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Method for preparation of sucrose-6-acetate from fructosyl transferase

A technology of fructosyltransferase and acetate, which is applied in the field of preparation of sucrose-6-acetate, can solve the problems of high cost, low conversion rate, and many operation steps, and achieve simple operation, low cost, and high separation efficiency Effect

Inactive Publication Date: 2015-07-15
ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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Problems solved by technology

Patent 200680034904.X uses immobilized Aureobasidium pullulans whole cells to catalyze sucrose and glucose-6-acetate to form sucrose-6-acetate, but the conversion rate is low, only 32%; Bacillus fixed proliferating cells and immobilized fructosyltransferase catalyze the synthesis of sucrose-6-acetate with a yield of 80%, but there are many operating steps, multiple strains and enzymes are required to participate in the reaction, and the cost is high

Method used

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  • Method for preparation of sucrose-6-acetate from fructosyl transferase
  • Method for preparation of sucrose-6-acetate from fructosyl transferase
  • Method for preparation of sucrose-6-acetate from fructosyl transferase

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Embodiment 1

[0047] Embodiment 1, established the method for separating and purifying fructosyltransferase:

[0048] The strain is: Aspergillus oryzae ZZ-01

[0049] The preparation of the culture medium is as follows. In a 1L culture medium: 200g of potatoes, 20g of glucose, 1000mL of water, heated to boiling on a heater for 20min, and filtered with 3 layers of gauze, discarding the filter residue; adding to the filtrate NaNO 3 2g, K 2 HPO 4 1g, KCl 1g, MgSO 4 0.5g, add water to 1000mL, adjust the pH value to 7.0; 28 ℃ constant temperature shaking culture for 48h, and the rotation speed is 200rpm / min, to obtain the bacterial liquid;

[0050] After the bacterial liquid was cultured for 48 hours, the culture was centrifuged at 14,000 rpm for 20 minutes, and the supernatant was collected. The collected supernatant was subjected to fractional precipitation (overnight) with 85% ammonium sulfate, and after centrifugation at 14,000 rpm for 20 min, the precipitate was resuspended in 0.02M ph...

Embodiment 2

[0054] Embodiment 2, the method for the immobilization enzyme of fructosyltransferase:

[0055] The magnetic chitosan microspheres were prepared by combining the inverse suspension method and the sol-gel method, and using this as a carrier, genipin as a cross-linking agent, immobilized the purified fructosyltransferase, the specific method is as follows:

[0056] The preparation of magnetic chitosan microspheres: in a capacity of 1000mL there-necked flask, add oil phase (150mL kerosene+3.5g Tween 80), start stirrer (stirring speed 600r / min), slowly add 90g magnetic chitosan solution ( The concentration of acetic acid is 2%, the concentration of chitosan (deacetylation degree 85-90%, Sinopharm Chemical Reagent Co., Ltd.) is 4%, Fe 3 o 4 content of 5%), stirred continuously for 1 hour to form a uniform water-in-oil emulsion, then quickly added to the condensate (V (2mol / L NaOH): V (ethanol) = 4: 1), kept magnetic stirring for 30 minutes, and stopped Stir, let stand overnight, ...

Embodiment 3

[0059] Embodiment 3: Utilize the method for immobilized fructosyltransferase to catalyze the synthesis of sucrose-6-acetate, with ionic liquid [Dmim][PF6] (99.9%, Shanghai Chengjie Chemical Co., Ltd.) and tert-butanol (anhydrous , Zibo Dehong Chemical Technology Co., Ltd.) mixed solvent as the reaction medium, with sucrose and glucose-6-acetate as the substrate, such as figure 2 shown.

[0060] The reaction system for the synthesis of sucrose-6-acetate catalyzed by immobilized fructosyltransferase is as follows: sucrose (99.5%, Shanghai Bioengineering Co., Ltd.) and glucose-6-acetate (99.9%, Zhengzhou Aoli Industrial Company) Molar ratio 4:1, immobilized fructosyltransferase 40mg / mL, reaction medium ionic liquid [Dmim][PF6] and organic solvent tert-butanol volume ratio 1:1, 5mM phosphate buffer (pH 6.0), 55°C Water bath reaction 20h. Under this condition, the yield of sucrose-6-acetate can reach 57.2g / L, and the yield can reach 86.2%, and the immobilized enzyme can be used ...

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Abstract

The invention relates to a method for preparation of sucrose-6-acetate from fructosyl transferase. The method includes the steps of: S1. preparation of fructosyl transferase; S2. preparation of immobilized fructosyl transferase; S3. catalytic synthesis of sucrose-6-acetate with fructosyl transferase in a nonaqueous phase reaction system; and S4. separation and purification of sucrose-6-acetate. The invention has the technical characteristics that: the invention establishes a fructosyl transferase separation and purification method; the invention establishes a fructosyl transferase immobilization method, magnetic chitosan microspheres are adopted as the carrier, and genipin is taken as the cross-linking agent; the invention establishes a method for catalytic synthesis of sucrose-6-acetate with immobilized fructosyl transferase, the ionic liquid [Dmin][PF6] and tert-butyl alcohol mixed solvent is taken as the reaction medium, and sucrose and glucose-6-acetate are adopted as the substrate to carry out catalytic synthesis of sucrose-6-acetate, the output and the yield respective reach 57.2g / L and 86.2%; and the invention establishes a separation and purification method for the product sucrose-6-acetate, the purity reaches 99.3%, and the recovery rate reaches 96.2%.

Description

technical field [0001] The invention relates to the technical field of a preparation method of sucrose-6-acetate, in particular to a method for preparing sucrose-6-acetate by using fructosyltransferase. Background technique [0002] Sucralose is the most perfect strong sweetener developed by humans so far, and sucrose-6-acetate is a key intermediate in the synthesis of sucralose. In addition, sucrose esters have been widely used in food, medicine, cosmetics and detergents due to their excellent application performance, good biodegradability and toxicological properties, and sucrose-6-acetate is an important sucrose ester. The catalytic synthesis of sucrose-6-acetate in the non-aqueous phase reaction system of the present invention has important industrial application value in the fields of bioengineering and medicine. [0003] At present, the synthesis methods of sucrose-6-acetate include chemical method and biological enzyme method. Chemical methods mainly include direct ...

Claims

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Application Information

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IPC IPC(8): C12P19/44C12N9/10C12N11/14C12N11/10C07H13/06C07H1/06C12R1/69
CPCY02P20/50Y02P20/54
Inventor 毛多斌魏涛张改红白冰黄申贾春晓杨靖
Owner ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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