M-preferred monosaccharide exo-algin lyase Aly-6 as well as coding gene and application thereof
A technology of alginate lyase and coding gene, which is applied in the direction of lyase, carbon-oxygen lyase, enzyme, etc., can solve the problem that there is no relevant report on the production characteristics of oligosaccharide products, and achieve the effect of stable physical and chemical properties and high activity
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[0060] Example 1. Extraction of genomic DNA of Flammeovirga yaeyamensis MY04 strain
[0061] The Flammeovirga yaeyamensis MY04 was inoculated into the liquid medium YT04, and under the conditions of 28 ° C and 200 rpm, it was shaken and cultivated to an absorbance value of 600 nm (OD). 600 ) is 1.2; take 10 mL of cultured bacterial solution, centrifuge for 15 min under 12,000 × g (g, gravitational constant), and collect the bacterial cell precipitate; use 10 mL of lysozyme buffer (10 mM Tris-HCl, pH 8.0) to suspend the bacterial cell , centrifuge at 12,000 rmp for 15 min, and collect the bacterial pellet.
[0062] The above-mentioned liquid culture medium YT04, the components per liter are as follows:
[0063] 10 g of tryptone, 5.0 g of yeast extract, and 30 g of sodium chloride were dissolved in water and made up to 1 L, pH 7.2.
[0064]In the above-mentioned thalline precipitation, add 6.0 mL of lysozyme buffer (purchased from Shanghai Sangon Bioengineering Co., Ltd.) to e...
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[0065] Example 2. Scanning and sequence analysis of the genome of Flammeovirga yaeyamensis MY04 strain.
[0066] The genomic DNA obtained in Example 1 was scanned and sequenced by pyrosequencing technology, which was completed by Shanghai Meiji Biotechnology Co., Ltd. The DNA sequencing results were analyzed with the online software of NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ). The NCBI website analysis software used is Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) blast.ncbi.nlm.nih.gov / Blast.cgi).
[0067] The results of analysis with the above biological software show that the genomic DNA of Flammeovirga yaeyamensis MY04 strain carries a alginate lyase encoding gene aly-6, the coding region of the gene aly-6 is 2238bp long, and the nucleotide sequence is as follows: shown in SEQ ID NO.1. The recombinant al...
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[0069] Example 3. Recombinant expression of gene aly-6 and truncated body aly-6Lm and aly-6Hpm in Escherichia coli BL21(DE3) strain
[0070] Using the genomic DNA prepared in Example 1 as a template, PCR amplification was performed. The primer sequences are as follows:
[0071] Forward primer Aly6-F for aly-6 amplification: 5'-gg CATATG CAGAACGGAAATTTAATTACTTCCG-3'(NdeI);
[0072] Reverse primer for aly-6 amplification Aly6-R:5'-gc TCTAGA AATTTCTTCCAATAGGTAAAACCCC-3'(XbaI);
[0073] Forward primer for aly-6Lm amplification Aly6Lm-F: 5'-gg CATATG CAGAACGGAAATTTAATTACTTCCGAG-3'(Nde I);
[0074] Reverse primer for aly-6Lm amplification Aly6Lm-R: 5'-gc TCTAGA CAAACTTCTTTTTTGGTAAGTCGTTGC-3'(Xba I);
[0075] Forward primer for aly-6Hpm amplification Aly6Hpm-F: 5’-gg CATATG TACTATTCAAGAGAATTGAAACTAGCC-3'(Nde I);
[0076] Reverse primer for aly-6Hpm amplification Aly6Hpm-R: 5'-gc TCTAGA AATTTCTTCCAATAGGTAAAACCCC-3'(Xba I);
[0077] The forward primer is underlined th...
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