CN106676157A - Application of legionella thalli in preparing amino acid profile transformation agent - Google Patents
Application of legionella thalli in preparing amino acid profile transformation agent Download PDFInfo
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- CN106676157A CN106676157A CN201710088482.4A CN201710088482A CN106676157A CN 106676157 A CN106676157 A CN 106676157A CN 201710088482 A CN201710088482 A CN 201710088482A CN 106676157 A CN106676157 A CN 106676157A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/12—Methionine; Cysteine; Cystine
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Abstract
The invention discloses an application of legionella thalli in preparing an amino acid profile transformation agent and use thereof and relates to the field of applied microbiology. The application comprises the steps of: nutrition adaptive screening of a strain, bacterial cultivation and proliferation, amino acid profile transformation and product recovery and the like. The agent disclosed by the invention is simple in process, low in cost, high in product yield and free of environmental pollution, so that the amino acid profile transformation agent has a good industrial application prospect.
Description
Technical field
The present invention relates to a kind of be converted into L-type aminoacid using legionella thalline and its metabolite by D-L type aminoacid
Method.
Background technology
In nature, the aminoacid for constituting protein is usually L-configuration.The method of Amino acid synthesis generally includes chemical conjunction
Cheng Fa, microbe fermentation method and enzyme process.Amino acid classes are more obtained by chemical synthesiss, but the racemic modification of synthesis（D-L
Aminoacid）The aminoacid that single configuration could be obtained through Chiral Separation is needed, and this process cost is sufficiently expensive.Fermentation
Method is the ability that a kind of utilization microorganism has various aminoacid needed for synthesis itself, and by fermentation technology the one of aminoacid is prepared
Plant common method.Although the method raw material is cheap, the amino acid products for preparing are single, and purity is not high, have association aminoacid to produce,
And the selection-breeding of fermented bacterium more trouble is not easy to control.Enzyme process be Organic substance is transformed into by various active enzyme needed for
A kind of method of aminoacid.Although this method can obtain the L-type aminoacid of 100 % purity, during inevitably result in the mistake of enzyme
It is living, and enzyme regeneration cost is still expensive.The present invention directly utilizes the legionella thalline wet product through nutrition screening, without the need for fine
Isolate and purify, you can efficiently realize that amino acid racemization body selectivity configuration splits, with biological method chemosynthesis are solved
Method later stage Chiral Separation is difficult, the problem of high cost.
The content of the invention
Technical problem：
To make up, existing Amino acid synthesis technical costss are expensive, and commercial production is worth low deficiency, the invention provides a kind of profit
With legionella thalline wet product as amino acid configuration transferring reagent, the D-L types aminoacid for directly obtaining to chemosynthesis carries out structure
Type is changed, and prepares highly purified l-amino acid.The product purity that the method process is simple, cost are relatively low, obtain is high, possesses wide
Industrial applications prospect.
Technical scheme：
The present inventor according to modern biotechnology theory, with industrial microorganism key technology and modern analysis detection technique, so as to
Complete the present invention.
It is an object of the invention to provide a kind of can be by a kind of amino acid converting biological method for L-type aminoacid of D-L types.Tool
Body technology process is as follows：
1. the condition of culture of antibacterial
The fluid medium of suitable legionella fast-growth is prepared in accordance with the following methods.Weigh 10g N- (2- acetylaminos) -2-
Aminoethane sulphonic acid (N- (2-Acetamido) -2-aminoethanesulfonic acid; ACES buffer)；10g ferment
During female extract (Yeast extract) is dissolved to 800mL distilled waters, pH value is adjusted to 6.9-7.0, high steam goes out
Bacterium, cools down standby.In addition 5g bovin serum albumin (Bovin serum albumin), the Guang ammonia of 0.4g L- half are weighed respectively
Acid hydrochloride (L-cysteine hydrochloride), 0.25g ferrous pyrophosphates (Ferrous pyrophosphate),
In being completely dissolved in 200 mL distilled waters, by the microporous filter membrane sucking filtration in 0.22 μm of aperture it is degerming after, be added to and above-mentioned sterilize
In standby solution, mixing is obtained final product.Flat board used by growth of the bacterium in solid agar adopts BCYE agar plates（Existing business
Product）.
