CN1431295A - Method for producing D-a amino acid series by using De's bacterium of Bakehuo in onions - Google Patents
Method for producing D-a amino acid series by using De's bacterium of Bakehuo in onions Download PDFInfo
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- CN1431295A CN1431295A CN 02151745 CN02151745A CN1431295A CN 1431295 A CN1431295 A CN 1431295A CN 02151745 CN02151745 CN 02151745 CN 02151745 A CN02151745 A CN 02151745A CN 1431295 A CN1431295 A CN 1431295A
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- kehuoshi
- amino acid
- cepecia
- burkholderia
- moral salmonella
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Abstract
A process for preparing the optically active D-alpha-amino acid series (D-Phe, D-Trap, D-Val, D-methionie, etc) from 5-substituted hydrantoin features use of Burkholderia cepecia JS-02 (CCTCC No.M202047) as splitting catalyst, which is obtained from soil. Its advantages are high output rate and high concentration of substrate.
Description
Technical field
D-a-amino acid is that a class has optically active compound, has been widely used at medicine, food and pesticide field.As D-phenylalanine, D-tryptophane, D-tyrosine and D-methionine(Met) is the important component of synthetic polypeptide medicaments; The D-L-Ala is the key ingredient of synthetic sweetener alitame; The D-Xie Ansuan is the important component of preparation optical activity sterilant etc.
Background technology
The present synthetic method of this class material adopts the chemical method for splitting of raceme usually.With the D-phenylalanine is example, the preparation method who adopts is at present: at first prepare the DL-phenylalanine through chemosynthesis, utilize the L-Aminoacylase method to split and obtain L-phenylalanine and N-acetyl-D-phenylalanine, obtain the D-phenylalanine through separating the N-acetyl-D-phenylalanine low temperature hydrolysis under the diluted acid effect that obtains; Other has the bibliographical information that adopts method of asymmetric synthesis to prepare the D-phenylalanine.But the low or used transition-metal catalyst of above preparation method or complex process and preparation yield costs an arm and a leg, and causes the preparation cost of D-phenylalanine can not satisfy application need.
In recent years, along with the progress of drug research, peptide medicament has become the focus in drug research field gradually, and D-a-amino acid becomes the restricted component of peptide medicament preparation owing to its unique metabolic characteristic; The development of food and agricultural chemicals has simultaneously also driven the demand of D-amino acid in foodstuff additive and chirality agricultural chemicals are synthetic.The amino acid whose low cost of D-, high yield technology of preparing become amino acid whose research emphasis.
Summary of the invention
The bacterial classification that the purpose of this invention is to provide the microorganism Bai Kehuoshi De Shi genus that a kind of utilization is screened and get from soil, the katalysis by Institute of Micro-biology produces enzyme replaces the glycolylurea compound with serial DL-5 and is converted into D-a-amino acids material, that is:
(R represents alkyl, phenyl, benzyl, indyl or other substituting groups)
Purpose of the present invention can reach by following measure:
Be used for the present invention and carry out microorganism onion bulkholderia cepasea (Burkholderia cepecia) JS-02 (being designated hereinafter simply as the JS-02 bacterial strain) that D-amino acid is produced, it has the ability that produces D-glycolylurea enzyme and N-carboxamide-D-amino acid lytic enzyme, can be used for the present invention and produce D-amino acid.This bacterial strain separated in the soil of Nanjing, China Jiangsu Province area and gets in calendar year 2001, it was identified through institute of microbiology of the Chinese Academy of Sciences on September 6th, 2002, be deposited in Chinese typical culture collection center then on December 16th, 2002, it abbreviates CCTCC as, and deposit number is CCTCCNO.M202047.
The JS-02 bacterial strain has following character
1, morphological specificity
On the ordinary nutrient agar substratum in 37 ℃ down cultivate 24 hours after, observe the form of mycelia and spore shape, surface tissue respectively with opticmicroscope and electron microscope.The result shows that this bacterium presents shaft-like, does not form gemma, has end to give birth to flagellum, is generally the 1-2 bar, can move.
