Method for semi-synthesizing mogroside V through enzyme catalysis
阅读说明：本技术 一种酶催化半合成罗汉果甜苷v的方法 (Method for semi-synthesizing mogroside V through enzyme catalysis ) 是由 李伟 黄华学 贺进军 黄�俊 宋谷良 江小龙 于 2021-08-16 设计创作，主要内容包括：本发明涉及一种酶催化半合成罗汉果甜苷V的方法,其特征在于,包括以下步骤：(1)将含有11-O-罗汉果甜苷V的底物和辅助底物投入缓冲溶液中,室温搅拌至全溶,得底物溶液；(2)将羰基还原酶、葡萄糖脱氢酶和辅酶投入上述底物溶液中,保温搅拌；(3)反应结束后调节pH值至碱性,过滤,滤液通过大孔吸附树脂柱,上柱毕,水洗至流出液为中性,再用醇洗脱,将醇洗脱液浓缩、真空干燥,得罗汉果甜苷V。本发明方法以11-O-罗汉果甜苷V为底物原料,采用了酶法辅酶再生,和外加试剂辅酶再生联用的方式,降低成本,按照本发明方法所得罗汉果甜苷V收率和纯度都高,反应条件温和,不使用易燃易爆、有毒有害的化工溶剂满足了工业化生产要求。(The invention relates to a method for semi-synthesizing mogroside V through enzyme catalysis, which is characterized by comprising the following steps: (1) adding a substrate containing 11-O-mogroside V and an auxiliary substrate into a buffer solution, and stirring at room temperature until the substrates are completely dissolved to obtain a substrate solution; (2) adding carbonyl reductase, glucose dehydrogenase and coenzyme into the substrate solution, and stirring while keeping the temperature; (3) adjusting pH to alkalinity after reaction, filtering, passing the filtrate through macroporous adsorbent resin column, washing with water until effluent liquid is neutral, eluting with alcohol, concentrating the alcohol eluate, and vacuum drying to obtain mogroside V. The method takes 11-O-mogroside V as a substrate raw material, adopts a mode of combining enzyme coenzyme regeneration and additional reagent coenzyme regeneration, reduces the cost, has high yield and purity of the mogroside V obtained by the method, has mild reaction conditions, does not use flammable, explosive, toxic and harmful chemical solvents, and meets the requirements of industrial production.)
1. A method for semi-synthesizing mogroside V through enzyme catalysis is characterized by comprising the following steps:
(1) adding a substrate containing 11-O-mogroside V and an auxiliary substrate into a buffer solution, and stirring at room temperature until the substrates are completely dissolved to obtain a substrate solution;
(2) adding carbonyl reductase, glucose dehydrogenase and coenzyme into the substrate solution, and stirring while keeping the temperature;
(3) adjusting pH to alkalinity after reaction, filtering, passing the filtrate through macroporous adsorbent resin column, washing with water until effluent liquid is neutral, eluting with alcohol, concentrating the alcohol eluate, and vacuum drying to obtain mogroside V.
2. The method as claimed in claim 1, wherein the concentration of 11-O-mogroside V in the substrate of step (1) is 60-150g/L, preferably 100-120 g/L; and/or
The auxiliary substrate is glucose, and the dosage of the glucose is 0.2-1 times of the weight of the substrate, preferably 0.3-0.5 times of the weight of the substrate.
3. The method according to claim 1, wherein the buffer solution in step (1) is potassium dihydrogen phosphate-sodium hydroxide buffer solution, the pH of the buffer solution is in the range of 5.8-8.0, and the amount of the buffer solution is 10-20 times (L/g) the weight of the substrate.
4. The method according to claim 1, wherein the carbonyl reductase is used in step (2) in an amount of 1 to 5 wt.%, preferably 2 to 3 wt.%, based on the weight of the substrate.
5. The method according to claim 1, wherein the coenzyme in step (2) is selected from NADPH or NADH, and the amount of the coenzyme is 0.5-2 wt%, preferably 1-1.5 wt% based on the weight of the substrate; and/or
The glucose dehydrogenase used in step (2) is used in an amount of 0.1% to 1%, preferably 0.2 to 0.3% by weight based on the weight of the substrate.
