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Method for preparing medicinal dextran and fructose by double-enzyme immobilization coupled with multi-stage membrane separation

A dextran and enzyme immobilization technology, applied in the direction of fermentation, etc., can solve the problems of low yield of target products, affecting the safe use of medicinal dextran, and complicated separation of follow-up products, so as to facilitate industrialization and application, reduce production costs, and reduce production cost. The effect of high material conversion rate

Inactive Publication Date: 2015-10-28
GUANGXI UNIV
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Problems solved by technology

At present, in China, there is a method of fermenting sucrose with bacteria or dextran sucrase to prepare dextran first, and then adding dextran-containing fermentation serum or dextranase to prepare dextran with different molecular weights, but the separation of subsequent products is more complicated. , and use traditional separation methods such as gauze filtration and ethanol fractionation to refine the product, which may affect the safe use of medicinal dextran, and the preparation method has a large room for improvement
In addition, the reaction between dextran sucrase and sucrose will quickly generate dextran with a molecular weight greater than 1 million in a short period of time. Although this method is outstanding in the conversion rate of sucrose, because the enzymatic hydrolysis of dextranase is random, the target product can be obtained rate is not high

Method used

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  • Method for preparing medicinal dextran and fructose by double-enzyme immobilization coupled with multi-stage membrane separation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Dextransucrase was fermented by Leuconostoc enterococci, the fermentation broth was centrifuged at 12,000r / min and 4°C for 20min, and the supernatant was obtained. The enzyme activity was detected by DNS method. Dextranase is derived from a commercial enzyme preparation (Amano Amano Enzyme Trading (Shanghai) Co., Ltd.), and the enzyme activity of dextranase is measured by the Hanes method combined with the actual situation of the present invention and the measurement conditions are slightly modified. It was determined that the enzyme activity of dextran sucrase before immobilization was 8.4U / mL, and the enzyme activity of dextranase was 22,000U / mL.

[0023] The double-enzyme immobilization method is to mix dextransucrase and dextranase with the ratio of enzyme activity of 10:1 (the final concentration of immobilized dextransucrase is 0.6U / mL) and pasteurized sodium alginate solution, seaweed The final concentration of sodium bicarbonate solution was 2.5% (w / v, g / 100mL)....

Embodiment 2

[0026] The acquisition of dextran sucrase and dextranase and the determination of enzyme activity are the same as in Example 1.

[0027] The double-enzyme immobilization method is to mix dextran sucrase and dextranase with the ratio of enzyme activity of 10:1 (the final concentration of immobilized dextran sucrase is 0.8U / mL) and pasteurized sodium alginate solution, seaweed The final concentration of sodium bicarbonate solution was 4.0% (w / v, g / 100mL). The mixed solution was degassed at 4°C for 2 hours, then vacuumed and degassed for 15 minutes, and then the mixed solution was dripped into 2 % sterile calcium chloride solution to prepare immobilized pellets, the immobilized pellets were rinsed with sterile water, and then put into 0.4% (v / v, mL / mL) glutaraldehyde solution for cross-linking After 45 minutes, they were taken out, washed with sterile water, and part of the immobilized pellets were taken to measure the enzyme activity of dextran sucrase and dextranase respectivel...

Embodiment 3

[0029] The acquisition of dextran sucrase and dextranase and the determination of enzyme activity are the same as in Example 1.

[0030] The double-enzyme immobilization method is to mix dextran sucrase and dextranase with the ratio of enzyme activity of 100:1 (the final concentration of immobilized dextran sucrase is 0.8U / mL) and pasteurized sodium alginate solution, seaweed The final concentration of sodium bicarbonate solution was 3.0% (w / v, g / 100mL). The mixed solution was degassed at 4°C for 2 hours, then vacuumed and degassed for 15 minutes, and then the mixed solution was dripped into 2 % sterile calcium chloride solution to prepare immobilized pellets, the immobilized pellets were rinsed with sterile water, and then cross-linked in 0.7% (v / v, mL / mL) glutaraldehyde solution After 40 minutes, remove them, wash them with sterile water, take some immobilized pellets to measure their dextransucrase and dextranase activities, and store the rest at 4°C for later use. The res...

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Abstract

The invention discloses a method for preparing pharmaceutical dextran and levulose by bi-enzyme immobilization coupled multistage membrane separation, which comprises the following steps: co-immobilizing dextran sucrase and dextranase in sodium alginate microspheres to obtain bi-enzyme-immobilized spheres; adding the bi-enzyme-immobilized spheres into a sucrose solution used as a substrate to form an immobilized bi-enzyme / sucrose reaction system; and after the reaction product is separated by a multistage membrane system composed of hyperfiltration membranes and a nanofiltration membranes, directly obtaining the pharmaceutical dextran in the hyperfiltration membrane separation stage, and obtaining the high-purity levulose solution in the last nanofiltration membrane separation stage, wherein the enzymes and macromolecular dextran contained in the trapped solution of the first-stage hyperfiltration membrane return to the immobilized bi-enzyme / sucrose reaction system to participate in the reaction again, thereby implementing cyclic utilization of the enzymes. The invention has the advantages of lower cost, environment friendliness, high safety and high efficiency, is simple to operate, implements oriented adjustable production of high-quality pharmaceutical dextran with target molecular weight, and obtains the high-purity high-added-value levulose byproduct.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals and biomass separation, and in particular relates to a method for preparing medicinal dextran and fructose by double-enzyme immobilization coupled with multi-stage membrane separation. Background technique [0002] Dextran is an exopolysaccharide produced by bacteria (such as Leuconostoc mesenteroides), and its main chain is composed of D-glucose units linked by α(1→6) glycosidic bonds. Dextran with different molecular weights has many clinical uses, such as being used as a plasma substitute, dredging microvessels, and preventing and treating thrombosis. "Chinese Pharmacopoeia" (2010 edition) includes three kinds of dextran and its derivative drugs including Dex-70 (average molecular weight about 70,000), Dex-40 (average molecular weight about 40,000), Dex-20 (average molecular weight about 20,000) There are 12 kinds in total. At present, most domestic preparations of dextran adopt the process ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/14C12P19/02C12P19/08
Inventor 陈山浦媛媛邹青松张义平姚晓麦王清王晓黄磊刘玫黄艳卢安根莫建光
Owner GUANGXI UNIV
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