2. the nutrition flexibility screening of strain
By legionella pneumophilia standard strain ATCC33152（Purchased from American Type Culture Collecti）It is inoculated in and is additionally supplemented with 10 %
In the aforesaid liquid culture of D-L type cysteine, in 37 DEG C, 5% CO2Cultivate in incubator, every 7 days, draw 1mL and pass on
Continue to cultivate into fresh above-mentioned culture medium, repeat aforesaid operations more than 10 times, you can obtain and have compared with homoamino acid configuration
The strain of conversion efficiency.The mutant strain is properly preserved in into -70 DEG C of refrigerators standby.
3. antibacterial is passed on and propagation
Above-mentioned operation is supported after the actication of culture of adaptability screening, the fresh training of the liquid without D-L type cysteine is inoculated in
In foster base (described in technique 1), in being placed in 37 DEG C of constant temperature track incubators, the h of 200rmp concussion and cultivates 48, to culture fluid
The middle ammonium sulfate for adding isopyknic 3mol/L so as in albumen or enzyme be deposited to bottom, then it is centrifuged in 10000 rmp
30 min, thalline and albumen therein or enzyme are deposited in ttom of pipe, will precipitate wet body and collect, and are preserved in -70 DEG C of refrigerators
It is standby.
4. amino acid configuration is changed and Product recycling
The racemic modification D-L type cysteine obtained to chemosynthesis is configured to aqueous solution, and it is heavy to be added thereto to above-mentioned wet body
Starch（Comprising the protein ingredient in thalline and metabolite or other compositions）, addition is 100 mg/mL, and solution is placed in
37 DEG C, the h of concussion and cultivate 24 in the shaking table of 200 rmp.Add isopyknic 3 mol/L ammonium sulfate in solution again, 10000
Rmp is centrifuged after 30 min, discards precipitation, and supernatant is processed by ion exchange, can harvest purity up to more than 97%
L-type cysteine.
Beneficial effect：
Racemate resolution method involved in the present invention, compared with prior art with following advantage：
1st, present invention firstly discovers that through legionella pneumophilia ATCC33152 there is D-L types aminoacid to carry out configuration conversion, can make
Standby highly purified l-amino acid.
2nd, method according to the present invention belongs to biological method, without using toxic chemical, to human body and environment almost without
Harm.
3rd, method process is simple according to the present invention, with low cost, is adapted to industrialized production, it is possible to resolve amino acid products valency
The prohibitively expensive problem of lattice.
Present invention is expanded on further by the following examples, and the scope of the present invention is not limited with embodiment.This
It is bright to there are various known replacements in the art or deform, on the premise of without departing from essential meaning of the present invention, at this
Within the protection domain of invention.
Description of the drawings：
Fig. 1：Manage the colonial morphology of the legionella pneumophilia for supporting adaptability screening.
Fig. 2：L-type cysteine electrochemistry differential pulse voltammetry（DPV）Curve.Curve 1：The L-type of 0.125mg/ml concentration
Cysteine solution；Curve 2：With the negative control solution without L-type cysteine that method is prepared.
Fig. 3：L-type cysteine Electrochemical Detection standard curve（Ipmax vs Cmg/ml）.
Fig. 4：Electrochemical method monitors the content of L-type cysteine.
Fig. 5：Nutrition flexibility screens the impact that the frequency changes yield to L-Cysteine.
Specific embodiment
Embodiment 1
Amino acid configuration conversion method according to the present invention is as follows：
1. the condition of culture of antibacterial
The fluid medium of suitable legionella fast-growth is prepared in accordance with the following methods.Weigh 10g N- (2- acetylaminos) -2-
Aminoethane sulphonic acid (N- (2-Acetamido) -2-aminoethanesulfonic acid; ACES buffer)；10g ferment
During female extract (Yeast extract) is dissolved to 800mL distilled waters, pH value is adjusted to 6.9-7.0, high steam goes out
Bacterium, cools down standby.In addition 5g bovin serum albumin (Bovin serum albumin), the Guang ammonia of 0.4g L- half are weighed respectively
Sour (L-cysteine hydrochloride), 0.25g ferrous pyrophosphates (Ferrous pyrophosphate), fully
In being dissolved in 200 mL distilled waters, by the microporous filter membrane sucking filtration in 0.22 μm of aperture it is degerming after, be added to it is above-mentioned sterilized it is standby
Solution in, mixing is obtained final product.