6. The method according to claim 1, wherein the coenzyme regenerating reagent is continuously supplemented in the step (2) at the beginning of the 6 th to 10 th hours of the reaction, or the coenzyme regenerating reagent is continuously supplemented when the initial enzyme activity is reduced to 60 to 80 percent, and the supplementation is stopped 2 to 5 hours before the reaction is finished; the coenzyme-regenerating agent is added in an amount of 1 to 10 wt%, preferably 3 to 5 wt%, based on the weight of the substrate.
7. The method of claim 6, wherein the coenzyme regeneration reagent is a mixture of phenazine methosulfate/phenazine ethosulfate, sodium hydrosulfite, and an organic amine in a molar ratio of 2-4:1-2: 1-1.3.
8. The method according to claim 7, wherein the organic amine is at least one selected from the group consisting of diethylamine, dipropylamine, trimethylamine, triethylamine, and tripropylamine.
9. The method according to claim 1, wherein the temperature of the incubation reaction in step (2) is 25-40 ℃, the stirring speed is 40-100rpm, and the incubation stirring time is 24-48 hours.
10. The method according to claim 1, wherein the alkali solution in step (3) is an aqueous solution of sodium hydroxide or potassium hydroxide, and the pH value is 10-12; the macroporous adsorption resin is nonpolar, low-polarity and medium-polarity macroporous adsorption resin, such as D101, AB-8, LX-T28 or HP 20.
Technical Field
The invention relates to a semisynthesis method of mogroside, in particular to a method for semisynthesis of mogroside V through enzyme catalysis.
Background
Mogroside as a sweetener is safe and nontoxic, and can be used for various foods in an unlimited way according to the national mandatory standard GB2760 food additive use standard.
Mogroside V, molecular formula: c60H102O291287.43, a specific cucurbitane tetracyclic triterpenoid saponin in the momordica grosvenori, is a main sweet component in the momordica grosvenori, and belongs to a low-calorie, non-nutritional and non-fermented natural high-potency sweetener.
11-O-mogroside V, aka: 11-oxo-Mogroside V (11-oxo-Mogroside V), molecular formula: c60H100O291285.43, which is also a cucurbitane tetracyclic triterpene saponin specific to fructus Siraitiae Grosvenorii, and is also a natural sweetener, but its sweetness is lower than that of mogroside V.
The number and the position of the tetracyclic triterpene skeleton and the substituted glycosyl of the 11-O-mogroside V and the mogroside V are completely consistent, but the substituents on the 11-site carbon atom are different, one is carbonyl and the other is hydroxyl. The subtle difference of the molecular structure leads to the great difference of sweetness between the two: the sweetness of mogroside V is 400 times that of sucrose, 300 times that of 11-O-mogroside V is 50-60 times that of sucrose.
At present, the quality standard of the momordica grosvenori extract product generally takes the content of mogroside V as an index (namely, the higher the content of mogroside V, the higher the value), and does not limit and require the content of 11-O-mogroside V. Therefore, the existence and nonexistence, much and little of 11-O-mogroside V in the fructus momordicae extract are not related to the actual value of the product. Generally, 11-O-mogroside V is present in the Lo Han Guo material in an amount of about 10% to 20% of the mogroside V content, which is the highest total mogroside content of the total mogrosides other than mogroside V. Therefore, the semisynthesis of the mogroside V by taking the 11-O-mogroside V as the starting raw material through an effective way has great significance.
The inventor has applied for the following two application related to the semi-synthesis of mogroside V in 2019:
CN201910643082.4 discloses a semi-synthesis method of mogroside V, which takes 11-O-mogroside V as a raw material and prepares the mogroside V by catalytic hydrogenation;
CN201910641262.9 discloses a semi-synthesis method of mogroside V, which takes 11-O-mogroside V as a raw material and prepares the mogroside V by reduction with sodium borohydride or potassium borohydride.
In all the chemical semi-synthesis methods, during the process of reducing the carbonyl on the 11-site carbon atom of the cucurbitane tetracyclic triterpene saponin into the hydroxyl, the alpha and beta isomerism is risked. Furthermore, as a food additive, it is preferable to use a safer synthesis method. If biological enzyme is used for catalyzing and reducing to prepare the mogroside V, similar risks do not exist.
