Microorganism capable of hydrolyzing glycosyl to hydroxyl and application thereof
A technology of microorganisms and taxanes, which is applied in the field of hydrolyzing glycosyl enzymes into hydroxyl groups to achieve good results
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Embodiment 1
[0046] Embodiment 1: Screening of xylosidase
[0047] Prepare agar plates: 1% xylan, 0.05% yeast extract, 0.05% tryptone, 1.5% agar. Suspend 0.2 g of soil sample or decayed wood sample in 40 ml of water, spread 50 microliters of 1:200 diluted suspension on each plate, and 50 microliters of 0.25 mg / ml 7-xylose-10-deacetyl paclitaxel. After incubation at room temperature (22° C.) for one week, 1 mM 4-methylumbelliferyl-β-D-xyloside was added dropwise on the grown colonies. Seventy-one colonies that fluoresce under UV light were picked and incubated at 28°C for 3-6 days.
[0048] Shake flask medium ingredients: 1% xylan, 0.2% yeast extract, 0.2% tryptone, 0.1% K 2 HPO 4 , 0.1% KH 2 PO 4 . Cultivate at 28°C, 175rpm. Two days later, 1 mg of 7-xylose-10-deacetylpaclitaxel or 7-xylose-10-deacetylbaccatin III dissolved in 0.2 ml of methanol was added to 10 ml of the culture medium, and the culture was continued for two days.
[0049] Another method is to culture for one day fi...
Embodiment 2
[0051] Example 2: Hydrolysis of 7-xylose-10-deacetyl paclitaxel
[0052] 1 liter of 1% birch wood xylan, 0.2% yeast extract, 0.2% tryptone, O.1% KH in a 4 liter Erlenmeyer flask 2 PO 4 and 0.1% K 2 HPO 4 , pH 7. Inoculate 20 milliliters of bacterial liquid into this medium, and culture in shake flasks at 30°C for 3 days. The cells were collected by centrifugation, washed with 50 ml of 50 mM potassium phosphate buffer (pH 7), and resuspended in this buffer after centrifugation. 1 mg of 7-xylose-10-desacetyl-paclitaxel (dissolved in 0.2 ml of methanol and 0.3 ml of water) was added to 1.5 ml of the cell suspension. Mix at 100 rpm for 21 hours at 30°C. The reaction was terminated by adding 2 mL of methanol and analyzed by HPLC. No 7-xylose-10-desacetyl-paclitaxel remained in the reaction solution, and 0.4 mg / ml 10-desacetyl-paclitaxel was produced (yield 100%). The reaction process of this example is as follows:
[0053]
Embodiment 3
[0054] Example 3: Hydrolysis of 7-xylose-paclitaxel
[0055] 0.5 mg of 7-xylose-paclitaxel (dissolved in 0.2 ml of methanol and 0.3 ml of water) was added to 1.5 ml of cell suspension (preparation method was the same as in Example 3). Mix at 100 rpm for 21 hours at 30°C. The reaction was terminated by adding 2 mL of methanol and analyzed by HPLC. 0.01 mg / ml 7-xylose-paclitaxel remained in the reaction solution, and 0.17 mg / ml paclitaxel was produced (yield 77%).
[0056] HPLC method
[0057] Column: Kromasil ODS (4.6×200mm, 5μm)
[0058] Mobile phase: 60% methanol / 40% water
[0059] Flow rate: 1ml / min
[0060] Column temperature: room temperature
[0061] Detection wavelength: 227nm
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