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Method for enhancing enzyme activity of maleate cis-trans isomerase by using fucose

An isomerase enzyme, cis-trans isomerization technology, applied in the field of enzyme chemistry, can solve the problems of many by-products, high cost of chemical synthesis, environmental pollution, etc., and achieves a simple and easy method, improves reaction efficiency, and saves enzyme consumption. Effect

Active Publication Date: 2016-04-20
ANHUI BBCA FERMENTATION TECH ENG RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the production of fumaric acid is mostly prepared from maleic anhydride by petrochemical methods. However, the chemical synthesis method has problems such as high cost, many by-products and possible environmental pollution, which makes people gradually turn to the environmentally friendly biological method. to prepare fumaric acid

Method used

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  • Method for enhancing enzyme activity of maleate cis-trans isomerase by using fucose
  • Method for enhancing enzyme activity of maleate cis-trans isomerase by using fucose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The mensuration of embodiment 1 maleic acid cis-trans isomerase enzyme activity

[0036] Take 450μl with pH8.4 reaction buffer solution (50mmol / L Na 2 HPO 4 -KH 2 PO 4 Buffer) suitably diluted enzyme solution (specific activity of enzyme solution is 50U / mg), add 50μl, 1mol / L maleic acid (KOH adjusted to pH8.4) solution, react at 37℃ for 20min, filter through 0.45μm organic filter Membrane filtration, the filtrate was determined by HPLC for fumaric acid content. Definition of enzyme activity: under the conditions of 37°C and pH 8.4, the amount of enzyme required to convert the substrate maleic acid into 1 μmol product fumaric acid per minute in the reaction system is defined as one enzyme activity unit 1U.

[0037] The HPLC chromatographic column is LaChromC18 (5μm4.6×250mm); the mobile phase is methanol aqueous solution with a volume fraction of 8%, and the flow rate is 0.6mL / min; the wavelength of the UV detector is 210nm, and the column temperature is 30°C.

Embodiment 2

[0038] The influence of embodiment 2 fucose concentration on maleic acid cis-trans isomerase enzyme activity

[0039] Fucose was added to the reaction system in Example 1, and the final concentrations of fucose were 0, 0.1, 0.3, 0.5, 0.7, and 1.0 mmol / L, respectively. Experimental results such as figure 1 shown. The results showed that when the concentration of fucose was 0.5mmol / L, the enzyme activity was the highest, which was 26% higher than that without adding fucose.

[0040] Definition of relative enzyme activity: Taking the enzyme activity measured when no fucose is added in the reaction system as the denominator, the relative enzyme activity measured at different fucose concentrations is obtained.

Embodiment 3

[0041] The influence of embodiment 3 fucose on maleic acid cis-trans isomerase reaction stability

[0042] Fucose was not added to the enzyme solution of the control group, 0.5mmol / L fucose was added to the enzyme solution of the test group, stored at room temperature, and the enzyme solution was taken every 8h to detect the enzyme activity according to the method in Example 1. The experimental results were as follows: figure 2 shown. It can be seen from the figure that the decay rate of the enzyme activity in the fucose-added group is slow, and the enzyme activity retention rate after 48 hours is 84.2%, while the enzyme activity retention rate in the no-fucose group is only 58% at this time

[0043] Definition of relative enzyme activity: the relative enzyme activity measured at different time points is obtained by taking the enzyme activity measured at 0h without adding fucose in the reaction system as the denominator.

[0044] Enzyme activity retention rate: (enzyme activit...

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Abstract

The invention relates to the field of zymochemistry, and particularly discloses a method for enhancing enzyme activity of maleate cis-trans isomerase by using fucose, which is implemented by adding fucose into an enzymolysis reaction system in which maleate cis-trans isomerase participates. The experiment is performed to optimize the addition concentration of the fucose, thereby maximally enhancing the enzyme activity of the maleate cis-trans isomerase. The method is simple and easy to implement, and can save the enzyme consumption and enhance the reaction efficiency in the maleate cis-trans isomerase reaction process.

Description

technical field [0001] The invention relates to the field of enzyme chemistry, in particular to a method for improving the enzyme activity of maleic acid cis-trans isomerase by using fucose. Background technique [0002] Fumaric acid, also known as fumaric acid and fumaric acid, is a naturally occurring organic acid widely used in food, medicine, chemical industry, coatings, plasticizers and other fields. For example, in food, it can be used as a sour agent and flavor enhancer with pure taste. In medicine, it can be used to produce antidote sodium dimercaptosuccinate, etc., and iron fumarate is widely used in medical treatment of human microcytic anemia. At present, the production of fumaric acid is mostly prepared from maleic anhydride by petrochemical methods. However, the chemical synthesis method has problems such as high cost, many by-products and possible environmental pollution, which makes people gradually turn to the environmentally friendly biological method. to ...

Claims

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Application Information

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IPC IPC(8): C12N9/90C12P7/46
CPCC12N9/90C12P7/46C12Y502/01001
Inventor 秦晴许鹏冯杰杨为华纪传侠娄雯
Owner ANHUI BBCA FERMENTATION TECH ENG RES
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