At present, no literature is published and reports about a method for semi-synthesizing mogroside V by adopting enzyme catalysis.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide the method for semi-synthesizing the mogroside V by enzyme catalysis, which has high yield, simple process and low cost and is suitable for industrial production.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for semi-synthesizing mogroside V by enzyme catalysis comprises the following steps:
(1) adding a substrate containing 11-O-mogroside V and an auxiliary substrate into a buffer solution, and stirring at room temperature until the substrates are completely dissolved to obtain a substrate solution;
(2) adding carbonyl reductase, glucose dehydrogenase and coenzyme into the substrate solution, and stirring while keeping the temperature;
(3) adjusting pH to alkalinity after reaction, filtering, passing the filtrate through macroporous adsorbent resin column, washing with water until effluent liquid is neutral, eluting with alcohol, concentrating the alcohol eluate, and vacuum drying to obtain mogroside V.
The chiral alcohol is prepared by adopting an enzyme catalysis method, so that the yield is high, the stereoselectivity is good, but the price of the enzyme is high, and the industrialized large-scale production cannot be carried out if the cost cannot be effectively controlled. The invention adopts the combination of an enzymatic method (glucose dehydrogenase) and a non-enzymatic method (coenzyme regeneration reagent), so that the enzymatic activity is kept at a higher level in the whole reaction process, the yield of the final product is high, the cost is reduced, and a brand-new method for synthesizing the mogroside V by taking 11-O-mogroside V as a raw material enzyme is provided.
Preferably, the concentration of 11-O-mogroside V in the substrate of step (1) is 60-150g/L, preferably 100-120 g/L.
Preferably, the co-substrate in step (1) is glucose, and the amount of glucose is 0.2-1 times, preferably 0.3-0.5 times the weight of the substrate.
Preferably, the buffer solution in the step (1) is potassium dihydrogen phosphate-sodium hydroxide buffer solution, the pH range of the buffer solution is 5.8-8.0, and the dosage of the buffer solution is 10-20 times (L/g) of the weight of the substrate.
Preferably, the carbonyl reductase described in step (2) may be of animal cell, plant cell or microorganism origin, and the amount of carbonyl reductase is 1-5 wt%, preferably 2-3 wt%, based on the weight of the substrate.
Preferably, the coenzyme described in step (2) is selected from NADPH or NADH, and the amount of the coenzyme is 0.5-2 wt%, preferably 1-1.5 wt%, based on the weight of the substrate.
Preferably, the glucose dehydrogenase in step (2) can be derived from animal cells, plant cells or microorganisms, and the glucose dehydrogenase is used in an amount of 0.1% to 1%, preferably 0.2 to 0.3% by weight based on the weight of the substrate.
If the dosage of the various enzymes is too small, the concentration of the enzymes in the reaction liquid is too low, so that the speed of catalytic reaction is reduced, and the production efficiency is low; if the amount of each enzyme is too large, waste is caused and the difficulty of post-treatment is increased.
Preferably, in the step (2), the coenzyme regeneration reagent is continuously supplemented from 6 h to 10h of the reaction, or the coenzyme regeneration reagent is continuously supplemented when the initial enzyme activity is reduced to 60-80%, and the coenzyme regeneration reagent is stopped being supplemented 2-5h before the reaction is finished. The coenzyme-regenerating agent is added in an amount of 1 to 10 wt%, preferably 3 to 5 wt%, based on the weight of the substrate. The coenzyme regeneration reagent is a mixture of phenazine methosulfate/phenazine ethosulfate, sodium hydrosulfite and organic amine according to the molar ratio of 2-4:1-2: 1-1.3. The organic amine is at least one of diethylamine, dipropylamine, trimethylamine, triethylamine and tripropylamine. The inventor unexpectedly finds that the coenzyme regeneration reagent compounded according to the proportion can make up the defect of insufficient enzyme activity in the middle and later stages of the reaction and stably and continuously enable the reaction to be carried out. Finally, the yield of the mogroside V is satisfactory, and the industrialized production can be realized.
Preferably, the temperature of the incubation reaction in step (2) is 25-40 ℃. If the reaction temperature is too low, the catalytic effect of various enzymes is reduced, and the catalytic reaction speed is slowed or the reaction is incomplete; if the reaction temperature is too high, various enzymes will be denatured and inactivated, which is also unfavorable for the reaction.
