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Method for preparing steviol through two-enzyme method

A technology of steviol and double-enzyme method, applied in the field of biosynthesis of natural compounds, can solve the problems of no mention of other uses, difficult separation and purification, low conversion efficiency, etc., and achieves mild conditions, high selectivity, and high selectivity. Effect

Active Publication Date: 2016-08-17
GUANGXI UNIV
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Problems solved by technology

Oliveira et al. used Gibberella to convert steviol glycosides through 7 days of fermentation at room temperature to obtain steviol, and the conversion efficiency was lower than 20% (Heleno de Oliveira, Bras, Ferreira da Trindade, Jose Luiz. Production of hydroxysteviol and its application as a plant growth regulator:Brazil,2004006278[P].2006-08-22.)
A Chinese patent discloses a method for preparing steviol by using Microbacterium pasteurianus (patent number: ZL201110159563.1). The conversion efficiency obtained when using whole cells for transformation is low, and the product steviol is mixed with bacteria and it is difficult to separate and purify
Compared with chemical and microbial methods, the enzymatic method of thoroughly hydrolyzing the glycosyl moiety in steviol glycosides to produce steviol has the advantages of rapid reaction, mild conditions, good selectivity, and environmental friendliness. However, obtaining steviol glycosides suitable for hydrolysis Compound production of steviol by highly active enzymes remains a challenge
A Chinese patent discloses a method for preparing steviol by using commercial Aspergillus niger β-glucosidase to catalyze the hydrolysis of stevioside (patent number: ZL 201210352461.6), but the reaction temperature is 70-75°C, maintaining a relatively high reaction temperature increases the The cost of production, and the need to use temperature programming in the reaction, the operation is relatively cumbersome
[0009] The inventor has previously applied for 2 Chinese patents "β-glucosidase gene encoding glycosyl hydrolase family 1 and its application" (patent number: ZL201310049608.9) and a Chinese patent "β-glucosidase gene encoding glycosyl hydrolase family 3 -Glucosidase Gene and Its Application" (patent number: ZL201210387271.8) discloses a β-glucosidase S-bgl4 from Streptomyces glycosyl hydrolase family 1 and a β-glucosidase S-bgl4 from Neosphingosine β-glucosidase Sbgl of the glycosyl hydrolase family 3, and the application of these two enzymes in the decomposition of cellobiose, but neither mentioned other uses of these two enzymes

Method used

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  • Method for preparing steviol through two-enzyme method

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preparation example Construction

[0036] The preparation of β-glucosidase S-bgl4 was carried out according to the method described in patent ZL201310049608.9, and the preparation of β-glucosidase Sbgl was carried out according to the method described in patent ZL201210387271.8.

[0037] HPLC analysis hydrolyzate condition:

[0038] Instruments and equipment: e2695 separation unit and 2998PDA detector of Waters Company.

[0039] Chromatographic column: C18 column (Alltima, 250×4.6 mm, 5 μm).

[0040] Chromatographic conditions: mobile phase consists of acetonitrile (A) and phosphate buffer (B, 10mM NaH 2 PO 4 The solution was adjusted to pH 2.6 with phosphoric acid, using a gradient elution procedure: 68% B (0-10min), 68 to 20% B (10-20min), 20% B (20-21min), 20 to 68% B (21-28min), finally 68% B (28-30min). The column temperature is 40°C, the flow rate is 1mL / min, and the detection wavelength is 210nm.

[0041] UPLC-MS analysis conditions for hydrolyzate:

[0042] Instruments and equipment: ACQUITY UPLC ...

Embodiment 1

[0047] A method for preparing steviol by a dual-enzyme method, comprising the following steps:

[0048] (1) Using steviol glycoside as a substrate, prepare it with water or a buffer to prepare a stevioside solution; the stevioside is stevioside (purity is 98%); the buffer is Teorell and Stenhagen buffer; 50 mM disodium phosphate, 33 mM citric acid and 50.7 mM boric acid were contained in the Teorell and Stenhagen buffer described;

[0049] (2) first add β-glucosidase S-bgl4, and react for 1 hour; the final concentration of the β-glucosidase S-bgl4 is 50 μg / mL; the pH value of the reaction is 5.0;

[0050] (3) After heating in a boiling water bath for 5 minutes to inactivate the enzyme, adjust the pH to 3.0 with concentrated hydrochloric acid;

[0051] (4) Add β-glucosidase Sbgl and react for 1 hour; the final concentration of the β-glucosidase Sbgl is 50 μg / mL; the pH value of the reaction is 3.0;

[0052] (5) After heating in a boiling water bath for 5 minutes to inactivate...

Embodiment 2

[0055] A method for preparing steviol by a dual-enzyme method, comprising the following steps:

[0056] (1) Using steviol glycoside as a substrate, prepare it with water or a buffer to prepare a stevioside solution; the stevioside is a stevioside extract containing stevioside (the content of stevioside is 80%); the buffer It is a Teorell and Stenhagen buffer; the Teorell and Stenhagen buffer contains 50mM disodium hydrogen phosphate, 33mM citric acid and 50.7mM boric acid;

[0057] (2) first add β-glucosidase S-bgl4, and react for 6 hours; the final concentration of the β-glucosidase S-bgl4 is 100 μg / mL; the pH value of the reaction is 10.0;

[0058] (3) After heating in a boiling water bath for 5 minutes to inactivate the enzyme, adjust the pH to 8.0 with concentrated hydrochloric acid;

[0059] (4) Add β-glucosidase Sbgl and react for 6 hours; the final concentration of the β-glucosidase Sbgl is 100 μg / mL; the pH value of the reaction is 8.0;

[0060] (5) After heating in ...

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Abstract

The invention belongs to the technical field of natural compound biosynthesis and particularly relates to a method for preparing steviol through a two-enzyme method. According to the method for preparing steviol through the two-enzyme method, stevioside is used as a substrate and prepared into a solution with water or a buffer solution, beta-glucosidase-S-bgl4 and Sbgl are added in sequence, a reaction is carried out for 1-12 h, and steviol can be obtained. The method is used for preparing steviol and has the advantages that selectivity, the conversion rate and the yield are high, conditions are mild, environment friendliness is achieved, and operation is easy.

Description

technical field [0001] The invention belongs to the technical field of biosynthesis of natural compounds, and specifically relates to a method for preparing steviol by using two β-glucosidases to synergistically catalyze the hydrolysis of stevioside. Background technique [0002] Steviol is the aglycon part of steviol glycosides, which has a tetracyclic diterpene skeleton structure. Steviol glycosides are a class of glycoside compounds with high sweetness extracted from the leaves of the plant Stevia rebaudiana Bertoni. Steviol glycosides can be used as food additives, and industrial production has been realized at present. my country's national food safety standards have announced 9 common glycoside compounds in stevioside, namely stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside Glycoside F, dulcoside A, rubusoside and steviolbioside (food additive steviol glycoside GB 8270-2014). [0003] Steviol has a wide range of physiological a...

Claims

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Application Information

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IPC IPC(8): C12P7/42
CPCC12P7/42
Inventor 杜丽琴王子龙庞浩郑芳芳周洁黄日波韦宇拓
Owner GUANGXI UNIV
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