阅读说明：本技术 一种酶法制备罗汉果苷iv和苷v的方法 (Method for preparing mogroside IV and mogroside V by enzyme method ) 是由 杨峰 李文娟 张琚政 孙泽文 蔡美玲 于 2019-11-12 设计创作，主要内容包括：本发明公开了一种酶法制备罗汉果苷IV和苷V的方法,具体是利用酶法催化罗汉果苷IIE、III生成高甜度的罗汉果苷IV、V,所述酶来源自UDP葡萄糖基转移酶UGT032,核苷酸DNA序列如SEQ NO.1所示,氨基酸PRT序列如SEQ No.2所示。先制备粗酶液,再用粗酶液分别催化罗汉果苷IIE、III生成罗汉果苷IV、V。本发明方法可改善罗汉果苷的口感,对提高罗汉果苷产业发展具有重要意义。(The invention discloses a method for preparing mogroside IV and mogroside V by an enzyme method, in particular to a method for catalyzing mogroside IIE and mogroside III by the enzyme method to generate high-sweetness mogroside IV and mogroside V, wherein the enzyme is derived from UDP glucosyltransferase UGT032, the nucleotide DNA sequence is shown as SEQ NO.1, and the amino acid PRT sequence is shown as SEQ NO. 2. Preparing crude enzyme solution, and catalyzing the mogrosides IIE and III to generate mogroside IV and V respectively by using the crude enzyme solution. The method can improve the mouthfeel of the mogroside and has important significance for improving the development of the mogroside industry.)

1. A method for preparing mogroside IV and mogroside V by an enzyme method is characterized in that: specifically, the mogrosides IIE and III are catalyzed by enzyme to generate mogrosides IV and V, the enzyme is derived from UDP glucosyltransferase UGT032, the nucleotide DNA sequence is shown as SEQ NO.1, and the amino acid PRT sequence is shown as SEQ NO. 2.

2. The enzymatic process of producing mogroside IV and glycoside V according to claim 1, characterized in that: preparing a crude enzyme solution, and catalyzing the mogrosides IIE and III to generate mogrosides IV and V respectively by using the crude enzyme solution;

the preparation method of the crude enzyme solution specifically comprises the steps of constructing an enzyme gene UGT032 onto pET22b, and naming the recombinant plasmid as pET22b-UGT 032;

then the recombinant plasmid is transformed into an escherichia coli competent cell Top10 to clone the plasmid, the thalli are collected by shaking culture, and the plasmid is extracted and stored at the temperature of minus 80 ℃;

and transforming the extracted plasmid into an escherichia coli expression host BL21p for induction and expression, collecting thalli, adding a bacteria breaking liquid according to 4 times of the weight of the thalli, and breaking cells by using a homogenizer to prepare a crude enzyme liquid.

3. The enzymatic process of producing mogroside IV and glycoside V according to claim 2, characterized in that: the method for producing mogrosides IV and V by using enzyme catalysis to obtain mogroside IIE and III comprises adding substrate mogroside IIE or III into a three-necked flask, adding crude enzyme solution, uridine diphosphate and sucrose synthase, continuously adding phosphate buffer solution with pH =7.5, stirring, and performing 35 deg.C water bath; after the reaction in water bath for 8-12h, the conversion rate of the glycoside IIE or III can be detected by HPLC.

Technical Field

The invention relates to a preparation method of mogrosides, in particular to a method for generating mogrosides IV and V by catalyzing mogrosides IIE and III by an enzyme method.

Background

Mogrosides is the most studied active component in momordica grosvenori, and has the obvious functions of diminishing inflammation, relieving cough, protecting liver, reducing blood sugar, resisting fatigue, enhancing immunity and the like. Mogroside is also an important natural sweetener, wherein mogroside V is the main component of the natural sweetener, the content of the mogroside V in the momordica grosvenori is most abundant, and the mogroside V accounts for about 0.57 percent of the dry fruit, and the content of the mogroside IV is only lower than that of the mogroside V. The sweetness of the mogrosides IV and V is 392 times and 425 times that of 5% sucrose water when the concentration of the mogrosides IV and V in water is ten thousandth. The mogrosides IIE and III have obvious bitter taste, and the removal or conversion of the mogrosides IIE and III into the glycoside IV and the glycoside V has important significance for improving the taste and the quality of the mogrosides.

Currently, enzymatic or chemical methods are used to synthesize mogroside V into glycoside IIIE, or enzymatic methods are used to prepare mogrosides 1a and 1 b. The enzymatic catalysis of the mogrosides IIE and III to generate the high-sweetness mogrosides IV and V has no report temporarily, and the development of the process has important significance for the development of the mogroside industry.

Disclosure of Invention

The invention aims to provide a method for preparing mogroside IV and mogroside V by an enzyme method, in particular to a method for catalyzing mogrosides IIE and III by the enzyme method to generate the high-sweetness mogrosides IV and V, which has important significance for improving the development of the mogroside industry.

The technical scheme for realizing the purpose of the invention is as follows:

the invention relates to a method for preparing mogroside IV and mogroside V by an enzyme method, in particular to a method for preparing mogroside IV and mogroside V by using enzyme catalysis of mogroside IIE and mogroside III, wherein the enzyme is derived from UDP glucosyltransferase UGT032, the nucleotide DNA sequence is shown as SEQ NO.1, and the amino acid PRT sequence is shown as SEQ NO. 2.

The invention relates to a method for generating mogrosides IV and V by catalyzing mogrosides IIE and III with enzyme, which comprises the steps of firstly preparing crude enzyme liquid, and then catalyzing the mogrosides IIE and III with the crude enzyme liquid to generate the mogrosides IV and V respectively.

The preparation method of the crude enzyme solution comprises the following specific steps: the enzyme gene UGT032 is constructed on pET22b, and the recombinant plasmid is named as pET22b-UGT 032;

then the recombinant plasmid is transformed into an escherichia coli competent cell Top10 to clone the plasmid, the thalli are collected by shaking culture, and the plasmid is extracted and stored at the temperature of minus 80 ℃;

and transforming the extracted plasmid into an escherichia coli expression host BL21p for induction and expression, collecting thalli, adding a bacteria breaking liquid according to 4 times of the weight of the thalli, and breaking cells by using a homogenizer to prepare a crude enzyme liquid.

The method for respectively catalyzing the mogrosides IIE and III to generate the mogrosides IV and V by using the crude enzyme solution comprises the steps of respectively adding a substrate of the mogroside IIE or III into a three-necked bottle, adding the crude enzyme solution, uridine diphosphate and sucrose synthase, continuously adding a phosphate buffer solution with the pH =7.5, stirring, and carrying out 35 ℃ water bath; after the reaction in water bath for 8-12h, the conversion rate of the glycoside IIE or III can be detected by HPLC.

The method has the beneficial effects that:

1. the method utilizes enzymes to catalyze the mogrosides IIE and III to generate the mogrosides IV and V, can generate the bitter mogrosides IIE and III into the high-sweetness mogrosides IV and V, improves the mouthfeel of the mogrosides, and has important significance for improving the development of the mogroside industry;

2. the enzyme is derived from UDP glucosyltransferase UGT032, which transfers glucose molecules on UDP-glucose (UDPG) molecules to mogroside IIE and glycoside III to generate glycoside IV and glycoside V with high sweetness;

3. the enzymatic method for preparing the mogroside has the advantages of mild reaction, simple operation and few byproducts, and is suitable for large-scale industrial production.

Drawings

FIG. 1 is a mass spectrum of recombinant plasmid pET22b-UGT032 according to the method of the present invention.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited thereto.