Preferably, the stirring speed in step (2) is 40-100 rpm. If the stirring speed is too slow, the probability of contacting the enzyme with the substrate is reduced, and the catalytic reaction speed is slowed or the reaction is incomplete; if the stirring speed is too high, the spatial structure of the enzyme is damaged by the excessively strong shearing force of the stirrer, so that the enzyme activity is reduced, and the catalytic reaction speed is also slowed or the reaction is incomplete.
Preferably, the reaction time in step (2) is 24 to 48 hours.
Preferably, the alkali liquor in the step (3) is an aqueous solution of sodium hydroxide or potassium hydroxide, and the pH value is 10-12. The reaction liquid is adjusted to alkaline pH value by alkali liquor, so as to terminate the reaction and inactivate enzyme.
In the step (3), the purpose of filtration is to remove various enzymes which have been inactivated and denatured, and to prevent the macroporous adsorbent resin column from clogging.
Preferably, the macroporous adsorbent resin in step (3) is a nonpolar, weakly polar and medium polar macroporous adsorbent resin, and the specific types thereof capable of adsorbing mogroside V without adsorbing other substances include, but are not limited to, D101, AB-8, LX-T28 and HP 20. The reaction solution is subjected to alkali regulation, enzyme deactivation and filtration, and then passes through a macroporous adsorption resin column, and the macroporous resin is used for adsorbing only the mogroside V in the filtrate and not adsorbing other components (glucose, gluconic acid, salt and the like) in the filtrate, so that the purposes of separation and purification are achieved.
The synthesis reaction formula of the method is as follows:
the method has the following beneficial effects:
(1) the method provides a brand new way for obtaining high-content mogroside V, namely enzyme-catalyzed semisynthesis; meanwhile, the by-product of the separation and purification of the mogroside V, namely the added value of 11-O-mogroside V, is improved to the maximum extent, and the comprehensive utilization mode of the mogroside V is enriched;
(2) the method adopts a mode of combining enzyme coenzyme regeneration and additional reagent coenzyme regeneration, reduces the cost, has high yield and purity of the mogroside V obtained by the method, and meets the requirement of industrial production;
(3) the method has the advantages of simple process, strong operability, low equipment requirement, mild reaction condition, low production cost, no use of flammable, explosive, toxic and harmful chemical solvents, safety, greenness, environmental protection and suitability for industrial production.
Detailed Description
The present invention will be further described with reference to the following examples. The starting materials or chemicals used in the examples of the present invention are, unless otherwise specified, commercially available in a conventional manner.
The 11-O-mogroside V used in the embodiment of the invention has a purity content of 97.5% (HPLC) and is purchased from Hunan Huacheng biological resource GmbH; the used macroporous adsorption resin is purchased from New science and technology materials GmbH of Xian blue and Xiao; the carbonyl reductase used was purchased from Suzhou Han enzyme Biotechnology GmbH, model KRED-101; the glucose dehydrogenase is purchased from Sancheng materials science and technology limited, Hubei province, and the specific activity is more than or equal to 150U/mg.
In the embodiment of the invention, the contents of 11-O-mogroside V and mogroside V are measured by adopting a high performance liquid chromatography external standard method.
Example 1
(1) Preparation of a substrate solution: adding a substrate (11-O-mogroside V, 10g) and an auxiliary substrate glucose (3 g) into 100ml of potassium dihydrogen phosphate-sodium hydroxide buffer solution (pH is 6.5), and stirring at room temperature until the substrate is completely dissolved to obtain a substrate solution;
(2) enzyme catalysis: adding 0.2g of carbonyl reductase, 0.1g of glucose dehydrogenase and 0.02g of NADPH into the substrate solution, keeping the temperature at 27 ℃, stirring at the speed of 60rpm, continuously supplementing a coenzyme regeneration reagent after reacting for 6 hours, and stopping supplementing until the reaction time reaches 25h, wherein the coenzyme regeneration reagent is phenazine methosulfate, sodium hydrosulfite and diethylamine according to the molar ratio of 3.5: 1.5: 1 and the addition amount of a coenzyme regeneration reagent is 0.3g, and the reaction is continued for 5 hours until the reaction is complete to obtain a reaction solution;
(3) and (3) post-treatment: after the reaction, the reaction solution is adjusted to pH 10 with potassium hydroxide aqueous solution, filtered, the filtrate is passed through a macroporous adsorption resin column (resin model: D101), and after the column is finished, the filtrate is washed with pure water until the effluent liquid of the macroporous adsorption resin column is neutral, then is eluted with ethanol, and the ethanol eluate is concentrated and dried in vacuum, so that 9.12g of mogroside V is obtained.
