Cellulosimicrobium cellulans strain and application thereof

Abstract

The invention relates to a cellulosimicrobium cellulans strain and application thereof. The strain is a Cellulosimicrobium cellulans strain F16 of which the preservation number is CCTCC M2013201 and the preservation date is in may, 2013; the Cellulosimicrobium cellulans strain F16 or a hydrolase generated by virtue of the fermentation of the Cellulosimicrobium cellulans strain F16 are capable of hydrolyzing a compound which contains a xyloside bond and a glucoside bond to obtain a product, namely a corresponding aglycone or an intermediate compound generated in the preparation process of the corresponding aglycone.

Description

technical field

[0001] The invention belongs to the field of biochemical industry, and in particular relates to a fibrosis cellulosic microbacteria strain and application thereof. Background technique

[0002] Many natural active compounds often exist in nature in the form of glycosides, and the active compounds can be released after the sugar group is removed. Such as the anticancer drug paclitaxel, which is mainly found in the bark of the yew plant. In the branches and leaves of yew, there are a large number of xylosylated paclitaxel precursors or their analogs of the same mother nucleus (called taxane compounds), such as 7-xylose paclitaxel and 7-xylose-10 Deacetylpaclitaxel (10DAXT), its accumulation in the branches and leaves of Taxus chinensis, Yunnan and Southern Taxus can be more than 10 times higher than that of paclitaxel, and the 7-xylosyl group can be hydrolyzed and then converted into paclitaxel by one step of acetylation .

[0003] Another typical natural pr...

Examples

Embodiment 1

[0035] Embodiment 1: the preparation of crude enzyme liquid

[0036] A medium containing 1% starch, 0.2% peptone, 0.2% yeast extract, and 0.2% dipotassium hydrogen phosphate (pH 7.0 before sterilization) was used as the seed medium.

[0037] Distribute 20 ml of the above-mentioned seed medium into 100 ml conical flasks, sterilize at 120 degrees for 15 minutes, inoculate the agar slant culture of Cellulomonas cellulosus F16 strain into it, and shake and cultivate it at 30 degrees for 2 days as seed liquid.

[0038] Put 100ml of 1% birch xylan, 0.2% yeast extract, 0.2% ammonium chloride, 0.1% NaH into a 500ml Erlenmeyer culture bottle 2 PO 4 and 0.1%Na 2 HPO 4 Medium, pH7, autoclaved at 121°C for 30min. Inoculate 1ml of seed liquid into this medium, shake the flask at 150rpm, and culture at 30°C for 5 days. Centrifuge to collect the supernatant, which is the crude enzyme solution.

Embodiment 2

[0039] Embodiment 2: the preparation of the hydrolysis crude enzyme liquid of astragaloside IV

[0040] Add 1ml of 50mM Tris-HCl buffer solution (pH7.5) to 0.9ml of the crude enzyme solution in Example 1, and then add 0.1ml of astragaloside IV methanol solution (concentration: 10mg / ml). After incubation at 100 rpm and 35°C for 8 hours, using cycloastragenol standard as a control, TLC analysis showed that almost all of astragaloside IV was converted into cycloastragenol.

Embodiment 3

[0041] Example 3: Preparation of compound CK by hydrolysis of ginsenoside Rb1

[0042] Add 1ml of 50mM Tris-HCl buffer solution (pH7.5) to 0.9ml of the crude enzyme solution in Example 1, and then add 0.1ml of methanol solution of ginsenoside Rb1 (concentration: 10mg / ml). 100 rpm, incubate at 35°C for 4 hours. Add 2ml of methanol to terminate the reaction, centrifuge at 15,000 rpm for 20 minutes, and take the supernatant for UPLC analysis. The ginsenoside Rb1 in the reaction solution was almost completely converted to produce compound K (ie, CK, with a yield of 75%), an incompletely hydrolyzed intermediate product and a small amount of final product PPD.