2, physiological and biochemical property
(1) gelatine liquefication, feminine gender
(2) starch hydrolysis, feminine gender
(3) nitrate reduction, feminine gender
(4) nitrite reduction, feminine gender
(5) denitrification, feminine gender
(6) fluorochrome, feminine gender
(7) water-soluble non-fluorescent colour element, feminine gender
(8) strict aerobic, the positive
(9) amphimicrobian, feminine gender
(10) catalase, the positive
(11) oxydase, the positive
(12) PHB, the positive
(13) methyl red test, feminine gender
(14) VP test, feminine gender
(15) arginine dihydrolase, feminine gender
(16) O/F test, oxidation
3, utilization of carbon source
The cultivation of microbes producing cellulase JS-02 bacterial strain: with monose or disaccharide as carbon source, as glucose, sorbyl alcohol, sucrose or lactose.Starch, alcohols and organic acid utilization are out of condition; As the nitrogenous source organic nitrogen source is good, and what effect was best is corn steep liquor and yeast extract paste; Co
2+To producing enzyme obvious facilitation is arranged.The enzyme that the JS-02 bacterial strain is produced is an inducible enzyme, and fermenting process must add the N-carboxamide phenylalanine of 0.1-0.5% as inductor; This bacterium is aerobic cultivation bacterium, under the aeration condition of 25-40 ℃ of pH5-9 temperature, and well-grown.
Use liquid culture when fermentation, general fermentation period was at 20-30 hour, and after the fermentation ends, centrifugal collection thalline is as enzyme process synthetic catalyzer.
In the enzyme process reaction process, DL-5 replaces glycolylurea asymmetric hydrolysis under the effect of enzyme, wherein only has 5 substituting group glycolylureas of D type that hydrolysis reaction can take place, and reaction produces D-a-amino acid.5 substituting group glycolylureas of L-type under reaction conditions automatically racemization promoted the process of hydrolysis reaction, formed hydrolysis, racemization, the reaction sequence of hydrolysis again, till reaction substrate is by complete hydrolysis.In reaction process, be the restraining effect of anti-block simultaneously, in reaction process, feed nitrogen and protect enzyme;
In the enzyme reaction process, the initial concentration that DL-5 replaces glycolylurea is 2-5%, pH7-10; Temperature of reaction 25-40 ℃; Reaction times is 20-75 hour.
After reaction finishes, centrifugal removal thalline and impurity, 55-60 ℃ of condensing crystal obtains the D-coarse amino acid under reduced pressure then, and recrystallization obtains the amino acid elaboration.
This preparation method's technology is simple, cost is low, product specific optical rotation height, and has good versatility.
Embodiment
Embodiment one:
Get glucose 20.0g, corn steep liquor 15.0g, NaCl3.0g, MgSO
47H
2O0.5g, KH
2PO
41.0g, CoCl
20.1g, N-carboxamide phenylalanine 1.5g, add water 1000ml, transfer PH7.0-7.2, the dissolving back is divided in the 250ml triangular flask of packing into, every bottled 50ml then adds 8 layers of gauze, after wrapping with kraft paper, 0.1MPa high pressure steam sterilization 25 minutes is standby.
With the bacterial classification of preserving in the refrigerator, after the taking-up, be connected on the fresh inclined-plane, cultivated 24 hours, insert in the above-mentioned liquid nutrient medium that makes, 32 ℃ of 180 rev/mins of shaking table shaking culture are after 24 hours, centrifugal 25 minutes of 5000 rev/mins of whizzers, collect thalline, make bacteria suspension (every 100ml bacteria suspension contains about wet thallus 40 grams).
Get DL-5-benzyl glycolylurea 3.0g and pack in the ready 500ml triangular flask, and add deionized water 100ml respectively, transfer PH9.0, in each triangular flask, add bacteria suspension 10ml then.After adding the plug jam-pack with nitrogen after with the replacement of oxygen in the triangular flask space, concussion reaction on shaking table.Reaction times was respectively 48 hours.Reaction finishes the back on 4000 rev/mins of whizzers centrifugal 30 minutes, removes thalline and impurity, and vacuum decompression 55-60 ℃ concentrates.After the alcohol washing, get D-phenylalanine 2.21g after the filtering for crystallizing oven dry, purity is 97.8%;
Embodiment two:
Substratum composition and conditions for sterilization are with example one.Spawn culture inserts after 14 hours in the liquid nutrient medium that makes, and 30 ℃ of 180 rev/mins of shaking table shaking culture are after 30 hours, and centrifugal 25 minutes of 5000 rev/mins of whizzers are collected thalline, make bacteria suspension (every 100ml bacteria suspension contains about wet thallus 40 grams).
Get DL-5-indole methyl glycolylurea 4.0g and pack in the ready 500ml triangular flask, and add deionized water 100ml respectively, transfer PH9.0, in each triangular flask, add bacteria suspension 10ml then.After adding the plug jam-pack with nitrogen after with the replacement of oxygen in the triangular flask space, concussion reaction on shaking table.Reaction times was respectively 42 hours.Reaction finishes the back on 4000 rev/mins of whizzers centrifugal 30 minutes, removes thalline and impurity, and vacuum decompression 55-60 ℃ concentrates.After the alcohol washing, get D-tryptophane 2.71g after the filtering for crystallizing oven dry, purity is 95.8%;
Embodiment three:
Substratum composition and conditions for sterilization are with example one.Spawn culture inserts after 18 hours in the liquid nutrient medium that makes, and 32 ℃ of 180 rev/mins of shaking table shaking culture are after 30 hours, and centrifugal 30 minutes of 5000 rev/mins of whizzers are collected thalline, make bacteria suspension (every 100ml bacteria suspension contains about wet thallus 40 grams).
