阅读说明：本技术 一种有机溶剂中酶法合成蔗糖-6-乙酯的方法 (Method for synthesizing sucrose-6-ethyl ester by enzyme method in organic solvent ) 是由 钱俊青 苟李红 赵长燕 郭辉 于 2020-07-02 设计创作，主要内容包括：本发明公开了一种有机溶剂中酶法合成蔗糖-6-乙酯的方法,所述方法为：将蔗糖加入有机溶剂中,再加入固定化脂肪酶和乙酸乙烯酯,混合均匀,在30-40℃水浴摇床转速120-180r/min,反应时间12-20h,过滤,滤饼是回收的固定化脂肪酶,滤液真空蒸馏回收溶剂,得到蔗糖-6-乙酯产品。本发明采用N,N-二甲基甲酰胺(DMF)作为酶法合成蔗糖-6-乙酯的主要溶剂,与叔戊醇或叔丁醇复合,与其他有机溶剂比,具有酶活保持较好,蔗糖溶解度可达10％,蔗糖酯化率高达85％以上,蔗糖-6-乙酯占总酯的比例85％以上,合成反应溶剂体系粘度较小,易回收的优点。(The invention discloses a method for synthesizing sucrose-6-ethyl ester in an organic solvent by an enzyme method, which comprises the following steps: adding sucrose into an organic solvent, adding immobilized lipase and vinyl acetate, uniformly mixing, carrying out reaction for 12-20h at the temperature of 30-40 ℃ at the rotating speed of 120-fold shaker for 180r/min, filtering, wherein the filter cake is the recovered immobilized lipase, and carrying out vacuum distillation on the filtrate to recover the solvent to obtain the sucrose-6-ethyl ester product. The method adopts N, N-Dimethylformamide (DMF) as a main solvent for synthesizing the sucrose-6-ethyl ester by an enzymatic method, is compounded with tert-amyl alcohol or tert-butyl alcohol, and has the advantages of better enzyme activity retention, sucrose solubility of up to 10 percent, sucrose esterification rate of up to more than 85 percent, sucrose-6-ethyl ester accounting for more than 85 percent of total esters, smaller viscosity of a synthesis reaction solvent system and easiness in recovery compared with other organic solvents.)

1. A method for synthesizing sucrose-6-ethyl ester by an enzyme method in an organic solvent is characterized by comprising the following steps: adding sucrose into an organic solvent, adding immobilized lipase and vinyl acetate, uniformly mixing, reacting for 12-20h in a water bath at 30-40 ℃ and at a shaker rotation speed of 120-; the organic solvent is formed by mixing 55-30% by volume of N, N-dimethylformamide and 45-70% by volume of tert-amyl alcohol or tert-butyl alcohol, and the total amount is 100%; the immobilized lipase is prepared by adsorbing and immobilizing lipase by macroporous resin.

2. The method for enzymatically synthesizing sucrose-6-ethyl ester in an organic solvent as claimed in claim 1, wherein the organic solvent is prepared by mixing 55-30 vol% of N, N-dimethylformamide and 45-70 vol% of t-amyl alcohol, the total amount being 100%.

3. The method for the enzymatic synthesis of sucrose-6-ethyl ester in the organic solvent as described in claim 1, wherein the volume of the organic solvent is added in an amount of 125-330ml/g based on the weight of sucrose; the mass ratio of the sucrose to the immobilized lipase is 1: 0.2-1.0; the mass ratio of the sucrose to the vinyl acetate is 1: 1-5.

4. The method for the enzymatic synthesis of sucrose-6-ethyl ester in an organic solvent as claimed in claim 1, wherein the lipase activity is 10-30 ten thousand units/g.

5. The process for the enzymatic synthesis of sucrose-6-acetate in an organic solvent as claimed in claim 4, wherein the lipase is Aspergillus niger lipase or Candida lipase.

6. The method for the enzymatic synthesis of sucrose-6-ethyl ester in an organic solvent as claimed in claim 1, wherein the macroporous resin type comprises D4006 type, D3520 type, D1300 type.

