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Method for preparing L-tryptophan by enzymatic conversion

A tryptophanase method and tryptophanase technology, applied in the direction of fermentation, etc., can solve the problems of unsuitability for large-scale industrial production, long synthesis route of synthetic tryptophan, harsh process conditions, etc., and achieve good economic benefits and environmental protection Benefits, solve the effect of high efficiency separation, high conversion rate and reaction rate

Active Publication Date: 2009-10-14
NANJING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But above-mentioned two kinds of methods synthesize tryptophan or synthetic route is long, or need to adopt expensive special raw material, or the route reaction step of constructing indole ring is many, involves many raw materials, and process condition is harsher, and operation difficulty is big, and is not suitable for large-scale industrial production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The method for preparing L-tryptophan by enzymatic conversion of L-serine derived from keratin, the preparation steps are as follows:

[0030] 1. The tryptophan synthase genetically engineered bacterial strain is cultivated in the following 1000ml medium (g / ml): corn steep liquor 0.5%, NaCl 0.5%, KH 2 PO 4 0.16%, MgSO 4 .7H 2 O 0.04%, FeSO 4 .7H 2 O 0.01%, beef extract 2%, maltose 0.5%, glucose 0.5%, IPTG 0.005%, pH 7.2. Shake the flask at 37°C for 12 hours, centrifuge at 4000rpm for 15 minutes to obtain 25g of wet cells.

[0031] 2. Wet cells are added in the 600ml conversion fluid that 0.1mol / L sodium carbonate damping fluid (pH9.5) prepares, and conversion fluid is the feather acid hydrolyzate that contains 3.8% L-serine (g / ml) (amino acid total content 15%), 22.8g indole and 0.05% OP solution (g / ml) 6mL, 37°C enzymatic reaction for 36h. The concentration of L-tryptophan in the reaction solution is 6.3%, and the molar conversion rate of L-serine is 87%.

[003...

Embodiment 2

[0035] The method for preparing L-tryptophan by enzymatic conversion of L-serine derived from keratin, the preparation steps are as follows:

[0036]1. Cultivate the tryptophanase genetically engineered bacteria in the following 1000ml medium (g / ml): corn steep liquor 0.5%, beef extract 2%, protein hydrolyzate 0.5%, maltose 0.5%, lactose 0.5%, IPTG 0.007% , K 2 HPO 4 0.5%, MgSO 4 .7H 2 O 0.03%, NaCl 0.5%, pH7.2. Shake the flask at 37°C for 14 hours, centrifuge at 4000 rpm for 15 minutes to obtain 23.8 g of wet cells.

[0037] 2. Wet cells are added in the 600ml conversion fluid that 0.1mol / L sodium carbonate buffer (pH9.5) prepares, and conversion fluid is the hair acid hydrolyzate (total amino acid content) that contains 4.2% L-serine (g / ml) 16%), 25.2g indole and 6mL of 0.5% Tween-80 (g / ml) solution, 39°C enzymatic reaction for 32h. The concentration of L-tryptophan in the reaction solution is 7.1%, and the molar conversion rate of L-serine is 90%.

[0038] 3. The sepa...

Embodiment 3

[0040] The method for preparing L-tryptophan by enzymatic transformation of plant source L-serine, the preparation steps are as follows:

[0041] 1. The tryptophan synthase genetically engineered strain is cultivated in the following 1000ml medium (g / ml): peptone 1%, beef extract 0.3%, maltose 0.5%, sucrose 0.3%, NaCl 0.5%, KH 2 PO 4 0.14%, K 2 HPO 4 0.5%, MgSO 4 .7H 2 O 0.05%, IPTG 0.005%, pH 7.0. Shake the flask at 37°C for 13 hours, centrifuge at 4000 rpm for 15 minutes to obtain 25.3 g of wet cells.

[0042] 2. Wet cells are added in the 600ml conversion solution prepared by 0.1mol / L boric acid buffer solution (pH9.0), the conversion solution is the molasses extract containing 2% L-serine (g / ml) (14% amino acid total content ), 12g of indole and 6mL of 0.05% CTAB (g / ml) solution, 39°C enzymatic reaction for 32h. The concentration of L-tryptophan in the reaction solution is 3.3%, and the molar conversion rate of L-serine is 87%.

[0043] 3. The separation and purific...

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Abstract

The invention belongs to the technical field of medicine and biology and particularly relates to a method for preparing L-tryptophan by enzymatic conversion. The method, of which L-tryptophan is prepared from mixed amino acids containing L-serine, comprises the following steps: mixing the high-activity genetic engineering bacteria of L-tryptophan synthetase or L-tryptophanase with the conversion solution of the mixed amino acid containing L-serine; carrying out the enzymatic reaction at the temperature of 30 to 45 DEG C; and then, separating the converted products by the isoelectric point crystallization method or the method combining isoelectric point crystallization and ion exchange resin to obtain high-purity L-tryptophan. The method solves the problems of the separation and overall development of L-serine in the mixed amino acids by obtaining L-serine products with wider applicable range and higher added value, therefore, the invention has the advantages of wide material availability, low price, convenient operation, short conversion time, low production cost and the like.

Description

1. Technical field [0001] The invention belongs to the technical field of medicine and biology, and in particular relates to an enzymatic conversion preparation method of L-tryptophan. 2. Background technology [0002] Tryptophan, also known as α-amino-β-indolepropionic acid, is an amino acid discovered and isolated by Hopkins and Cole in 1902. It has two isomers, D- and L-, and only L-tryptophan exists naturally. L-tryptophan plays an important role in the growth, development and metabolism of humans and animals, so it is widely used in the fields of medicine, food and feed. [0003] According to current literature reports, the preparation method of L-tryptophan mainly contains following three kinds: [0004] 1. Fermentation method [0005] The fermentation method to produce L-tryptophan can be roughly divided into: direct fermentation method and precursor addition fermentation method. [0006] The commonly used L-tryptophan-producing bacteria in the direct fermentation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/22
Inventor 焦庆才赵根海刘均忠张飞刘茜肖国安王先兵曾庆群
Owner NANJING UNIV
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