydrous CaCl 2 Dissolve in 1L deionized water.

[0104] Trace elements: FeSO 4 ·7H 2 O4.8g / L, MnSO 4 ·H 2 O2.4g / L, ZnSO 4 ·7H 2 O2.4g / L, CuSO 4 ·5H 2 O0.96g...

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Abstract

The invention relates to alginate lyase gene algp and its preparation method and expression and discloses alginate lyase of Vibrio vulnificus origin, which has good protein stability and high bioactivity. The invention also discloses alginate lyase gene algp, a carrier and a host cell containing the alginate lyase gene algp, and a method of recombinant expression of the alginate lyase. The technical scheme enables large-scale production of recombinant alginate lyase at lower cost.

Description

technical field [0001] The invention relates to the field of bioengineering, more specifically, the invention relates to a alginate lyase gene algp and its preparation method and expression. Background technique [0002] The vast ocean occupies 71% of the earth's area, and it is a huge treasure house, containing a huge amount of biological resources. Seaweed is one of the important marine plants. It is a natural enricher of inorganic matter and a producer of many marine active substances. Seaweed polysaccharide is a large class of important active substances in seaweed. At present, the most widely used seaweed polysaccharides in the world are alginate, carrageenan and agar gum. [0003] Alginate is a water-soluble acidic polysaccharide derived from the cell wall of brown seaweed plants, which is mainly composed of α-L-guluronic acid and β-D-mannuronic acid. With the discovery of research, the degradation products of alginate have special chemical activity and biological ac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/63C12P19/00
Inventor 周祥山沈伟蔡孟浩张元兴
Owner EAST CHINA UNIV OF SCI & TECH
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