Streptomyces producing glucosidase and its application in biotransformation to prepare cucurbitacin b

A technology of streptomyces and glycosidase, applied in biochemical equipment and methods, methods based on microorganisms, microorganisms, etc., can solve problems such as inability to be absorbed by the human body, difficulty in hydrolysis, etc., and achieve safe use, simple nutritional requirements, and high conversion rate Effect

Active Publication Date: 2015-08-05
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compounds connected by glycosidic bonds need to be metabolized into aglycones before they can be absorbed by the human body. However, due to the lack of corresponding enzymes in the human body, it is difficult to hydrolyze them and can hardly be absorbed by the human body.

Method used

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  • Streptomyces producing glucosidase and its application in biotransformation to prepare cucurbitacin b
  • Streptomyces producing glucosidase and its application in biotransformation to prepare cucurbitacin b
  • Streptomyces producing glucosidase and its application in biotransformation to prepare cucurbitacin b

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Enrichment, isolation, screening and mutagenesis of glycosidase-producing microorganisms

[0031] Add about 2g of melon stalks to a 250mL conical flask, add an appropriate amount of sewage from the river, stir evenly, and place in a 30°C incubator for 7 days to enrich the glycosidase-producing microorganisms.

[0032] Dilute the above-mentioned enriched material covered with hyphae with sterile water, spread it on the plate medium, and cultivate it in a biochemical incubator at 30° C. for 3 days. Pick spores of colonies with different shapes and colors and inoculate them on the slant medium, and culture the slant at a constant temperature of 30°C for 3 days to obtain spore-rich slant strains.

[0033] Use an inoculation loop to pick out the spores of the slant strains of the above-mentioned strains, insert them into 50mL enzyme-producing medium (packed in a 250mL triangular bottle), ferment and cultivate them at 28°C and 180r / min for 5 days, and then use 4 la...

Embodiment 2

[0039] Example 2: Glycosidase Fermentation for Bioconversion Process

[0040] Using Streptomyces RW-2 as the enzyme-producing strain, the ability to convert cucurbitacin B glycosides into cucurbitacin B was significantly improved after optimization of the medium composition and fermentation conditions, and the content of cucurbitacin B was increased by 2.71 times compared with that before the optimization. It has increased by 45.7% before optimization, and the preferred preparation method is as follows:

[0041] (1) The Streptomyces RW-2 strain preserved on the slant of the test tube was inoculated on the slant medium, and the slant was cultured in a biochemical incubator at 30°C for 3 days. The slant medium is prepared according to the following composition and method: wash and peel the potatoes, cut them into small pieces, weigh 200g, add 1000mL of tap water and boil for 30min, filter the potato pieces with 4 layers of gauze, make up the filtrate to 1000mL, and then add sucr...

Embodiment 3

[0045] Embodiment 3: biotransformation method improves the content of cucurbitacin B in total cucurbitacin

[0046] The melon stalks, a commercially available Chinese herbal medicine, were dried in an oven at 85°C for 24 hours, and pulverized with a pulverizer. Add 200mL of 70% ethanol to 10g of melon stalk powder, extract in a water bath at 40°C for 2.5h, then ultrasonically extract for 0.5h, filter through 4 layers of gauze, and collect the filtrate. After the filtrate was evaporated to dryness under reduced pressure, it was dissolved in 40 mL of methanol to obtain the total cucurbitacin solution (the content of cucurbitacin B was 1.68 g / L).

[0047] Take 9 mL of the crude glucosidase solution prepared in Example 2 in a 50 mL Erlenmeyer flask, add 1 mL of the above-mentioned total cucurbitacin solution, seal the Erlenmeyer flask with plastic wrap, and biotransform in a water-bath shaking box at 32° C. and 180 r / min 16h. The conversion solution was extracted twice with 10 m...

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Abstract

The invention provides streptomycete (Streptomyces sp.) RW-2 used as a glycosidase producing bacterium and application of the streptomycete in preparing cucurbitacin B during biotransformation of cucurbitacin B-2-O glucoside. A bacterial strain is preserved in China Center for Type Culture Collection, the address of the China Center for Type Culture Collection is Wuhan University in Wuhan, China, the postcode is 430072, the preservation number is CCTCC NO:M2013330, and the preservation date is July 17th, 2013. The streptomycete has the beneficial effects that (1) the streptomycete is a common bacterium in the fermentation industry and is safe to use without toxicity and harms; (2) the nutritional requirements of the streptomycete RW-2 are simple, the growth speed of the streptomycete RW-2 is rapid, and the streptomycete RW-2 is easily cultivated; (3) the glycosidase prepared by the streptomycete RW-2 is extracellular enzyme, thallus is removed by separating the extracellular enzyme to obtain crude enzyme, or the crude enzyme can be used as a biotransformation system after being diluted; (4) after biotransformation treatment on total cucurbitacin extracted from muskmelon pedicel, the cucurbitacin B-2-O glucoside can be almost entirely transformed into the cucurbitacin B, and the content of the cucurbitacin B can be increased by 1-2.7 times.

Description

(1) Technical field [0001] The invention relates to a strain of streptomyces producing glucosidase and its application in biotransformation to prepare cucurbitacin B. (2) Background technology [0002] Melon stalk is the mature dried fruit stalk of Cucumis melo, an annual herbaceous vine of Cucurbitaceae. It is also called melon pedicle, melon diced, and bitter clove. Chinese Pharmacopoeia, a 1977 edition contains varieties, bitter in taste, cold in nature, enters the spleen and stomach meridians. It has the functions of spitting phlegm and food, dispelling dampness and reducing jaundice. In modern clinics, it is mostly used to treat indigestion, food poisoning, epilepsy, acute and chronic hepatitis and cirrhosis. [0003] The medicinal ingredients of melon stalks are cucurbitacins. Cucurbitacins are a class of tetracyclic triterpenoids. More than 40 species have been found, such as cucurbitacin B, cucurbitacin D and cucurbitacin E. They have the function of protecting the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N9/24C12P33/00C12R1/465
Inventor 梅建凤李莎金航应国清王鸿易喻陈建澍
Owner ZHEJIANG UNIV OF TECH
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