7. The method for enzymatically synthesizing sucrose-6-ethyl ester in an organic solvent as described in claim 1, wherein said immobilized lipase is prepared by the following method: dissolving lipase in deionized water, adding disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate, uniformly stirring to form 0.05mol/L enzyme solution of phosphate buffer solution with the pH value of 7.4, adding macroporous resin, keeping the temperature at 20-35 ℃, stirring at 80-100 rpm, adsorbing for 1-3h, filtering, and drying a filter cake in vacuum until the water content is 3-5%, thereby obtaining the immobilized lipase.

8. The method for the enzymatic synthesis of sucrose-6-ethyl ester in organic solvent as claimed in claim 7, wherein the volume usage of said deionized water is 150-500ml/g based on the weight of lipase; the mass ratio of the lipase to disodium hydrogen phosphate dodecahydrate to sodium dihydrogen phosphate is 1:0.5-1.5 and 1: 1: 0.5-1.0; the weight ratio of the lipase to the macroporous resin is 1: 2-4.

9. The method for enzymatically synthesizing sucrose-6-ethyl ester in an organic solvent as claimed in claim 7, wherein the macroporous resin is pretreated before adding the enzyme solution, and the pretreatment method comprises: adding 10 times mass of 95% ethanol aqueous solution with volume concentration into the macroporous resin, shaking and washing for 24 hours at room temperature and 150r/min, replacing the 95% ethanol aqueous solution twice in the middle, washing for five times by using distilled water with twice mass each time after the shaking and washing are finished, and performing suction filtration to obtain a filter cake, namely the pretreated macroporous resin.

(I) technical field

The invention relates to a method for synthesizing sucrose-6-ethyl ester in an organic solvent by an enzyme method.

(II) background of the invention

The sucrose-6-ethyl ester is synthesized by a chemical method in N, N-Dimethylformamide (DMF) solvent at home and abroad, the sucrose is dissolved in the DMF solvent and undergoes an esterification reaction with trimethyl orthoacetate, and also can undergo an esterification reaction under the catalysis of dibutyltin oxide. The chemical method has great influence on the environment, and the lipase catalyzes sucrose esterification to belong to green production, so the development prospect is ideal.

The sucrose-6-ethyl ester is a key intermediate for producing the non-caloric high-power sweetener sucralose, so that a green and safe technology for synthesizing the sucrose-6-ethyl ester by lipase catalysis is developed at home and abroad. In order to better dissolve sucrose and simultaneously enable lipase to exert catalytic activity, the reported research adopts a mixture of an organic solvent and water as a reaction solvent or an ionic liquid as the reaction solvent, but the sucrose esterification rate is below 70%. Dimethyl sulfoxide composite medium chain alcohol is used as a sucrose esterification reaction solvent, and the effect of synthesizing sucrose-6-ethyl ester by lipase catalysis is good. However, the influence of dimethyl sulfoxide on enzyme activity is serious, the solubility of sucrose in dimethyl sulfoxide is not high enough, and the viscosity of dimethyl sulfoxide is high, so that the operation difficulty in the reaction process is increased. Therefore, the research on the ideal organic solvent for enzymatic synthesis of sucrose-6-ethyl ester is a hot spot of the technical invention at home and abroad.

Disclosure of the invention

The invention aims to improve the esterification efficiency of synthesizing sucrose-6-ethyl ester by an enzyme method in an organic solvent, provides a method for synthesizing sucrose-6-ethyl ester by an enzyme method in an organic solvent, replaces the prior process for synthesizing sucrose-6-ethyl ester by a chemical method, is applied to the production of non-caloric high-power sweetener sucralose, can greatly reduce the influence of the production process on the environment, and realizes the green modification of sucralose production.

The technical scheme adopted by the invention is as follows:

the invention provides a method for synthesizing sucrose-6-ethyl ester in an organic solvent by an enzyme method, which comprises the following steps: adding sucrose into an organic solvent, adding immobilized lipase and vinyl acetate, uniformly mixing, reacting for 12-20h (preferably for 16h at 30 ℃ and 160 r/min) in a water bath at 30-40 ℃ and at a shaker rotation speed of 120-; the organic solvent is formed by mixing 55-30% by volume of N, N-dimethylformamide and 45-70% by volume of tert-amyl alcohol or tert-butyl alcohol, and the total amount is 100%; the immobilized lipase is prepared by adsorbing and immobilizing lipase by macroporous resin.

