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Method for producing dextran and fructose by recombinant enzyme process

A technology of dextran and recombinant enzymes, which is applied in the catalytic synthesis of dextran and fructose. Recombinant dextran sucrase uses gap oscillation in the field of enzyme catalysis, which can solve pollution and other problems, and achieve the effects of solving pollution, high activity and high conversion rate

Inactive Publication Date: 2011-11-02
六安华源制药有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to use the lower cost IPTG and lactose joint induction method to prepare free recombinant dextran sucrase, and then start from the substrate sucrose based on this recombinant enzyme Dextran and fructose are prepared in one step by the enzyme-catalyzed method of interstitial oscillation. This production method replaces the process of producing dextran by Leuconostoc enteroformis, avoids bacteria that appear during the production process of bacteria, eliminates miscellaneous proteins, and improves product quality; Realize multiple functions of one enzyme, recover fructose, reduce cost, and solve the environmental pollution problem of by-product fructose in the original production process of dextran

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] (1) Combined induction to prepare recombinant dextran sucrase fermentation

[0030] The genetically engineered strain BL21 (DE3) / pET28-dexYG was transferred from the slant to 20mL of fresh LB medium (containing 50 μg / mL of Kanna), cultivated overnight at 37°C and 300r / min for 16h, and then followed 1% of the inoculum was inoculated into medium B for fermentation. Culture the fermentation broth at 37°C and 300r / min, take 1mL of the fermentation broth every hour, dilute it 10 times with sterile water, and measure the OD 600 .

[0031] Dextransucrase was prepared by the combined induction method of IPTG and lactose, when OD 600 When = 3.0, add IPTG to a final concentration of 0.09 mmol / L and lactose to a final concentration of 2.5 g / L, and induce culture at 25°C and 300 r / min for 4 hours. The fermented liquid was centrifuged at 4°C and 12000r / min for 15 minutes to obtain bacteria cells, then added pH5.4 acetate buffer solution, ultrasonically crushed for 10 minutes, and...

Embodiment 2

[0039] (1) Combined induction to prepare recombinant dextran sucrase fermentation 2

[0040] The genetically engineered strain BL21 (DE3) / pET28-dexYG was transferred from the slant to 20mL of fresh LB medium (containing 50ug / mL of Kanna), cultivated overnight at 37°C and 300r / min for 16h, and then followed the 1 % of the inoculum was inoculated into medium B for fermentation. Culture the fermentation broth at 37°C and 300r / min, take 1mL of the fermentation broth every hour, dilute it 10 times with sterile water, and measure the OD 600 .

[0041] When OD 600 When = 3.5, dextran sucrase was prepared by the joint induction method of IPTG and lactose to a final concentration of IPTG of 0.12 mmol / L and a final concentration of lactose of 3.0 g / L, and induced culture at 28°C and 300 r / min for 5 hours. The fermented liquid was centrifuged at 4°C and 12,000r / min for 15 minutes to obtain bacterial cells, then added to pH 5.4 acetate buffer, ultrasonically crushed for 10 minutes, and...

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Abstract

The invention discloses a method for producing dextran and fructose by a recombinant enzyme process. The method comprises the following steps of: (1) culturing free recombinant dextransucrase by using a genetically engineered bacterial strain BL21 (DE3) / pET28-dexYG; and (2) catalytically synthesizing dextran and fructose with the recombinant dextransucrase. The invention provides a method for preparing recombinant dextransucrase by an IPTG (isopropyl beta-D-1-thiogalactopyranoside) / lactose combined induction method through using genetically engineered bacterial strain BL21 (DE3) / pET28-dexYG. The method greatly reduces the enzyme preparation cost in the single IPTG induction method and is favorable for large-scale production. Besides, by use of the intermittent vibratory enzyme catalysis method, the dextran and fructose are catalytically synthesized in one step, so as to increase the conversion efficiency, achieve the effects of multiple purposes of one enzyme and fructose recovery, reduce the cost and solve the problem of environmental pollution due to fructose as the byproduct of the dextran production process in the prior art.

Description

technical field [0001] The present invention relates to a method for co-inducing low-cost preparation of recombinant dextran sucrase and a method for producing biological polysaccharides and monosaccharides by enzymatic catalysis, in particular to an enzyme-catalyzed method in which recombinant dextran sucrase adopts gap oscillation to catalyze the synthesis of dextran and fructose way. Background technique [0002] Dextran sucrase (Dextransucrase, EC 2.4.1.5) is produced by Leuconostoc membranosa ( Leuconstoc mesenteriodes ) produced glucosyltransferases (Glucosyltransferases), which are composed of 1250 to 1600 different amino acids, with a molecular weight of about 170KDa. have important applications. The catalysis of this enzyme uses sucrose as the substrate to catalyze the transfer of the D-glucose group in the sucrose molecule to form D-glucosyl-Enzyme, and release fructose, resulting in two important products: ① Catalytic synthesis of dextran with different molecula...

Claims

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Application Information

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IPC IPC(8): C12P19/18C12P19/08C12P19/02
Inventor 张洪斌胡雪芹崔维华杨运根衣学福
Owner 六安华源制药有限公司