A kind of N-acetylglucosamine deacetylase and its coding gene and application
A technology of glucosamine and acetylamino, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., and achieves the effect of good application value
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Embodiment 1
[0063] Embodiment 1, the acquisition of gene TpDac and the construction of expression vector
[0064] The gene shown in positions 1-801 of SEQ ID NO.1 is named gene TpDac, and the protein encoded by the gene is named protein TpDac, and its amino acid sequence is shown in SEQ ID NO.2.
[0065] According to the DNA sequence information shown in SEQ ID NO.1, the target gene is artificially synthesized.
[0066] The upstream primer TpDac-up (5'-ATGGCGTTCGAGGAGTTTG-3') and the downstream primer TpDac-down (5'-TTAGATAAGCTCGGCAAATGG-3',) were designed, and the target DNA fragment was amplified by PCR. The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 2 min, and 30 cycles; finally, extension at 72°C for 10 min. PCR products were recovered by 1% agarose gel electrophoresis and verified by sequencing.
[0067] The present invention also relates to a recombinant expressi...
Embodiment 2
[0072] Embodiment 2, expression and purification of recombinant N-acetylglucosamine deacetylase (TpDac)
[0073] The present invention also relates to the construction of a recombinant host cell for expressing TpDac, which comprises the nucleotide encoding TpDac described in the present invention. A host cell can be any cell useful in the recombinant production of proteins of the present invention, such as prokaryotic or eukaryotic cells.
[0074] The prokaryotic host cell can be any gram-positive or gram-negative bacterium, including but not limited to: Bacillus, Clostridium, Lactobacillus, Streptomyces, Staphylococcus, Escherichia coli, Pseudomonas, Paenibacillus.
[0075] Eukaryotic host cells can be mammalian, insect, plant or fungal cells, including but not limited to: filamentous fungi (Aspergillus, Mucor, Rhizopus, Penicillium, etc.), yeasts (Pichia pastoris, Candida spp. yeast, etc.).
[0076] In one embodiment, the expression host cell selected in the present inven...
Embodiment 3
[0083] Example 3, the application of recombinant N-acetylglucosamine deacetylase (TpDac) in the enzymatic preparation of glucosamine
[0084] Recombinant N-acetylglucosamine deacetylase (TpDac) hydrolyzes N-acetylglucosamine Reaction conditions: pH 8.0, 40°C, substrate concentration 1-3%, enzyme addition 0.5mg / L, hydrolysis time 1h. The volume of the hydrolyzate is 5L, and the stirring speed is 80-120rpm / min. Samples were taken at 0, 5, 10, 20, 40, and 60 minutes respectively, and hydrochloric acid was added to inactivate the enzyme to terminate the reaction.
[0085] Thin layer chromatography (TLC) monitoring of hydrolyzate
[0086] The hydrolyzate was analyzed using a Kieselgel 60 silica gel plate (Merck), and the developing solution was isopropanol:ammonia:water (15:7.5:1, v / v / v). Spot 1 μL of the sample on the silica gel plate, spread the silica gel plate with a spreading agent, dry it, and evenly apply the color developer anisaldehyde: ethanol: concentrated sulfuric aci...
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