2. the nutrition flexibility screening of strain
By legionella pneumophilia standard strain ATCC33152（Purchased from American Type Culture Collecti）It is inoculated in and is additionally supplemented with 10 %
In the aforesaid liquid culture of D-L type cysteine, in 37 DEG C, 5% CO2Cultivate in incubator, every 7 days, draw 1mL and pass on
Continue to cultivate into fresh above-mentioned culture medium, repeat aforesaid operations more than 10 times, you can obtain and have compared with homoamino acid configuration
The strain of conversion efficiency.The mutant strain is properly preserved in into -70 DEG C of refrigerators standby.
3. antibacterial is passed on and propagation
Above-mentioned operation is supported after the actication of culture of adaptability screening, the fresh training of the liquid without D-L type cysteine is inoculated in
In foster base (described in technique 1), in being placed in 37 DEG C of constant temperature track incubators, the h of 200rmp concussion and cultivates 48, to culture fluid
The middle ammonium sulfate for adding isopyknic 3mol/L so as in albumen or enzyme be deposited to bottom, then it is centrifuged in 10000 rmp
30 min, thalline and albumen therein or enzyme are deposited in ttom of pipe, will precipitate wet body and collect, and are preserved in -70 DEG C of refrigerators
It is standby.
4. amino acid configuration is changed and Product recycling
The racemic modification D-L type cysteine obtained to chemosynthesis is configured to aqueous solution, and it is heavy to be added thereto to above-mentioned wet body
Starch（Comprising the protein ingredient in thalline and metabolite or other compositions）, addition is 100 mg/mL, and solution is placed in
37 DEG C, the h of concussion and cultivate 24 in the shaking table of 200 rmp.Add isopyknic 3 mol/L ammonium sulfate in solution again, 10000
Rmp is centrifuged after 30 min, discards precipitation, and supernatant is processed by ion exchange, can harvest purity up to more than 97%
L-type cysteine.
Embodiment 2
According to above-mentioned technique（Described in technique 2）The nutrition flexibility screening of strain is carried out, the legionella pneumophilia bacterial strain for being obtained exists
Colony growth form on BCYE agar plates, is shown in Fig. 1.
Embodiment 3
The content of L-Cysteine in electrochemical methods monitoring transformation process
1. electrochemical behavior of the L-type cysteine on gold disc electrode
1.1. experimental technique：Prepare certain density L-type cysteine, qualitative detection its in three-electrode system（Working electrode：
Gold disc electrode φ=5mm；Reference electrode：Saturated calomel electrode；To electrode：Platinum filament）In electrochemical behavior.
1.2. result and conclusion：See Fig. 2, DPV results show that L-Cysteine there are electrochemical signals on gold disc electrode surface
Response, therefore be expected to build the content quantitative analysis method of the material.
Type cysteine content bioassay standard curve
2.1. experimental technique：0.5,0.25,0.125,0.0625,0.0313 is prepared respectively .... wait the L-type of 2 times of dilutions
Aqueous cystein solution, carries out according to the method described above electrochemical gaging, and records maximum peak current（Ipmax）Value.With material concentration
For abscissa, Ipmax For vertical coordinate, standard curve is made.
2.2. result and conclusion：Fig. 3 is seen, by standard curve it can be seen that Ipmax It is in line with the concentration of L-type cysteine
Sexual intercourse.This method may be used to the changes of contents of L-type substance cysteine in detection sample.
According to electrochemistry DPV IpmaxThe changes of contents of L-type cysteine in value characterization processes
3.1. experimental technique：According to technique, the racemic modification D-L type cysteine that chemosynthesis are obtained is configured to into aqueous solution,
And the mg/mL of wet body 100 described in technique thereto, 37 DEG C are placed in, the h of concussion and cultivate 24 in the shaking table of 200 rmp.When different
Between section is separately sampled carries out the detection of electrochemistry DPV, the Ip that detection is obtainedmaxValue
Substitute into linear equation：Ipmax=32.468C+0.37642;Calculate the Guang ammonia of L-type half of differential responses time point in solution
The content of acid.
