Food grade thermophilic arabinose isomerase expressed from gras, and tagatose manufacturing method by using it
A technology of arabinose and isomerase, which is applied in the field of food-grade thermophilic arabinose isomerase expressed by GRAS and its use in the preparation of tagatose, which can solve the problems of inability and low expression level
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Embodiment 1
[0035] Example 1: Cloning of arabinose isomerase
[0036] Cultivation of Geobacillus stearothermophilus DSM 22 under aerobic conditions. Centrifuge at 8000xg for 10 minutes to recover the cultured cells. Genomic DNA was extracted from the obtained cells by using the cell culture DNA Midi Kit (Qiagen, USA). By using oligonucleotides 5′-TCTAGAATGATGCTGTCATTACGTCCTTATGAATTTTG-S′ (SEQ.ID.NO:1) and 5′-TCTAGATTACCGCCCCCGCCAAAACACTTCGTTCC-S′ (SEQ.ID.NO: 2) inserted by using the oligonucleotides having the sequence of Xbal and BamHi restriction enzyme sites As a primer, genomic DNA was used for polymerase chain reaction (PCR). PCR product 1 was obtained by amplifying a 1494 bp DNA containing the arabinose isomerase gene from Geobacillus stearothermophilus. Again by using the oligonucleotides 5′-CCCGAT ATCATGCTGT-CATT ACGTCCTTATG-S′ (SEQ.ID.NO: 3) and 5′-TGCACTGCAGTTACCGCCCCCG CCAAAACAC-3′ (SEQ. ID.NO: 3) inserted into the oligonucleotides with EcoRV and PstI restriction enzyme site s...
Embodiment 2
[0040] Example 2: Expression of arabinose isomerase in Bacillus
[0041] The recombinant strain Bacillus subtilis GSAlB-1 (preservation number: KCCM10788P) prepared in the above Example 1 was inoculated on LB medium (Bacto-tryptone 10g / L, Bacto-yeast extract) containing 20 μg / mL chloramphenicol 5g / L, NaCl 10g / L), and then shake flask culture at 230rpm at 37°C for 12 hours. Produce pre-culture solution. Then the pre-culture broth was inoculated into the main medium with the same composition at a concentration of 0.1%, followed by shaking flask culture at 37°C at 230rpm until the OD 600 Reach 1 to induce the expression of recombinant arabinose isomerase. In order to determine the enzymatic activity of the expressed arabinose isomerase, the culture solution was centrifuged at 12000×g for 10 minutes, and the cells were recovered. The cells were suspended in 50 mM Tris-HCl (pH 8.2) buffer, followed by sonication (170 watts, cooling on ice at intervals of 1 second / 2 minutes) to lyse...
Embodiment 3
[0044] Example 3: Expression of recombinant arabinose isomerase in coryneform bacteria
[0045] The recombinant strain Corynebacterium glutamicum GSAIC-1 (preservation number: KCCM10789P) prepared in Example 1 was inoculated into MB medium (Bacto-tryptone 10g / L, Bacto) containing 10 μg / mL kanamycin -Yeast extract 5g / L, NaCl 10g / L, soy peptone 5g / L), and then shake flask culture at 30°C at 200 rpm for 24 hours to prepare a pre-culture solution. The obtained pre-culture solution was inoculated into the main medium with a concentration of 1%, and then cultured in a shake flask at 30°C at 200 rpm until the OD 600 Achieve 0.1 to induce the expression of recombinant arabinose isomerase. In order to determine the enzymatic activity of the expressed arabinose isomerase, the culture solution was centrifuged at 8000xg for 10 minutes, and the cells were recovered. The cells were suspended in 50 mM Tris-HCl (pH 7.0) buffer, followed by ultrasonic lysis. Centrifuge again at 8000xg for 12 m...
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