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Process of synthesizing gamma-D-glutamine acyl L-tryptophane by enzyme method

A technology of enzymatic synthesis and tryptophan, which is applied in fermentation and other directions, can solve the problems of high operating costs, side reactions, and low conversion rate, and achieve high recovery, simple method, and good purity

Inactive Publication Date: 2007-07-18
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 1. The above patents all have disadvantages such as long process route, high operating cost, low yield and purity, which increases the cost of operation and energy consumption, is not conducive to large-scale production, and weakens the market competitiveness of the product
[0006] 2. The substrates of the above patents all use L-glutamine as the donor of the enzyme, which will cause side reactions and generate redundant tripeptides and tetrapeptides
[0007] 3. The reaction time for the enzymatic preparation of dipeptide in the above patents is too long, and the product will start to be hydrolyzed after accumulating to a certain amount, resulting in a low final conversion rate
However, after searching, there is no report on the method of enzymatically synthesizing γ-D-glutamyl-L-tryptophan at home and abroad.

Method used

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  • Process of synthesizing gamma-D-glutamine acyl L-tryptophane by enzyme method

Examples

Experimental program
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Effect test

preparation example 1

[0037] Preparation Example 1: Preparation and Purification of γ-Glutamyl Transpeptidase

[0038] Take 15g of sucrose, 30g of corn steep liquor, 15g of peptone, K 2 HPO 4 15g, MgSO 4 0.5g, pH 7.5, add tap water to make 1L liquid culture medium, prepare 3.5L according to this recipe, put it into a 5L fermenter and put it into a 0.1MPa high-pressure steam sterilization for 25 minutes for later use. At the same time, divide 50mL of medium into 500mL, add 8 layers of gauze, wrap it in kraft paper, and sterilize it together with the fermenter for later use.

[0039] After taking out the bacterial species preserved in the refrigerator—Bacillus subtillis NX-2 (preservation number is CGMCCNo.0833), connect it to fresh slant medium (slant medium (g / L): peptone 10, beef extract 3. On NaCl5, agar 20), activate the culture for 24 hours, insert the seed medium (seed medium (g / L): glucose 15, corn steep liquor 10, peptone 10, K 2 HPO 4 2. MgSO 4 0.25) shake culture at 33° C. and 220 r...

Embodiment 1

[0042] Get the gamma-glutamyl transpeptidase 0.02U that preparation example 1 obtains, add 0.0292g gamma-D-glutamine, L-tryptophan 0.0489g, Tris (trishydroxymethylaminomethane)-HCl (pH9.0 , 0.2mol / L) 20mL, after mixing evenly, stir and react for 5h, and the reaction temperature is 38°C. After the reaction finishes, analyze and detect with high performance liquid chromatography system (chromatographic separation condition: chromatographic column is Kromasil KR100-5C18 post, and detection wavelength is 220nm, mobile phase is 75%, 20mmol / L KH 2 PO 4 , 25% methanol; flow rate: 1 mL / min), the conversion rate of γ-D-glutamyl-L-tryptophan was 58%, and the purity was 98%.

[0043] After the reaction, the reaction solution was micro-filtered to remove enzymes, and then KTA TM The explorer 100 protein purification instrument was used for separation and purification. The separation and purification conditions were as follows: the chromatographic column was a reversed-phase C18 column...

Embodiment 2

[0045] Get 0.5U of γ-glutamyl transpeptidase obtained in Preparation Example 1, add 0.0584g γ-D-glutamine, 0.122g L-tryptophan, NaHCO 3 -NaOH (pH10.5, 0.1mol / L) 20mL, after mixing evenly, stir and react for 6h, the reaction temperature is 37°C. After the reaction finishes, analyze and detect with high performance liquid chromatography system (chromatographic separation condition: chromatographic column is Kromasil KR100-5C18 post, and detection wavelength is 220nm, mobile phase is 75%, 20mmol / L KH 2 PO 4 , 25% methanol; flow rate: 1 mL / min), the conversion rate of γ-D-glutamyl-L-tryptophan was 65%, and the purity was 98.4%.

[0046] After the reaction, the reaction solution was micro-filtered to remove enzymes, and then KTA TM The explorer 100 protein purification instrument was used for separation and purification. The separation and purification conditions were as follows: the chromatographic column was a reversed-phase C18 column, the detection wavelength was 214nm, the...

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PUM

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Abstract

The present invention relates to enzyme process of synthesizing gamma-D-glutamyl-L-tryptophane. Gamma-glutamyltranspeptidase is first added into the substrate solution comprising L-tryptophane in 8-50 mmol / L and gamma-D-glutamie in 5-35 mmol / L to produce the peptide-converting reaction preparing gamma-D-glutamyl-L-tryptophane in 8 hr, and the prepared gamma-D-glutamyl-L-tryptophane is then separated and purified. The present invention has simple process, low cost, less side reactions, high gamma-D-glutamyl-L-tryptophane converting rate, high yield and high product purity, and may be used in industrial production.

Description

technical field [0001] The invention relates to an enzymatic synthesis method, in particular to a method for synthesizing γ-D-glutamyl-L-tryptophan by using γ-glutamyl transpeptidase. Background technique [0002] The immune system is a network of cells adapted to protect an organism against pathogens and cells that are recognized as not "self". Once activated, the immune system recruits a wide variety of cells and components to enhance designated effector functions to eliminate "non-self" entities in the body. Lymphocytes are cells of the immune system capable of specifically recognizing and selectively eliminating foreign entities, as compared to other cells of the immune system, such as neutrophils, which are thought to be nonspecific in responding to invaders , Lymphocytes bring specificity, diversity, memory and self / non-self recognition to the immune response. There are two main groups of lymphocytes: B lymphocytes and T lymphocytes. The normal development, maturati...

Claims

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Application Information

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IPC IPC(8): C12P13/22
Inventor 姚忠汪前荀志金徐虹韦萍
Owner NANJING UNIV OF TECH
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