3.2. result and conclusion：See Fig. 4, during transformation experiment, the content of L-type substance cysteine being continuously increased,
Indicate DL type cysteine and be gradually transforming into single L-type conformation, after reaching 18 hours, substantially without increase, show conversion
Reaction is basically completed.
Embodiment 4
The nutrition flexibility screening experiment of strain changes the impact of yield to L-Cysteine
3.1. experimental technique：According to technique 2, by difference pass on the screening frequency (0,1,2...10,11,12) harvest bacterium respectively
Kind, then according to technique 3 and 4 carries out aminoacid transition experiment, further according to the method needed for example containing for L-type cysteine is calculated
Amount, 13 batches of the frequency are passed on by being calculated difference（Batch）The yield of product（Yield）Percentage ratio.
3.2. result and conclusion：Fig. 5 is seen, by curve as can be seen that the yield percent of L-type cysteine is with passing on
The increase of the frequency and increase, reach after passing on for 10 times, yield percent reaches higher level, and subsequent increasing degree is very low.
Therefore, process choice passes at least 10 times.
Claims (3)
1. legionella thalline is used as the application for preparing amino acid configuration transferring reagent, it is characterised in that legionella thalline is used as one
Amino acid configuration transferring reagent is planted, D-L type aminoacid is completely converted into into L-type aminoacid；Wherein described legionella thalline is
Legionella pneumophilia ATCC33152.
2. used as the application for preparing amino acid configuration transferring reagent, its feature exists legionella thalline as claimed in claim 1
In described legionella thalline is bacterial strain Jing after Secondary Culture, the wet product obtained after centrifugation.
3. legionella thalline wet product as claimed in claim 2, it is characterised in that it includes legionella thalline, enzyme, albumen, core
The composition produced during acid and growth metabolism.
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| CN201710088482.4A CN106676157A (en) | 2017-02-20 | 2017-02-20 | Application of legionella thalli in preparing amino acid profile transformation agent |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0346115B2 (en) * | 1982-06-10 | 1991-07-15 | Ajinomoto Kk | |
| CN1592789A (en) * | 2001-11-23 | 2005-03-09 | Dsmip财产有限公司 | Process for the preparation of an enantiomerically enriched alpha-amino acid |
| CN101974605A (en) * | 2010-11-12 | 2011-02-16 | 南开大学 | Method for preparing D-cystine by using microorganism enzymic method to split DL-cysteine |
| CN102417900A (en) * | 2011-11-17 | 2012-04-18 | 天津启仁医药科技有限公司 | ATC racemase and coding gene thereof, and application of recombinant expression protein thereof |
-
2017
- 2017-02-20 CN CN201710088482.4A patent/CN106676157A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0346115B2 (en) * | 1982-06-10 | 1991-07-15 | Ajinomoto Kk | |
| CN1592789A (en) * | 2001-11-23 | 2005-03-09 | Dsmip财产有限公司 | Process for the preparation of an enantiomerically enriched alpha-amino acid |
| CN101974605A (en) * | 2010-11-12 | 2011-02-16 | 南开大学 | Method for preparing D-cystine by using microorganism enzymic method to split DL-cysteine |
| CN102417900A (en) * | 2011-11-17 | 2012-04-18 | 天津启仁医药科技有限公司 | ATC racemase and coding gene thereof, and application of recombinant expression protein thereof |
Non-Patent Citations (5)
| Title |
|---|
| EWANN FANNY等: "Cysteine Metabolism in Legionella pneumophila:Characterization of an L-Cystine-Utilizing Mutant", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
| PRICE CHRISTOPHER T. D.等: "Amoeba host-Legionella synchronization of amino acid auxotrophy and its role in bacterial adaptation and pathogenic evolution", 《ENVIRONMENTAL MICROBIOLOGY》 * |
| WARREN WJ等: "Growth of Legionnaires Disease Bacterium (Legionella pneumophila) in Chemically Defined Medium", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| 胡建强等: "氨基酸手性拆分研究进展", 《食品与药品》 * |
| 高吉: "氨肽酶B酶法拆分DL-氨基酸研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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Application publication date: 20170517 |