Novel fructose-4-epimerase and method for preparing tagatose using same
A technology for epimerase and fructose, which is applied in the field of novel fructose-4-epimerase and the preparation of tagatose by using it, can solve problems such as difficulty in supplying lactose, and achieve the effect of excellent heat resistance
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[0056] The preparation method of the present invention may further include separating and / or purifying the produced tagatose. Isolation and / or purification can be performed using any method commonly used in the art, such as, but not limited to, dialysis, precipitation, adsorption, electrophoresis, ion exchange chromatography, and fractional crystallization. Purification can be performed using these methods alone or in combination.
[0057] In addition, the preparation method of the present invention may further include decolorizing and / or desalting the prepared tagatose before or after separation and / or purification. Higher quality tagatose can be obtained by decolorizing and / or desalting.
[0058] According to another embodiment, the production process of the present invention may further comprise crystallizing tagatose after conversion, separation and / or purification, or decolorization and / or desalting. Crystallization of tagatose can be performed by using any crystallizat...
Embodiment 1
[0065] Example 1: Preparation of recombinant expression vectors comprising hypothetical protein genes and transformed microorganisms
[0066] In order to discover a new type of heat-resistant fructose-C4-epimerase, the gene information of the hypothetical protein whose function has not been revealed was obtained from Litorilinea aerophile, and the vector and transformed microorganism.
[0067] Specifically, from the gene sequences of Litorilinea aerophile registered in the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the National Center for Biotechnology Information (NCBI) in the United States, the gene sequence of the hypothetical protein was selected based on the amino acid sequence of the microorganism (Bioneer Corporation, Korea) (SEQ ID NO: 1) and nucleotide sequence (SEQ ID NO: 2), the recombinant vector pBT7-C-His-CJ_LiA_F4E was prepared, which contains the nucleotide sequence of the protein and can be expressed in Escherichia coli.
[0068] Escherichia coli BL21...
Embodiment 2
[0069] Embodiment 2: Preparation and purification of protein
[0070]In order to prepare protein, inoculate the transformed strain Escherichia coli BL21(DE3) / CJ_LiA_F4E prepared in Example 1 into a culture tube containing LB liquid medium supplemented with ampicillin as an antibiotic, and perform seed culture in a shaking incubator at 37°C , until the absorbance at 600 nm reaches 2.0. The culture solution obtained from the seed culture was inoculated into a culture bottle containing a liquid medium supplemented with LB and lactose as a protein expression regulator, for main culture. The seed culture and the main culture were performed at a stirring speed of 180 rpm and 37°C. Subsequently, the culture solution was centrifuged at 8,000 rpm and 4° C. for 20 minutes, and strains were collected therefrom. The collected strains were washed twice with 50 mM Tris-HCl buffer solution (pH 8.0), and resuspended in 50 mM NaH containing 10 mM imidazole and 300 mM NaCl 2 PO 4 buffer sol...
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