CN1300304C - 5-amino-4-carbamyl imidazole nucleoside producing bacteria and preparing method thereof - Google Patents
5-amino-4-carbamyl imidazole nucleoside producing bacteria and preparing method thereof Download PDFInfo
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- CN1300304C CN1300304C CNB2005100344043A CN200510034404A CN1300304C CN 1300304 C CN1300304 C CN 1300304C CN B2005100344043 A CNB2005100344043 A CN B2005100344043A CN 200510034404 A CN200510034404 A CN 200510034404A CN 1300304 C CN1300304 C CN 1300304C
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Abstract
The present invention provides a 5-amino-4-carbamyl imidazole nucleoside producing bacterium and a method for preparing 5-amino-4-carbamyl imidazole nucleoside. The microbial bacterial strain arranged in the present invention is withered grass gemma bacillus XG15 CCTCC M 205040 after breeding. The bacterial strain is used as fermenting bacterial strain, and saccharine materials are used for fermentation, wherein the fermentation for 50 to 80 hours by swinging a bottle can produce 10-20 g/l of 5-amino-4-carbamyl imidazole nucleoside. The present invention provides a feasible method for preparing 5-amino-4-carbamyl imidazole nucleoside (AICAR) by industrial fermentation.
Description
Technical field
The present invention relates to the microorganism strains of a kind of 5-of generation amino-4-carbamyl imidazole nucleoside and utilize this strain fermentation to produce the method for 5-amino-4-carbamyl imidazole nucleoside (hereinafter to be referred as AICAR).
Background technology
Guanylic acid of Xiao Shouing and sodium salt sodium guanylate thereof have the effect that promotes leukocytic hyperplasia, promotes vascular smooth muscle diastole and activation liver function to human body in the market, be used for the leukopenia that a variety of causes causes clinically, also can be used for acute, chronic hepatitis and toxic hepatitis, simultaneously, sodium guanylate mixes the market demand of I+G (Sodium Inosinate+sodium guanylate) of the food freshener made from Sodium Inosinate increasing, and guanylic acid generally is to be made by the organic chemistry synthetic method by 5-amino-4-carbamyl imidazole nucleoside (AICAR); Simultaneously, AICAR also can be used as the raw material of Synthetic 2-mercaptoinosine and isoprinosine, and 2-mercaptoinosine and isoprinosine have the effect that strengthens animal immune as fodder additives, and consumption is increasing at present; But present 5-amino-4-carbamyl imidazole nucleoside (AICAR) is to be produced by chemical synthesis by various organic materialss, and there are shortcomings such as cost pollutent higher, big for environment pollution and that produce is difficult in this method.
Summary of the invention
The purpose of this invention is to provide a kind of 5-amino-4-carbamyl imidazole nucleoside and produce bacterium and adopt this strain fermentation to prepare the method for 5-amino-4-carbamyl imidazole nucleoside, to avoid producing 5-amino-existing problem of 4-carbamyl imidazole nucleoside (AICAR) with chemical synthesis.
5-amino provided by the present invention-4-carbamyl imidazole nucleoside generation bacterium is subtilis (Bacillussubstilis) XG15 through seed selection, and this bacterial strain is preserved in Chinese typical culture collection center (CCTCC), is numbered: CCTCC M205040.This bacterial strain is hereinafter to be referred as subtilis XG15 CCTCC M 205040.
Bacterial strain provided by the present invention is that to produce bacterium GMI-741 with the Bacillus subtilus inosine be starting strain, multiplefactor mutagenic and breedings such as employing thalline nitrosoguanidine, ethyl sulfate, ultraviolet ray, lithium chloride go out a strain, have subtilis (Bacillus substilis) XG15 of adenine deaminase disappearance, non-accurate purine defective type, this bacterial strain shake flask fermentation can produce 5-amino-4-carbamyl imidazole nucleoside (AICAR) 20.3g/L in 72 hours.
This subtilis XG15 CCTCC M 205040 on the preservation inclined-plane 28～34 ℃ cultivated 12～18 hours, the preservation slant culture is creamy white, its suitable culture condition is: 1. culture (meat soup plate or preservation slant medium with): peptone 3～6g/l, extractum carnis 10～18g/l, yeast extract paste 5～10g/l, agar 15～25g/l.2. culture temperature: 25 ℃～35 ℃.Culture pH value 6.5～7.5; In the liquid activation medium, 28 ℃～34 ℃, to cultivate 12～18 hours, every milliliter of bacterium number is 2.0 * 10
9More than, the about 6-8 of pH value, there is the oyster white bacterial sediment static for some time of the liquid after the cultivation, its suitable growth condition is: 1. culture: glucose 10～30g/l, urea 3～6g/l, corn steep liquor 4～10g/l, peptone 5～15g/l, yeast extract paste 5～15, guanine 0.010～0.040.2. culture temperature is 25～35 ℃.3. cultivate pH value 6.0～8.0.4. cultural method: the inclined-plane seed of cultivating 18-24 hour inserts in the 500ml triangular flask that 20ml liquid activation medium is housed (0.07Mpa sterilize 10min.), and 32 ℃ in reciprocating type cradle vibrate cultivation 12-20 hour.
