Iota-carrageenan degrading enzyme gene as well as preparation method and application thereof

A technology of carrageenase and carrageenan, which is applied in botany equipment and methods, microorganism-based methods, biochemical equipment and methods, etc., and can solve the problems of limited application, solubility and absorption effects, and excessive molecular weight of carrageenan and other problems, to achieve the effect of single product, good stability and high expression level

Active Publication Date: 2014-05-07
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing research results have shown that carrageenan has a variety of biological activities, such as anticoagulant, anti-virus, immune regulation, anti-oxidation, etc. its application in the field of medicine

Method used

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  • Iota-carrageenan degrading enzyme gene as well as preparation method and application thereof
  • Iota-carrageenan degrading enzyme gene as well as preparation method and application thereof
  • Iota-carrageenan degrading enzyme gene as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Isolation of iota-carrageenase gene cgiA2_Ce

[0025] Multiple carrageenase genes and their gene sequences were obtained by whole-genome sequencing of Flavobacterium KL-A, including κ-, λ-, and ι-carrageenan genes, and cgiA2_Ce is one of the ι-carrageenase genes. Use the biological software Primer5.0 to design upstream primers (5'-3') and downstream primers (5'-3') and use genomic DNA as a template to perform PCR reactions. The PCR reaction composition is as follows (50 μL reaction system): upstream and downstream 2 μL of each primer, 1 μL of DNA template, 5 μL of 10×Buffer, 5 μL of dNTP Mix, 0.5 μL of Ex Taq, ddH 2 O 34.5 μL. The reaction conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52.4°C for 30 s, extension at 72°C for 1 min and 20 s, and final extension at 72°C for 5 min, a total of 30 cycles. After the reaction, the PCR product was recovered to obtain the iota-carrageenase gene cgiA2_Ce.

Embodiment 2

[0026] Embodiment 2: Construction of Escherichia coli cloning vector pGEM-T-cgiA2_Ce

[0027] Ligate cgiA2_Ce to the vector pGEM-T, and use the DNA Ligation Kit to ligate. The ligation system (10 μL) is as follows: 2×Buffer 5 μL, DNA fragment 3 μL, pGEM-T vector 1 μL, T4 ligase 1 μL, and ligate overnight at 4°C. The connection solution is pGEM-T-cgiA2_Ce, which is used to transform Escherichia coli DH5α.

Embodiment 3

[0028] Example 3: Construction of Escherichia coli recombinant strain DH5α-PGEM-T-cgiA2_Ce

[0029] Add 100 μL of thawed competent E.coli DH5α and 10 μL of the connection solution obtained in Example 2 to a 1.5 mL Eppendorf tube, ice-bath for 30 minutes, heat shock at 42°C for 90 seconds, ice-bath for 30 minutes, add 900 μL of LB medium, and culture with shaking at 37°C After 1 h, the bacterial solution was mixed with 4 μL TPTG and 40 μL X-gal, spread on the LB plate containing 100 μg / mL ampicillin, and cultured overnight at 37°C. Pick a single white colony for PCR detection, and those showing a 1.5 kb specific band in agarose gel electrophoresis are positive transformed colonies, that is, E. coli colonies containing pGEM-T-cgiA2_Ce.

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Abstract

The invention relates to an iota-carrageenan degrading enzyme gene as well as a preparation method and an application thereof and relates to iota-carrageenanase. An iota-carrageenanase gene cgiA2_Ce is a carrageenanase gene for coding Cellulophaga sp.KL-A. An iota-carrageenan recombinase CgiA2_Ce can be applied to iota-carrageenan degradation, degradation products are two iota-carrageenan oligosaccharides, and the degrees of polymerization of the two iota-carrageenan oligosaccharides are respectively 2-4 saccharide and 6-10 saccharide. The degradation products can be applied in preparation of antibacterial agents, antiviral agents, immunomodulators, antioxidants and the like, particularly antibacterial agents, antiviral agents, immunomodulators, antioxidants and the like for mariculture, so that the survival rate of prawns can be increased by 20%-30%; and a recombined protein of the iota-carrageenanase gene cgiA2_Ce can be obtained through a gene engineering technology.

Description

technical field [0001] The present invention relates to iota-carrageenase, in particular to a iota-carrageenase gene cgiA2_Ce derived from Cellulophaga sp.KL-A and its preparation method and application. Background technique [0002] Carrageenan (Carrageenan), also known as staghorn gum, carrageenan, etc., is a high-molecular hydrophilic polysaccharide extracted from red algae, with repeated α-(1→4)-D-galactopyranose -β-(1→3)-D-galactopyranose (or 3,6 inner ether-D-galactopyranose) disaccharide units are connected as the basic skeleton. According to whether it contains 3,6 inner ether-D-galactopyranose, the content of sulfate group, and the position of sulfate group in the molecule, carrageenan is mainly divided into three groups: κ, λ, and ι. [0003] Carrageenan is widely used in the food industry because of its excellent thermoreversible gelation, anti-protein gel, hydrophilic and non-toxic properties. At present, carrageenan has been widely used in jelly, soft candy, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/56C12N9/42C12P19/14A61K31/7016A61K31/702A61K31/737A61P31/04A61P31/12A61P37/02A61P39/06C12R1/01
CPCY02A50/30
Inventor 邵宗泽单大鹏古铮李旭应见喜
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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