The content of mogroside V obtained in this example was 96.3% and the yield of mogroside V was 90.08% as determined by High Performance Liquid Chromatography (HPLC) external standard method.
Example 2
(1) Preparation of a substrate solution: adding a substrate (11-O-mogroside V, 12g) and an auxiliary substrate (glucose 5g) into 100ml of potassium dihydrogen phosphate-sodium hydroxide buffer solution (pH is 7.0), and stirring at room temperature until the substrates are completely dissolved to obtain a substrate solution;
(2) enzyme catalysis: adding 0.36g of carbonyl reductase, 0.18g of glucose dehydrogenase and 0.03g of NADPH into the substrate solution, keeping the temperature at 35 ℃, stirring at 40rpm, continuously supplementing a coenzyme regeneration reagent after reacting for 8 hours, and stopping supplementing until the reaction reaches 30h, wherein the coenzyme regeneration reagent is phenazine ethosulfate, sodium hydrosulfite and triethylamine are added according to the molar ratio of 3: 1.3: 1 and the addition amount of a coenzyme regeneration reagent is 0.5g, and the reaction is continued for 5 hours until the reaction is complete to obtain a reaction solution;
(3) and (3) post-treatment: after the reaction, the pH value of the reaction solution is adjusted to 11 by using an aqueous solution of sodium hydroxide, the reaction solution is filtered, the filtrate passes through a macroporous adsorption resin column (the resin model: AB-8), the filtrate is washed by pure water until the effluent liquid of the macroporous adsorption resin column is neutral, then the ethanol is used for elution, and the ethanol eluent is concentrated and dried in vacuum to obtain 11.25g of mogroside V.
The content of mogroside V obtained in this example was 97.1% and the yield of mogroside V was 93.36% as determined by High Performance Liquid Chromatography (HPLC) external standard method.
Example 3
(1) Preparation of a substrate solution: adding a substrate (11-O-mogroside V, 10g) and an auxiliary substrate (glucose 5g) into 100ml of potassium dihydrogen phosphate-sodium hydroxide buffer solution (pH 7.5), and stirring at room temperature until the substrates are completely dissolved to obtain a substrate solution;
(2) enzyme catalysis: adding 0.5g of carbonyl reductase, 0.2g of glucose dehydrogenase and 0.03g of NADPH into the substrate solution, keeping the temperature at 30 ℃, stirring at 80rpm, continuously supplementing a coenzyme regeneration reagent after reacting for 8 hours, and stopping supplementing until the reaction reaches 30h, wherein the coenzyme regeneration reagent is phenazine ethosulfate, sodium hydrosulfite and triethylamine in a molar ratio of 4: 1.5: 1 and the addition amount of a coenzyme regeneration reagent is 0.5g, and the reaction is continued for 5 hours until the reaction is complete to obtain a reaction solution;
(3) and (3) post-treatment: after the reaction, the pH value of the reaction solution is adjusted to 10 by potassium hydroxide aqueous solution, the solution is filtered, the filtrate passes through a macroporous adsorption resin column (the resin model is LX-T28), after the solution is loaded on the column, the solution is washed by pure water until the effluent of the macroporous adsorption resin column is neutral, then the ethanol is used for elution, and the ethanol eluent is concentrated and dried in vacuum, so that 9.82g of mogroside V is obtained.
The content of mogroside V obtained in this example was 95.2% and the yield of mogroside V was 95.88% as determined by High Performance Liquid Chromatography (HPLC) external standard method.
Example 4
The other conditions and procedure were the same as in example 1 except that no coenzyme-regenerating reagent was added in step (2). 7.41g of mogroside V is finally obtained, the content of mogroside V is 96.4%, and the yield is 73.26%.
Example 5
The other conditions and procedure were the same as in example 1 except that the coenzyme-regenerating reagent was not supplemented in step (2), and the amount of glucose dehydrogenase used was 0.5 g. 8.93g of mogroside V is finally obtained, the content of mogroside V is 95.8%, and the yield is 87.74%.
By comparing the results of examples 1, 4 and 5, it was found that the amount of glucose dehydrogenase used can be reduced, the yield can be improved and the cost can be reduced by adding a coenzyme-regenerating reagent.