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Process for producing L-methionine, aminoacylase strain and aminoacylase

The technology of aminoacylase and production process is applied in the field of L-methionine production process, aminoacylase strains and aminoacylase, and can solve the problems of difficulty in screening high-yielding strains, unsuitability for industrial production, and high preparation cost, Achieve considerable social and economic benefits, save foreign exchange, and efficiently generate aminoacylase

Inactive Publication Date: 2006-10-11
新疆威仕达实业(集团)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of purification and immobilization of rice koji acylase, the loss of enzyme activity is large, the yield of enzyme is low, and the preparation cost is high
As early as 1957, Japan's Tchiro and Chibata etc. had studied the properties of Escherichia coli acylase, but because the enzyme activity produced by wild strains was very low, it was not suitable for industrial production, and, due to metabolic pathways, etc. It is very difficult to screen high-yield strains, so far, it is impossible to produce L-methionine by microbial fermentation technology at home and abroad

Method used

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  • Process for producing L-methionine, aminoacylase strain and aminoacylase
  • Process for producing L-methionine, aminoacylase strain and aminoacylase
  • Process for producing L-methionine, aminoacylase strain and aminoacylase

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1, the preparation of aminoacylase

[0044] (1) Preparation of highly active aminoacylase

[0045] 1. The genetic characteristics of the genetically engineered strain XW9707 of the present invention:

[0046] Biotin, sodium succinate double deficient, ampicillin resistance (Apr + ), with pALM118 plasmid carrying Arg - L gene.

[0047] 2. Compatibility of medium.

[0048] No. 1 agar medium

[0049]

[0050]

[0051]

[0052] No. 2 Bacterial Medium

[0053] The ingredients are the same as Medium 1, but no agar is needed. The final concentration of ampicillin was 0.05 g / l.

[0054] 3) Sodium Potassium Phosphate Buffer

[0055] 1 mole KH 2 PO 4 pH=6.8-7.0

[0056] Adjust the pH with 10M NaOH solution.

[0057] 4) Borate buffer

[0058] 0.1 mole H 3 BO 3 pH=9.7.

[0059] 5) Cobalt chloride solution

[0060] 0.02 mol CoCl 2 Soluble in distilled water.

[0061] No. 6 Enzyme Suspension Buffer

[0062] Sodium Potassium Phosphate Buffer...

Embodiment 2

[0099] The generating reaction of embodiment 2, L-methionine

[0100] The culture of the CGMCC.No.0368 strain obtained in Example 1 is treated with toluene (2%) for more than 30 minutes to obtain activated aminoacylase, and 1000 units of strain treatment or aminoacylase are added to 1 liter of reaction solution. Acylase, 50g N-acetyl-D, L-methionine, adding sodium hydroxide to adjust the pH to 6.5. The reaction liquid was fully stirred, and the enzymatic hydrolysis reaction was carried out under the condition of 37°C. The enzymolysis time is controlled until the content of L-methionine in the enzymolysis solution no longer increases, about 12-16 hours. Water-insoluble matter was removed by centrifugation, and then concentrated under reduced pressure to 1 / 20 (50 ml) of the original volume. An equal volume (50 ml) of cold absolute ethanol was added to the concentrate and the resulting mixture was left overnight at 4°C. The precipitated crystals were collected by filtration an...

Embodiment 3

[0101] The generating reaction of embodiment 3, L-methionine

[0102] The culture solution obtained in Example 1 was centrifuged to obtain bacterial cells. The bacterial cells were suspended in a phosphate buffer (0.1 M potassium phosphate, sodium, pH 6.8). Treat the suspension with ultrasound or 2% toluene to activate the enzyme activity. Then add the activated suspension to 5 times equal volume of 3% sodium alginate sol and stir evenly, then slowly add it dropwise into 0.5M calcium chloride solution to form solidified pellets, and place it in a refrigerator at 4°C overnight , remove the calcium chloride solution, add water to wash 2-3 times, and then put the immobilized enzyme into a glass tube to make an enzyme column. Then N-acetyl-D, L-methionine solution (50 g / l adjusted to pH 6.5 with sodium hydroxide) was passed through the enzyme column at 37° C. to obtain 20 g / l L-methionine solution. Then concentrate under reduced pressure to 1 / 20 of the original volume, then add...

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Abstract

The invention relates to the process for preparing L-methionine by using escherichia coli gene engineering bacterium (storage number CGMCC No. 0368), the invention also relates to the escherichia coli aminoacylase strain and method for preparation, and aminoacylase originated from the escherichia coli genetic engineering bacterium. The process for preparing the L-methionine comprises using N-acetyl-D, L-methionine as raw material, and using the aminoacylase, cultured thalline or the thalline treatment article as enzyme source for enzymolysis reaction to produce the L-methionine.

Description

Technical field: [0001] The invention relates to a production process of L-methionine, an aminoacylase strain and a preparation method thereof, and an aminoacylase. Specifically, the present invention relates to a production process for producing L-methionine by using Escherichia coli aminoacylase enzymatic hydrolysis reaction, an Escherichia coli aminoacylase strain and a preparation method thereof, and the aminoacylase. Background technique: [0002] Amino acid is the basic unit of protein in organisms and the basic substance for maintaining life. L-methionine is one of the "eight essential amino acids" containing sulfur. It plays an extremely important role in human health and has been widely used in medicine and health. , food additives, daily cosmetics and other fields. The chemical synthesis technology of D, L-methionine (D, L-Met) is very mature, the production cost is relatively low, and the world's annual output can reach more than 200,000 tons. It is mainly used ...

Claims

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Application Information

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IPC IPC(8): C12P13/12C12N1/21C12N9/78
Inventor 魏东杨新平王炜崔卫东石玉瑚穆斯坦帕冯蕾张慧涛李晨华冯怀蓉
Owner 新疆威仕达实业(集团)股份有限公司
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