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A kind of enzymatic conversion preparation method of 2-methyl-1-tryptophan

A technology of enzymatic transformation and tryptophan synthase, applied in the field of 2-methyl-L-tryptophan transformation, can solve problems such as being unsuitable for large-scale industrial production, high cost of L-serine, and many reaction steps, etc. , to achieve good economic and social benefits, low prices and low costs

Active Publication Date: 2016-08-31
宿州学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the production of 2-methyl-tryptophan in my country mainly comes from chemical synthesis, which has many reaction steps, difficult operation, high production cost, and is not suitable for large-scale industrial production.
On the other hand, in the industrial production of amino acids in my country, a large amount of hair acid hydrolyzate rich in L-serine that cannot be directly used in food and medicine will be produced. The direct separation of the mixed amino acid feed solution to prepare L-serine has high cost and low efficiency.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] This embodiment includes the following steps:

[0023] (1) 1000mL Escherichia coli ATCC15489 is cultivated and fermented in the culture medium, and the fermented liquid is centrifuged to obtain 15g wet thallus, joins in the 500mL transformation liquid, contains the hair acid hydrolyzate of 21g of L-serine in the transformation liquid, 26.2g of 2 - methyl indole, 0.2g / L of pyridoxal phosphate and 0.005g / L of methyl alcohol, pH 8.0, 37 ℃ enzymatic reaction for 12h, 2-methyl-L-tryptophan in the conversion liquid after the reaction is 42.7g, the molar conversion rate to L-serine is 98%;

[0024] (2) Centrifuge the transformation solution at 4000r / min for 10min, remove bacterial cells, heat, decolorize with activated carbon, filter with suction, adjust the pH of the filtrate to about 5.5, let stand to precipitate precipitation, filter with vacuum, and dry to obtain 39.5g of 2-methyl - The crude L-tryptophan was washed with 95% ethanol, vacuum filtered, and dried to obtain 3...

Embodiment 2

[0027] This embodiment includes the following steps:

[0028] (1) Cultivate and ferment 1000mL of Pseudomonas aeruginosa CGMCC NO: 1.1129 in the culture medium, centrifuge the fermentation broth to obtain 18g of wet bacteria, add it to 500mL of transformation liquid, which contains 11g of L-serine, 13.1g of 2 -Methylindole, 0.2g / L pyridoxal phosphate and 0.5g / L diethyl ether, pH8.0, 37°C enzymatic reaction for 16h, after the reaction, the 2-methyl-L-tryptophan in the conversion solution was 22.4 g, the molar conversion rate to L-serine is 98%;

[0029] (2) Centrifuge the transformation solution at 3000r / min for 20min, remove bacterial cells, heat, decolorize with activated carbon, filter with suction, adjust the pH of the filtrate to about 5.5, let stand to precipitate precipitation, filter with vacuum, and dry to obtain 20.1g of 2-methyl - The crude L-tryptophan was washed with 95% ethanol, vacuum filtered, and dried to obtain 18.6 g of refined 2-methyl-L-tryptophan with a p...

Embodiment 3

[0032] This embodiment includes the following steps:

[0033] (1) Cultivate and ferment 1000mL of Pseudomonas stutzeri CGMCC NO:1.202 in the culture medium, centrifuge the fermentation broth to obtain 20g of wet bacteria, add it to 500mL of transformation liquid, and the transformation liquid contains 21g of L-serine hairy acid Hydrolyzate, 26.2g 2-methylindole, 0.2g / L pyridoxal phosphate and 0.01g / L acetone, pH 8.0, 37°C enzymatic reaction for 15h, after the reaction, 2-methyl-L -Tryptophan is 42.9g, and the molar conversion rate to L-serine is 98.5%;

[0034](2) Centrifuge the transformation solution at 5000r / min for 10min, remove the bacterial cells, heat, decolorize with activated carbon, filter with suction, adjust the pH of the filtrate to about 5.5, let it stand for precipitation, filter with vacuum, and dry to obtain 39.9g of 2-methyl - The crude product of L-tryptophan was washed with 95% ethanol, vacuum filtered, and dried to obtain 36.3 g of refined 2-methyl-L-tryp...

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Abstract

The invention discloses an enzymatic conversion preparation method of 2-methyl-L-tryptophan. The bacterial strain with tryptophan synthase activity is cultivated and fermented in a culture medium, and the fermented liquid is centrifuged to obtain tryptophan-containing synthetic Wet cells of enzymes; the above-mentioned wet cells are added to the conversion liquid, which includes: feed liquid containing L-serine, pyridoxal phosphate and 2-methyl indole, ether, methanol, ethanol, acetone One of them; 2-methyl-L-tryptophan is obtained by enzymatic reaction at 20-50°C, pH 5-12, and isoelectric point crystallization. The present invention adopts a specific bacterial strain containing tryptophan synthase, which can efficiently express tryptophan synthase when cultured in a preferred medium, so that the enzymatic synthesis of 2-methyl-L-tryptophan has a higher catalytic rate and The conversion rate, the L-serine molar conversion rate reaches over 98%, the enzymatic synthesis reaction conditions are mild, the enzyme stereoselectivity is strong, the catalytic efficiency is high, and it is suitable for industrial production.

Description

technical field [0001] The present invention relates to a conversion method of 2-methyl-L-tryptophan, in particular to an enzymatic conversion preparation method of 2-methyl-L-tryptophan. Background technique [0002] 2-methyl-L-tryptophan is a non-protein amino acid, L-2-methyl tryptophan is an important intermediate in the synthesis of antibiotic thiostrepton, Chinese invention patent: CN101914605, N-fluorene The synthesis method of methoxycarbonyl-2-methyl tryptophan discloses that the oligopeptide compound containing L-2-methyl tryptophan can make parent body cells (somatotrope) release auxin, and mammals can induce the release of growth hormone After the compound is added, the auxin in the animal body is improved, and the growth rate of the animal is accelerated. 2-Methyl-L-tryptophan can be used in the fields of medicine and food, etc., and has very broad application prospects. [0003] Chinese invention patent: CN101914605, the synthesis method of N-fluorenylmethoxy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/22C12R1/19C12R1/385C12R1/38C12R1/125
Inventor 徐礼生高贵珍卓馨曹稳根赵亮袁维风吴超
Owner 宿州学院