[0043] UPLC method:

[0044] Chromatographic column: Kromasil ODS (2.1×150mm, 3μm)

[0045] Mobile phase: acetonitrile: water (0→30min: 20:80→95:5 gradient elution)

[0046] Flow rate: 0.4ml / min

[0047] Column temperature: room temperature

[0048] Detection wavelength: 203nm

technical field

[0001] The invention belongs to the field of biochemical industry, and in particular relates to a fibrosis cellulosic microbacteria strain and application thereof. Background technique

[0002] Many natural active compounds often exist in nature in the form of glycosides, and the active compounds can be released after the sugar group is removed. Such as the anticancer drug paclitaxel, which is mainly found in the bark of the yew plant. In the branches and leaves of yew, there are a large number of xylosylated paclitaxel precursors or their analogs of the same mother nucleus (called taxane compounds), such as 7-xylose paclitaxel and 7-xylose-10 Deacetylpaclitaxel (10DAXT), its accumulation in the branches and leaves of Taxus chinensis, Yunnan and Southern Taxus can be more than 10 times higher than that of paclitaxel, and the 7-xylosyl group can be hydrolyzed and then converted into paclitaxel by one step of acetylation .

[0003] Another typical natural pr...

Examples

Embodiment 1

[0035] Embodiment 1: the preparation of crude enzyme liquid

[0036] A medium containing 1% starch, 0.2% peptone, 0.2% yeast extract, and 0.2% dipotassium hydrogen phosphate (pH 7.0 before sterilization) was used as the seed medium.

[0037] Distribute 20 ml of the above-mentioned seed medium into 100 ml conical flasks, sterilize at 120 degrees for 15 minutes, inoculate the agar slant culture of Cellulomonas cellulosus F16 strain into it, and shake and cultivate it at 30 degrees for 2 days as seed liquid.

[0038] Put 100ml of 1% birch xylan, 0.2% yeast extract, 0.2% ammonium chloride, 0.1% NaH into a 500ml Erlenmeyer culture bottle 2 PO 4 and 0.1%Na 2 HPO 4 Medium, pH7, autoclaved at 121°C for 30min. Inoculate 1ml of seed liquid into this medium, shake the flask at 150rpm, and culture at 30°C for 5 days. Centrifuge to collect the supernatant, which is the crude enzyme solution.

Embodiment 2

[0039] Embodiment 2: the preparation of the hydrolysis crude enzyme liquid of astragaloside IV

[0040] Add 1ml of 50mM Tris-HCl buffer solution (pH7.5) to 0.9ml of the crude enzyme solution in Example 1, and then add 0.1ml of astragaloside IV methanol solution (concentration: 10mg / ml). After incubation at 100 rpm and 35°C for 8 hours, using cycloastragenol standard as a control, TLC analysis showed that almost all of astragaloside IV was converted into cycloastragenol.

Embodiment 3

[0041] Example 3: Preparation of compound CK by hydrolysis of ginsenoside Rb1

[0042] Add 1ml of 50mM Tris-HCl buffer solution (pH7.5) to 0.9ml of the crude enzyme solution in Example 1, and then add 0.1ml of methanol solution of ginsenoside Rb1 (concentration: 10mg / ml). 100 rpm, incubate at 35°C for 4 hours. Add 2ml of methanol to terminate the reaction, centrifuge at 15,000 rpm for 20 minutes, and take the supernatant for UPLC analysis. The ginsenoside Rb1 in the reaction solution was almost completely converted to produce compound K (ie, CK, with a yield of 75%), an incompletely hydrolyzed intermediate product and a small amount of final product PPD.

[0043] UPLC method:

[0044] Chromatographic column: Kromasil ODS (2.1×150mm, 3μm)

[0045] Mobile phase: acetonitrile: water (0→30min: 20:80→95:5 gradient elution)

[0046] Flow rate: 0.4ml / min

[0047] Column temperature: room temperature

[0048] Detection wavelength: 203nm

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