Alginate lyase, host cells capable of secreting alginate lyase and application of alginate lyase and host cells

A technology of alginate lyase and host cells, which is applied in the field of host cells secreting alginate lyase and alginate lyase, which can solve the problems of large molecular weight of precursor protein, low enzyme activity and low expression of alginate lyase , to achieve the effect of wide application prospect and high safety

Active Publication Date: 2018-04-13
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the highest enzyme activity of the alginate lyase secreted by this method is only 35-40U/mL, which has the problem of low enzyme activity.
[0006] CN 106995811 A discloses an alginate lyase, its preparation method and application. The alginate lyase is stable in nature and has a specific enzyme activity as high as 4600U/mL. However

Method used

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  • Alginate lyase, host cells capable of secreting alginate lyase and application of alginate lyase and host cells
  • Alginate lyase, host cells capable of secreting alginate lyase and application of alginate lyase and host cells
  • Alginate lyase, host cells capable of secreting alginate lyase and application of alginate lyase and host cells

Examples

Experimental program
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Effect test

Embodiment 1

[0069] Example 1 Construction of the first plasmid

[0070] (1) The upstream and downstream of the D-alanine racemase gene (dal) on the Bacillus subtilis 1A751 chromosome respectively select a 1000bp fragment as a homology arm, carry out PCR amplification, and connect two sections of homology arms by recombinant PCR, Obtain the dalU-dalD fragment of the knockout D-alanine racemase gene;

[0071] (2) Insert the lethal factor gene (mazF) of Escherichia coli behind the xylose promoter xylA' of the pAX01 vector, and obtain a fragment containing xylR-xylA'-macF-erm through PCR amplification;

[0072] (3) Insert the dalU-dalD fragment and the xylR-xylA'-macF-erm fragment into the multiple cloning site of the pUC19 plasmid to construct the first plasmid pUC-dalU-dalD to obtain the knockout D-alanine racemase gene -xylR-xylA'-macF-erm.

Embodiment 2

[0073] Example 2 Preparation of D-alanine racemase gene knockout strain

[0074] (1) transforming the first plasmid pUC-dalU-dalD-xylR-xylA'-macF-erm into Bacillus subtilis 1A751, and using erythromycin to select the bacterial strain in which the first plasmid is inserted into the chromosome;

[0075] (2) Use xylose as a growth pressure to screen secondary recombined plasmids and strains in which the dal gene has been knocked out. For strains that have not undergone secondary recombination, xylose is used as an inducer of the xylose promoter to induce the expression of macF, Lethal was performed to obtain D-alanine racemase gene knockout Bacillus subtilis 1A751-Δdal.

Embodiment 3

[0076] Example 3 Construction of the second plasmid

[0077] (1) PCR amplification obtains the D-alanine racemase gene of Bacillus subtilis 1A751 and its promoter fragment, utilizes this fragment to replace the kanamycin resistance gene on the Bacillus subtilis expression plasmid pMA5, obtains pMA5- dal plasmid;

[0078] (2) Gene fusion of the amplified alginate lyase gene and signal peptide gene was performed by overlapping PCR, and connected between NdeI and BamHI of the pMA5-dal plasmid to obtain the second plasmid pMA5-dal secreting alginate lyase -Aly.

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Abstract

The invention provides alginate lyase, host cells capable of secreting alginate lyase and application of the alginate lyase and the host cells. The alginate lyase has any one of amino acid sequences shown as (I) and (II): (I) an amino acid sequence shown as SEQ ID NO. 1; (II) an amino acid sequence obtained by carrying out substitution, deletion or addition of 1 to 20 amino acid residues on the amino acid sequence shown as SEQ ID NO. 1; the obtained amino acid sequence has the activity of the alginate lyase. According to the alginate lyase, whole genes of the alginate lyase are truncated to reduce the molecular weight, so that the expression amount of the host cells on the alginate lyase is remarkably improved; the secreted alginate lyase has high activity, good stability and strong degradation capability on sodium alginate.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to an alginate lyase, a host cell secreting the alginate lyase and an application thereof. Background technique [0002] Sodium alginate is a by-product of extracting iodine and mannitol from brown algae kelp or sargassum, and is a natural polysaccharide formed by linking β-D-mannuronic acid and α-L-guluronic acid . Studies have found that alginate oligosaccharides, the degradation products of sodium alginate, have the functions of lowering blood sugar, anti-inflammation, anti-tumor, anti-coagulation, immune regulation and growth regulation, and have been widely used in the fields of medicine, food and feed. [0003] The preparation of fucoidan oligosaccharides is mainly carried out by acid degradation, oxidative degradation, ultrasonic degradation and enzymatic degradation. However, these methods generally have problems such as low efficiency and low yield. [0004] The...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/75C12N15/74C12N1/21C12P19/12C12P19/04C12P19/00A23L33/10A23L33/13C05F11/00A61K8/66A61Q19/00
CPCA23L29/06A23L33/10A23L33/13A23V2002/00A61K8/66A61K2800/10A61Q19/00C05F11/00C12N9/88C12N15/74C12N15/75C12P19/00C12P19/04C12P19/12C12Y402/02011A23V2200/30
Inventor 朱玥明孙媛霞陈朋曾艳门燕杨建刚
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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