Furthermore, the organic solvent is prepared by mixing 55-30% by volume of N, N-dimethylformamide and 45-70% by volume of tertiary amyl alcohol, and the total amount is 100%.

Further, the volume adding amount of the organic solvent is 125-330ml/g (preferably 160-250ml/g) based on the weight of the sucrose; the mass ratio of the sucrose to the immobilized lipase is 1: 0.2-1.0 (preferably 1: 0.5-0.7); the mass ratio of the sucrose to the vinyl acetate is 1: 1-5 (preferably 1: 2-4).

Further, the immobilized lipase is prepared as follows: dissolving lipase in deionized water (stirring to dissolve the lipase, centrifuging to remove insoluble components, controlling the concentration of the enzyme in the enzyme solution to be 2-4mg/ml, adding disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate, stirring uniformly to form 0.05mol/L enzyme solution of phosphate buffer solution with the pH value of 7.4, adding macroporous resin, preserving the temperature at 20-35 ℃, adsorbing for 1-3h (preferably 30 ℃, 90 revolutions/min and adsorbing for 2h) under the stirring of 80-100 revolutions/min, filtering, drying a filter cake in vacuum (preferably 40 ℃) to the water content of 3-5% (preferably 4.0%) to obtain the immobilized lipase, wherein the volume consumption of the deionized water is 150 ml/g (preferably 160 ml/g) and the mass ratio of the lipase to the disodium hydrogen phosphate dodecahydrate and the sodium dihydrogen phosphate is 1:0.5-1.5 (preferably 1:0.7-1.1) and 1: 0.5-1.0.0 (preferably 1: 0.5-0.8); the weight ratio of the lipase to the macroporous resin is 1: 2-4. The macroporous resin model comprises D4006 model, D3520 model and D1300 model.

The macroporous resin is pretreated before enzyme liquid is added, and the pretreatment method comprises the following steps: adding 10 times mass of 95% ethanol aqueous solution with volume concentration into the macroporous resin, shaking and washing for 24 hours at room temperature (25-30 ℃) under the condition of 150r/min, replacing the 95% ethanol aqueous solution twice in the middle, washing for five times by using distilled water with twice mass each time after the shaking and washing are finished, and performing suction filtration to obtain a filter cake, namely the pretreated macroporous resin.

The lipase activity is 10-30 ten thousand units/g, and aspergillus niger lipase or candida lipase is preferred.

Compared with the prior art, the invention has the following beneficial effects: the method adopts N, N-Dimethylformamide (DMF) as a main solvent for synthesizing the sucrose-6-ethyl ester by an enzymatic method, is compounded with tert-amyl alcohol or tert-butyl alcohol, and has the advantages of better enzyme activity retention, sucrose solubility of up to 10 percent, sucrose esterification rate of up to more than 85 percent, sucrose-6-ethyl ester accounting for more than 85 percent of total esters, smaller viscosity of a synthesis reaction solvent system and easiness in recovery compared with other organic solvents. The solvent has small influence on enzyme activity, has proper viscosity and boiling point, and is suitable for the reaction operation of synthesizing sucrose-6-ethyl ester by an enzyme method.

(IV) description of the drawings

FIG. 1 is a standard curve of bovine serum albumin.

FIG. 2 is a liquid chromatogram of sucrose-6-ethyl ester prepared in example 1.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

in the embodiment of the invention, sucrose is purchased from chemical reagent engineering research and development center (chemical purity) in Guangdong province, and N, N-dimethylformamide and tert-amyl alcohol are purchased from national medicine group chemical reagent company Limited (chemical purity); vinyl acetate was purchased from fine chemicals, llc (chemical purity) of monhua, jiang su; the room temperature of the invention is 25-30 ℃.