Facultative incision type recombinant alginate lyase rAly-1 as well as coding gene and application thereof

A technology of alginate lyase and coding gene, applied in the direction of lyase, carbon-oxygen lyase, recombinant DNA technology, etc., to achieve broad application prospects, good temperature stability and pH stability

Inactive Publication Date: 2017-02-15
WUTONG AROMA CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant report on the facultative endo-type alginate lyase

Method used

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  • Facultative incision type recombinant alginate lyase rAly-1 as well as coding gene and application thereof
  • Facultative incision type recombinant alginate lyase rAly-1 as well as coding gene and application thereof
  • Facultative incision type recombinant alginate lyase rAly-1 as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1, Extraction of Flammeovirga yaeyamensis MY04 strain genomic DNA

[0048] 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.

[0049] The above-mentioned liquid medium YT04 has the following components per liter:

[0050] Tryptone 10g, yeast extract 5.0g, sodium chloride 30g, dissolved in water and adjusted to 1L, pH 7.2.

[0051] 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

[0052] Example 2. Genome scanning and sequence analysis of Flammeovirga yaeyamensis MY04 strain.

[0053] Genomic DNA prepared in Example 1 was scanned and sequenced by Shanghai Meiji Biotechnology Co., Ltd. using pyrosequencing technology. 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).

[0054] The results analyzed by the above-mentioned biological software show that the genomic DNA of the Flammeovirga yaeyamensis MY04 strain carries an encoding gene aly-1 of alginate lyase, and the coding region of the gene aly-1 is 1335 bp long, and the nucleotide sequence is as follows: Shown in SEQ ID NO.1. The recombinant alginate lyase rAly-1 encoded by ...

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 ...

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Abstract

The invention relates to facultative incision type recombinant alginate lyase rAly-1 as well as a coding gene and application thereof. An amino acid sequence of the facultative incision type recombinant alginate lyase rAly-1 is as shown by SEQ ID NO. 2; a nucleotide sequence of the coding gene of the facultative incision type recombinant alginate lyase rAly-1 is as shown by SEQ ID NO. 1. According to the facultative incision type recombinant alginate lyase rAly-1 as well as the coding gene and the application thereof, the facultative incision type recombinant alginate lyase rAly-1 is obtained from a genome of flammeovirga yaeyamensis MY04 for the first time; not only can the lyase be used for degrading a guluronic acid segment from alginate, but also can be used for degrading a mannuronic acid segment. The lyase can be used for thoroughly degrading alginate polysaccharide in any M / G ratio, and efficiently preparing alginate oligosaccharide with a lower polymerization degree.

Description

technical field [0001] The invention relates to a facultative endo-cutting recombinant alginate lyase rAly-1 and its coding gene and application, belonging to the technical field of genetic engineering. Background technique [0002] Alginate is a linear acidic polysaccharide, its main chain molecules are linear, composed of mannuronic acid (Mannuronate, M) and its C-5 differential isomer guluronic acid (Guluronate, G), through glycosidic bonds Linked continuously or alternately. There are uniform poly-M segments, poly-G segments, and hybrid MG or GM segments in the alginate molecule. Alginate is mainly produced from kelp, sargassum and other large marine eukaryotic algae that are used for medicine and food, and is the main component of cell wall and intercellular matrix. In addition, the opportunistic pathogen Pseudomonas aeruginosa and some soil microorganisms such as brown nitrogen-fixing bacteria can also secrete alginate, the difference is that the alginate derived fro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12P19/12C12P19/00C12N15/70C12N1/21C12R1/01C12R1/19
CPCC12N9/88C12N15/70C12N2800/101C12P19/00C12P19/12C12Y402/02003C12Y402/02011
Inventor 韩文君程媛媛古静燕王丹丹刘会会李福川高长健李新卫洁
Owner WUTONG AROMA CHEM CO LTD
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