Method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase

A technology of glucosidase and catalytic hydrolysis, which is applied in the field of biosynthesis of organic compounds, can solve problems such as poor thermal stability, and achieve the effects of reduced thermal inactivation, high yield, and mild reaction conditions

Active Publication Date: 2012-12-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Although enzyme catalysis has many advantages, poor thermal stability has become an unavoidable problem in the process of using enzymes.

Method used

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  • Method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase
  • Method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase
  • Method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase

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Experimental program
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Effect test

Embodiment 1

[0019] Disperse 15 g St (commercially available, 97% pure) in 55 o C in 100 mL deionized water, put in 55 o Shake in C water bath shaker for 0.5 h, the shaker speed is 150 rpm; add β - Glucosidase enzyme solution 800 U / g St; react until the conversion rate of St no longer rises. St conversion was 99.0%. After cooling the reaction solution, filter it with suction, wash the filter cake with mother liquor and deionized water respectively, and recrystallize with 20% methanol aqueous solution to obtain the steviol product. The HPLC purity of the final product was greater than 98%. The reaction required a total of 8 hours.

[0020] The product is analyzed by LC-MSMS and NMR, the results are shown in the attached Figure 1-3 . The molecular ion peak of the main component in LC-MSMS (317.4, [M-H] - ) can correspond to steviol, and the NMR of the hydrolyzate can further confirm that the product is steviol.

[0021] The conversion rate of St can be determined by the HPLC externa...

Embodiment 2

[0027] Dissolve 20 g of 90% St (stevia extract, containing 10% RA) in 55 o C in 100 mL deionized water, put in 55 o Shake in C water bath shaker for 0.5 h, the shaker speed is 150 rpm; add β -Glucosidase enzyme solution 600 U / g St; rise to 75 after 3 h reaction o C continues to react until the conversion rate of St no longer increases. Then recrystallize with 20% methanol aqueous solution to obtain steviol product. The reaction required a total of 3.5 hours.

Embodiment 3

[0029] Disperse 15 g St in 50 mM 100 mL pH 5.0 acetate buffer in 55 o Shake in C water bath shaker for 0.5 h, the shaker speed is 150 rpm; add β - Glucosidase enzyme solution 700 U / g St; rise to 70 after 3h reaction o C continues to react until the conversion rate of St no longer increases. The reaction takes 4 hours in total. Then recrystallize with 20% methanol aqueous solution to obtain steviol product.

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Abstract

The invention discloses a method for preparing steviol by carrying out catalytic hydrolysis on stevioside by beta-glucosidase, belonging to the field of biosynthesis technology of organic compounds. The method takes the beta-glucosidase derived from aspergillus niger as a catalyst, and can be used for preparing the steviol by carrying out catalytic hydrolysis on the stevioside in single stevioside or stevia rebaudiana extract through temperature programming; the method has the advantages of being high in conversion rate and selectivity, simple in separation and the like; and the enzyme activity loss can be reduced by the temperature programming reaction, so that the utilization rate of enzyme can be improved. The invention provides a new method for preparing the steviol, which is efficient and environment-friendly.

Description

technical field [0001] [0002] The invention belongs to the technical field of biosynthesis of organic compounds, and specifically relates to a method derived from Aspergillus niger (Aspergillus niger) of β - A method for preparing steviol by catalyzing the hydrolysis of stevioside by temperature programming of glucosidase. Background technique [0003] Stevioside (13-[(2-O- β -D-Glucopyranosyl- β -D-glucopyranosyl)oxy]kaur-16-en-18-oic acid β -D-glucopyranosyl ester, stevioside, hereinafter referred to as St) is derived from stevia ( Stevia rebaudiana Bertoni), a tetracyclic diterpenoid compound, is a sweetener with a bitter aftertaste. There are three kinds of glycosidic bonds in the St molecule, which can be hydrolyzed to obtain steviol, isosteviol, steviol monoglycoside, steviolbioside, rubusoside or a mixture thereof; the content of each component of the hydrolyzate depends on the hydrolysis conditions: for example, using inorganic Alkali-catalyzed hydr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P15/00
Inventor 夏咏梅方云万会达
Owner JIANGNAN UNIV
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