Get DL-5-methylthio ethyl glycolylurea 3.0g and pack in the ready 500ml triangular flask, and add deionized water 100ml respectively, transfer PH9.0 in each triangular flask, to add bacteria suspension 10ml then.After adding the plug jam-pack with nitrogen after with the replacement of oxygen in the triangular flask space, concussion reaction on shaking table.Reaction times was respectively 36 hours.Reaction finishes the back on 4000 rev/mins of whizzers centrifugal 25 minutes, removes thalline and impurity, and vacuum decompression 55-60 ℃ concentrates.After the alcohol washing, to filter and get D-methionine(Met) 2.42g after the collection crystallization is dried, purity is 97.7%
Embodiment four:
Substratum composition and conditions for sterilization are with example one.Spawn culture inserts after 18 hours in the liquid nutrient medium that makes, and 32 ℃ of 180 rev/mins of shaking table shaking culture are after 30 hours, and centrifugal 25 minutes of 5000 rev/mins of whizzers are collected thalline, make bacteria suspension (every 100ml bacteria suspension contains about wet thallus 40 grams).
Get DL-5-sec.-propyl glycolylurea glycolylurea 3.0g and be respectively charged in the ready 500ml triangular flask, and add deionized water 100ml respectively, in each triangular flask, add bacteria suspension 10ml behind the accent PH9.0.After adding the plug jam-pack with nitrogen after with the replacement of oxygen in the triangular flask space, concussion reaction on shaking table.Reaction times was respectively 72 hours.Reaction finishes the back on 4000 rev/mins of whizzers centrifugal 25 minutes, removes thalline and impurity, and vacuum decompression 55-60 ℃ concentrates.After the alcohol washing, to filter and get D-Xie Ansuan 1.86g after the collection crystallization is dried, purity is 96.5%
Claims (3)
1. onion Bai Kehuoshi moral Salmonella (Burkholderia cepecia) JS-02, it is in China's typical culture collection center preservation, and its deposit number is: CCTCC NO.M202047.
2. the cultural method of an onion Bai Kehuoshi moral Salmonella (Burkholderia cepecia) JS-02, it is characterized in that in the fermentation culture process of onion Bai Kehuoshi moral Salmonella (Burkholderia cepecia) JS-02, with monose or disaccharide as carbon source, organic nitrogen source is a nitrogenous source, adds inductor N-carboxamide phenylalanine and growth stimulant metal ion Co
2+
3. one kind is utilized onion Bai Kehuoshi moral Salmonella (Burkholderia cepecia) JS-02 to prepare the amino acid whose method of serial D-a-, it is characterized in that utilizing onion Bai Kehuoshi moral Salmonella (Burkholderia cepecia) JS-02, produce the katalysis of enzyme by Institute of Micro-biology, serial DL-5 is replaced the glycolylurea compound be converted into D-a-amino acids material, wherein, DL-5 replaces the glycolylurea compound as reaction substrate, and its concentration is at 2-5%; Be reflected in the aqueous solution and carry out; Be reflected under the condition of inflated with nitrogen protection and carry out.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021517452A CN1164736C (en) | 2002-12-30 | 2002-12-30 | Method for producing D-a amino acid series by using De's bacterium of Bakehuo in onions |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021517452A CN1164736C (en) | 2002-12-30 | 2002-12-30 | Method for producing D-a amino acid series by using De's bacterium of Bakehuo in onions |
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| CN1431295A true CN1431295A (en) | 2003-07-23 |
| CN1164736C CN1164736C (en) | 2004-09-01 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104862B (en) * | 2007-06-06 | 2010-05-19 | 河北科技大学 | Method for synthesizing D-arylglycine by using heterogeneous enzyme to catalytically hydrolyzing 5-arylhydantoin |
-
2002
- 2002-12-30 CN CNB021517452A patent/CN1164736C/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101104862B (en) * | 2007-06-06 | 2010-05-19 | 河北科技大学 | Method for synthesizing D-arylglycine by using heterogeneous enzyme to catalytically hydrolyzing 5-arylhydantoin |
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| CN1164736C (en) | 2004-09-01 |
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Granted publication date: 20040901 Termination date: 20101230 |