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Truncated recombinant alginate lyase rAly1-T185N, and encoding gene and application thereof

A technology of alginate lyase and encoding gene, which is applied in the directions of lyase, application, genetic engineering, etc., to achieve the effect of improving the degradation rate and improving economic benefits

Active Publication Date: 2017-06-09
WUTONG AROMA CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the difference is that after the facultative Aly2 is truncated, the enzyme's propensity to the polyM fragment substrate can be relatively increased; but after the same truncation operation on the G-specific Aly5, this substrate propensity is not seen Significant changes in
Then, why the non-catalytic region can only affect the substrate preference of this facultative alginate lyase, but has little effect on the specific alginate lyase? For facultative alginate lyases, how does the non-catalytic region affect the catalytic region and thus change the substrate preference? At present, there are only reports of related experimental phenomena, but no detailed mechanism discussion

Method used

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  • Truncated recombinant alginate lyase rAly1-T185N, and encoding gene and application thereof
  • Truncated recombinant alginate lyase rAly1-T185N, and encoding gene and application thereof
  • Truncated recombinant alginate lyase rAly1-T185N, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1, Extraction of Flammeovirga yaeyamensis MY04 strain genomic DNA

[0049] Flammeovirga yaeyamensis MY04 was inoculated into liquid medium YT04, and cultured with shaking at 28°C and 200 rpm until the absorbance value at 600 nm (OD 600 ) is 1.2; take 10mL of cultured bacteria, centrifuge at 12,000×g (g, earth’s gravitational constant) for 15min, and collect the bacterial sediment; use 10mL of lysozyme buffer (10mM Tris-HCl, pH 8.0) to suspend the bacterial , and centrifuged at 12,000rmp for 15min to collect the cell pellet.

[0050] The above-mentioned liquid medium YT04 has the following components per liter:

[0051] Tryptone 10g, yeast extract 5.0g, sodium chloride 30g, dissolved in water and adjusted to 1L, pH 7.2.

[0052] Add 6.0 mL of lysozyme buffer solution (purchased from Shanghai Sangon Bioengineering Co., Ltd.) to each tube to obtain about 7.0 mL of bacterial liquid, add 280 μL of lysozyme solution with a concentration of 20 mg / mL, Make the final ...

Embodiment 2

[0053] Example 2. Genome scanning and sequence analysis of Flammeovirga yaeyamensis MY04 strain.

[0054] The scanning and sequencing of the genomic DNA prepared in Example 1 was carried out by pyrosequencing technology, which was completed by Shanghai Meiji Biotechnology Company. The DNA sequencing results were analyzed with the online software of NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ) website. The analysis software used on the NCBI website is Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0055] The results analyzed by the above biological software showed that the genomic DNA of the Flammeovirga yaeyamensis MY04 strain carried a gene aly-1 encoding alginate lyase, and the online analysis by BLASTp software showed that the protein of Aly-1 was speculated The C-terminus contains the putative catalytic domai...

Embodiment 3

[0056] Embodiment 3, recombinant expression of gene aly-1 in Escherichia coli BL21 (DE3) bacterial strain

[0057] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:

[0058] Forward primer Aly1-F: 5'-cgc GGATCC AACAATAAAGTAGAGGACGAG-3' (BamH I);

[0059] Reverse primer Aly1-R: 5'-ccg CTCGAG TATAAGTTTCTTTTAATTCTATAG-3' (Xho I);

[0060] The underlined mark in the forward primer Aly1-F is the restriction endonuclease BamH I site, and the underlined mark in the reverse primer Aly1-R is the restriction endonuclease Xho I site. The high-fidelity DNA polymerase PrimeSTAR HS DNAPolymerase 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.

[0061] PCR reaction conditions: pre-denaturation at 95°C for 4min; denaturation at 94°C for 40s, annealing at 60°C for 30s, extension at 72°C for 75...

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Abstract

The invention relates to truncated recombinant alginate lyase rAly1-T185N, and an encoding gene and application thereof. The amino acid sequence of the truncated recombinant alginate lyase rT185N is as shown in SEQ ID NO.2; the nucleotide sequence of the encoding gene of the truncated recombinant alginate lyase rT185N is as shown in SEQ ID NO.1. 185 amino acid residues are cut off from the N end of the full-length alginate lyase rAly-1 to obtain T185N truncated enzyme; the expression quantity of the rT185N can reach 1259+ / -2.2 mg / L fermentation liquid, the specific enzyme activity can reach 2895+ / -10.3 U / mg, the expression quantity is increased by about 2.6 times compared with that of full-length protein rAly-1, the aims of increasing yield, saving energy and improving economic benefit are fulfilled, and rT185N can realize one-step purification of fusion protein through nickel column separation.

Description

technical field [0001] The invention relates to a truncated recombinant alginate lyase rAly1-T185N and its coding gene and application, belonging to the technical fields of genetic engineering technology and protein expression. Background technique [0002] Alginate is a linear acidic polysaccharide formed by mannuronic acid (Mannuronate, M) and its C-5 differential isomer guluronic acid (Guluronate, G) linked by glycosidic bonds, and there is a uniform poly-M in the molecule. segment, polyG segment, and heterozygous MG or GM segment. Alginate widely exists in the cell wall and intercellular matrix of kelp, sargassum and other medicinal and edible large marine brown algae. At present, the main application in the fields of daily chemical industry, food and medicine is alginate derived from seaweed. In-depth research on glycobiology found that alginate oligosaccharides have important biological activities. For example, alginate oligosaccharides have important functions such...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12P19/00C12P19/12
CPCC12N9/88C12P19/00C12P19/12
Inventor 韩文君程媛媛王丹丹刘会会古静燕李福川李新卫洁
Owner WUTONG AROMA CHEM CO LTD
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