Method for transglycosylating using whole cell
A whole-cell, galactooligosaccharide technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., to achieve the effect of saving production costs
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Embodiment 1
[0068] Sulfolobus solfataricus (Sulfolobus solfataricus 98 / 2) was cultivated in the following medium for 24 hours under the condition of 80°C. The medium was composed of 0.1% yeast extract, 0.1% casamino acid, 0.3% dipotassium hydrogen phosphate, 0.25% ammonium sulfate, 0.02% MgSO 4 ·7H 2 O, 0.02% CaCl 2 2H 2 Trace elements (1.8mg MnCl 2 4H 2 O, 4.5 mg Na 2 B 4 o 7 10H 2 O, 0.22mg ZnSO 4 ·7H 2 O, 0.05 mg CuCl 2 2H 2 O, 0.03 mg Na 2 MoO 4 2H 2 O, 0.03mg VOSO 4 2H 2 O, 0.01 mg CoSO 4 ·7H 2 O / L) and adjust the final pH value to 4. 500 μg of genomic DNA was isolated from the above cells (Genomic DNAextraction kit; produced by RBC Corporation), and then the genomic DNA isolated above was used as the main type, and then PCR was performed using the primers of SEQ ID NO: 2 and SEQ ID NO: 3 , and then, the PCR product was treated with restriction enzymes NdeI and AvaI, and then ligated on the vector pET24a (produced by Novagen) treated with the same restriction enzy...
Embodiment 2
[0087] Select one to smear on the LB agar (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5% agar) medium that contains Kanamycin (Kanamycin) 20mg / L concentration and convert glutamic acid Coryneform bacterium bacterium colony (colony), then, bacterium colony is inoculated into in the 500ml Erlenmeyer flask that has added the 50mlLB culture medium that has been sterilized, and under the condition of 30 ℃ according to the stirring speed of 200rpm 24 hours, thus cultivate species ( seed) medium.
[0088] A fermenter (Jar-fermentor; manufactured by KoBioTech Co.) can be used for this culture, and the composition of the medium is shown in Table 1.
[0089] 【Table 1】
[0090] composition Concentration (g / l) glucose 40 Peptone 28 yeast extract 15 ammonium sulfate 7 Potassium dihydrogen phosphate 2.5 Sodium chloride 0.5 CaCl 2 2H 2 o 10 MgSO 4 ·7H 2 o 0.5
[0091] Inoculate 50ml of the seed mediu...
Embodiment 3
[0095] In order to determine the activity of β-galactosidase, 4-nitrophenyl-β-D-galactopyranoside was dissolved in 0.1M phosphate buffer (pH 6.0) to use it as a substrate, and The reaction was carried out at 80° C., and then the enzyme reaction was stopped with 10% (w / v) borax (Disodium tetraborate) for coloring. The activity of the enzyme can be confirmed by the concentration of free O-nitrophenol in the absorbance at 420nm, that is, the concentration of β-galactosidase 1unit freeing 1 μmol of O-nitrophenol per minute under the above conditions Quantity is confirmed.
[0096] In addition, SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis) uses a separating gel to adjust the concentration of acrylamide to 10%, and then uses a stacking gel to make acrylamide The concentration was adjusted to 8%. The SDS-PAGE buffer can use a mixed solution with a pH of 8.3 consisting of 25 mM Tris-base, 192 mM glycine, and 0.1% SDS. The supernatant of the cell disruption was...
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