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Multifunctional algal polysaccharide lyase gene and application thereof

A seaweed polysaccharide and lyase technology, applied in the field of genetic engineering, can solve the problems of low activity, poor enzyme stability, hindering the production and application of seaweed oligosaccharides, and achieve high activity effect

Inactive Publication Date: 2018-08-17
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, most strains producing seaweed polysaccharide lyase have low activity and poor enzyme stability, which hinders the production and application of seaweed oligosaccharides

Method used

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  • Multifunctional algal polysaccharide lyase gene and application thereof
  • Multifunctional algal polysaccharide lyase gene and application thereof
  • Multifunctional algal polysaccharide lyase gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Extraction of Genomic DNA of Pseudoalteromonas carrageenovora ASY5 Strain

[0036] Inoculate Pseudoalteromonas carrageenovora ASY5 into artificial seawater medium, and culture with shaking at 25°C and 180r / min until OD 600 1 to 1.5, take 1 mL of the culture liquid, and use Dongsheng Biological Bacterial Genome Rapid Extraction Kit (silica membrane spin column method) to extract the genomic DNA of Pseudoalteromonas carrageenovora ASY5 strain.

[0037] The above-mentioned artificial seawater medium configuration method is as follows:

[0038] Beef extract 10g, tryptone 10g, distilled water 250mL, artificial seawater 750mL (NaCl 37.51g, KCl 1.03g, CaCl 2 1.61g, MgCl 2 ·6H 2 O 6.4g, NaHCO 3 0.15g, MgSO 4 ·7H 2 O4.67g, distilled water 1000mL). After dissolving the beef extract and tryptone, adjust the pH to 7.8, heat and boil for 10 minutes, adjust the pH to 7.3 after cooling, then mix with artificial seawater, and sterilize at 121°C for 20 minutes. Add ...

Embodiment 2

[0039] Example 2: Sequence Analysis

[0040] Use NCBI's BLAST program to search for homology of nucleic acid sequences and amino acid sequences in the GenBank database, use the signal peptide prediction tool SignalP 4.0 of the SMART database to predict whether there is a signal peptide in the multifunctional seaweed polysaccharide lyase, and use the protparam tool of ExPASy to predict proteins The physical and chemical properties of the protein, using SWISS-MODEL for three-dimensional modeling of protein structure.

[0041] The results analyzed by the above-mentioned biological software showed that the coding region of the gene Alg823 was 2259 bp long, and the nucleotide sequence was shown in SEQ ID NO.1. The multifunctional seaweed polysaccharide lyase Alg823 encoded by the gene Alg823 contains 752 amino acids in total, and its amino acid sequence is shown in SEQ ID NO.2. Online analysis with BLAST software showed that the N-terminal contained the catalytic domain conserved ...

Embodiment 3

[0042] Embodiment 3: the recombinant expression of gene Alg823 in Escherichia coli BL21 (DE3) bacterial strain

[0043] Using the genomic DNA obtained in Example 1 as a template, carry out PCR amplification, and the primer sequences are as follows:

[0044] Forward primer Alg823-F:5'-CCG GAATTC AAAGATTATTTTGTAGA-3' (EcoRI)

[0045] Reverse primer Alg823-R:5'-CGC GGATCC CCCCGCCTTATTTAAAA-3' (BamHI)

[0046]The underlined forward primer Alg823-F is the restriction endonuclease EcoRI site, and the reverse primer Alg823-R is underlined the restriction endonuclease BamHI site. The high-fidelity DNA polymerase PrimeSTAR HS used was purchased from China Dalian Biotech Co., Ltd., and the PCR reaction reagents used were operated according to the product instructions provided by the company.

[0047] The PCR conditions are: 95°C, 5min; 94°C, 45s; 50.7°C, 45s, 72°C, 1min, 30 cycles, and finally 72°C, 10min.

[0048] The PCR product and the pET-28a(+) plasmid were double-digested w...

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Abstract

The invention discloses a multifunctional algal polysaccharide lyase gene, multifunctional algal polysaccharide lyase, a genetic engineering bacterium with the multifunctional algal polysaccharide lyase and a method for constructing the genetic engineering bacterium. Nucleotide and amino acid sequences of the multifunctional algal polysaccharide lyase are shown as SEQ ID NO.1 and SEQ ID NO.2. Themethod includes steps of firstly, utilizing DNA (deoxyribonucleic acid) of genomes of marine bacteria as a template and amplifying multifunctional algal polysaccharide lyase gene sequences by the aidof primers; secondly, cloning the multifunctional algal polysaccharide lyase gene into plasmids to obtain recombinant vectors; thirdly, carrying out transformed cloning on the recombinant vectors in hosts; fourthly, carrying out transformed expression on the recombinant vectors in hosts to obtain the multifunctional algal polysaccharide lyase production genetic engineering bacterium. The multifunctional algal polysaccharide lyase gene, the multifunctional algal polysaccharide lyase, the genetic engineering bacterium and the method have the advantage that the multifunctional algal polysaccharide lyase is high in activity for sodium alginate, k-carrageenan, l-carrageenan and agar and can be widely used in the field of chemical engineering, agriculture, food and feed addition, medicines, algahereditary engineering and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a multifunctional seaweed polysaccharide lyase gene and its application. Background technique [0002] Polysaccharides are widely distributed in nature and are important components of living organisms. They play an important role in controlling cell division, regulating cell growth and maintaining normal metabolism of life. Therefore, studies related to polysaccharides have attracted more and more attention. As the medicinal potential of polysaccharides from marine organisms is gradually developed, seaweed has the largest number and variety of marine plants, and the polysaccharide content accounts for more than 50% of the dry weight, making it the most promising type of active substance. Seaweed polysaccharide is a high molecular carbohydrate composed of multiple identical or different monosaccharide groups connected by glycosidic bonds, which has high ap...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P19/02C12P19/12C12R1/19
CPCC12N9/88C12N15/70C12P19/02C12P19/12
Inventor 肖安风曾洁杨秋明倪辉姜泽东陈艳红伍菱
Owner JIMEI UNIV
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