Incision difunctional alginate lyase Aly2 generating various monosaccharide products, encoding gene of Aly2 and application of Aly2

A technology of alginate lyase and coding gene, which is applied in the field of endo-type bifunctional alginate lyase Aly2 and its coding gene, can solve the problems that no endo-type alginate lyase has been found, and achieve stable physical and chemical properties of the enzyme, The effect of high degradation activity and broad application prospects

Active Publication Date: 2018-05-18
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the current reports on endo-type alginate lyase, in terms of oligosaccharide production characteristics, no endosaccharides that can produce both saturated monosaccharide products such as M and G and unsaturated Δ monosaccharide products have been found. cleaved alginate lyase

Method used

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  • Incision difunctional alginate lyase Aly2 generating various monosaccharide products, encoding gene of Aly2 and application of Aly2
  • Incision difunctional alginate lyase Aly2 generating various monosaccharide products, encoding gene of Aly2 and application of Aly2
  • Incision difunctional alginate lyase Aly2 generating various monosaccharide products, encoding gene of Aly2 and application of Aly2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1, Extraction of Flammeovirga yaeyamensis MY04 strain genomic DNA

[0046] Inoculate Flammeovirga yaeyamensis MY04 into the liquid medium, and shake it to OD at 28°C and 200rpm 600 1.2; take 10mL of the cultured bacteria, centrifuge at 12,000rpm for 25min, collect the bacterial pellet, wash with 10mL of lysozyme buffer (10mM Tris-HCl pH 8.0), centrifuge at 12,000rpm for 25min, collect the bacterial precipitation.

[0047] The components per liter of the above-mentioned liquid culture medium are as follows:

[0048] Tryptone 10g, yeast extract 5g, NaCl 30g, dilute water to 1L, pH value is 7.2.

[0049] Add 6.0mL of lysozyme buffer solution to each tube to obtain about 7.0mL of bacterial liquid in the above bacteria precipitation, add 280μL of lysozyme with a concentration of 20mg / mL respectively, and the final concentration is about 800μg / mL; after 1.0h of ice bathing, , incubate at 37°C for 2h until the solution is viscous; add 0.41mL of 100mg / ml SDS, 30μL of...

Embodiment 2

[0050] Example 2 Genome scanning and sequence analysis of Flammeovirga yaeyamensis MY04 strain.

[0051] The large molecular weight genomic DNA prepared in Example 1 was sequenced (Meiji Biological Company). The sequencing results were analyzed with the software on NCBI (National Center for Biotechnology Information, http: / / www.ncb1.nlm.nih.gov / ). The NCBI analysis software used is Open Reading Frame Finder (ORF Finder, http: / / www.ncb1.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi .nlm.nih.gov / Blast.cgi).

[0052] NCBI analysis results show that the genome of Flammeovirga yaeyamensis MY04 strain carries an alginate lyase gene aly2, the coding region of which is 1620 bp long, and its nucleotide sequence is shown in SEQ ID NO.1. The alginate lyase Aly2 encoded by the gene aly2 contains 539 amino acids in total, and its amino acid sequence is shown in SEQ ID NO.2. Online analysis with BLAST software showed that Aly2 had 30% homolog...

Embodiment 3

[0054] Recombinant expression of the gene of embodiment 3, Aly2 in Escherichia coli

[0055] Using the large molecular weight genomic DNA prepared in Example 1 as a template, PCR amplification was performed.

[0056] Primers are as follows:

[0057] Forward primer 30aAly2-F: 5'-C GGATCC CAACAACCTATCGTTATTGTAAAC-3' (BamH I);

[0058] Reverse primer 30aAly2-R: 5'-G CTCGAG TTTTATTTGGTGTATAAGTGGTTTTTAAC-3' (Xho I);

[0059] The underlined forward primer is the restriction endonuclease BamH I, and the underlined reverse primer is the restriction endonuclease Xho I site. Primerstar HS DNA polymerase was purchased from Bao Biological Company, and the PCR reaction system was operated according to the product instructions provided by the company.

[0060] PCR reaction conditions: pre-denaturation at 95°C for 4 min; denaturation at 95°C for 40 s, annealing at 60°C for 40 s, extension at 72°C for 2 min, 30 cycles; extension at 72°C for 10 min, stabilization at 4°C for 15 min.

[...

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Abstract

The invention relates to incision difunctional alginate lyase Aly2 generating various monosaccharide products, an encoding gene of the Aly2 and an application of the Aly2. The amino acid sequence of the Aly2 is as shown in SEQ ID NO. 2, and the nucleotide sequence of the encoded Aly2 is as shown in SEQ ID NO. 1. The specific activity of prepared gene engineering recombinant protein rAly2 for alginate, polyguluronic acid and polymannuronic acid is 2025U/mg, 3672U/mg and 1324U/mg, so that the gene engineering recombinant protein rAly2 is a difunctional enzyme. The enzyme is an incision enzyme, and final main products are unsaturated disaccharide and unsaturated trisaccharide when an alginate polysaccharide substrate is thoroughly degraded. When different oligosaccharide substrates are degraded, various monosaccharide products such as saturated M and G or unsaturated delta can be generated. Besides, the rAly2 is stable in biochemical property, is a potential tool enzyme, can be applied tothe field of medicines, food and the like and has a wide application prospect.

Description

technical field [0001] The invention relates to an endo-type bifunctional alginate lyase Aly2 capable of producing various monosaccharide products, its coding gene and application, and belongs to the technical field of genetic engineering. Background technique [0002] Alginate is an important acidic polysaccharide composed of β-D-mannuronic acid (β-D-Mannuronate, M) and α-L-guluronic acid (α-L-Guluronate, G) arranged alternately Linear anionic polysaccharides formed sequentially through β-1,4 glycosidic linkages. In the alginate molecule, these two sugar units are usually randomly arranged and combined into polymannuronate (PM), polyguluronate (PG) or glycerin / paleoalternating blocks [1] . Natural alginate mainly comes from macroalgae, sargassum, kelp and other large marine brown algae with the same source of medicine and food. As a structural component of marine brown algae, the content of alginate can reach more than 40% of the dry weight. In addition, some bacteria, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12P19/00C12P19/12
CPCC12N9/88C12P19/00C12P19/12C12Y402/02
Inventor 韩文君彭春娥李福川程媛媛王庆彬路丹荣
Owner SHANDONG UNIV
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