The preparation method of 5-amino provided by the present invention-4-carbamyl imidazole nucleoside is characterized in doing fermentation strain with subtilis XG15CCTCC M 205040, utilizes saccharine material to ferment.Said saccharine material can be glucose, starch double-enzyme hydrolysis sugar (with saccharifying enzyme and α-Dian Fenmeishuixie) etc., shake flask fermentation 50-80 hour, can produce 5-amino-4-carbamyl imidazole nucleoside (AICAR) 10-20g/l.It is produced the AICAR nucleosides for industrial fermentation a kind of feasible method is provided.
It is as follows to utilize this subtilis XG15 CCTCC M 205040 shake flask fermentations to prepare the specific embodiment of 5-amino-4-carbamyl imidazole nucleoside (AICAR):
(1) slant strains activation: cultured preservation subtilis (Bacillus substilis) XG15 CCTCC M205040 inclined-plane kind is lined on the activated inclined plane of new preparation and carry out actication of culture.Described activated inclined plane is composed as follows: glucose 10～30g/l, urea 3～6g/l, corn steep liquor 4～10g/l, peptone 5～15g/l, yeast extract paste 5～15, guanine 0.010～0.040, agar 20-25g/l, pH6.5-7.5.Cultivated 18-24 hour for 28-34 ℃.
The cultured preservation inclined-plane kind preservation time is the longest above half a year.
(2) liquid of bacterial classification activation: cultured activated inclined plane kind is connect encircle in the liquid activation medium, make the bacterium number of liquid strain reach 2.0 * 10
9Individual/more than the ml.Described liquid activation medium is composed as follows: glucose 10～30g/l, urea 3～6g/l, corn steep liquor 4～10g/l, peptone 5～15g/l, yeast extract paste 5～15, guanine 0.010～0.040, initial pH6.5-7.5.The liquid activation medium is loaded in the 250ml triangular flask, and the triangular flask loading amount is 20ml, and in 28-34 ℃ of reciprocating type shaking table shaking culture 12-18 hour, shaking speed was 80-110r/m, and stroke is 65-75mm.The bacterium number of liquid strain can reach 2.0 * 10
9Individual/more than the ml.
(3) shake flask fermentation: by volume per-cent is 2～10% inoculum sizes, inserts in the saccharine material fermention medium, in reciprocating type shaking table shaking culture, treats that residual sugar drops to 3g/l when following, stops to ferment.The available 500ml triangle of described fermention medium (forming with the liquid activation medium) is bottled, and loading amount 20ml is in 32-38 ℃, stroke 65-80mm, the reciprocating type shaking table shaking culture of rotating speed 90-130r/m 60-80 hour treats that residual sugar drops to 3g/l when following, stops fermentation.
Suitable fermention medium composition following (calculating): sugar 100～140 by every liter of contained grammes per square metre g/l of substratum, corn steep liquor 10～20, yeast powder 15～20, ammonium sulfate 8～15, potassium primary phosphate 1～3, lime carbonate 10～30, urea 2～6, sal epsom 0.5～1.5, pH6.5～7.5, wherein lime carbonate is weighed into separately in the triangular flask, and urea is sterilization separately.
(4) the shake flask fermentation mash that fermentation is finished is centrifugal, gets supernatant liquor, promptly gets to contain 5-amino-4-carbamyl imidazole nucleoside fermentation liquid.
To containing 5-amino-4-carbamyl imidazole nucleoside fermentation liquid, can adopt following ply of paper to analyse and analyze AICAR nucleosides content: after shaking bottle under the fermented liquid, with the whizzer of 4000r/m rotating speed centrifugal 10 minutes, with No. 3 chromatographic papers of 50ul microsyringe point, ammonium sulfate is made developping agent, exhibition layer 5～10 hours, oven dry, with the uv analyzer AICAR nucleosides spot of breaking forth, cut spot, soaked 1-4 hour with the HCl of 0.001mmol/l.With 752 ultraviolet spectrophotometers in 260
NmThe wavelength place measures ultraviolet absorption value, calculates fermentation liquid and contains AICAR nucleosides amount.
Maximum characteristics are the nucleosides with Production by Microorganism Fermentation AICAR among the present invention, more in the past than the cost of the chemical synthesis that adopted usually bigger reduction are arranged, and little to the pollution of environment; The present invention produces the AICAR nucleosides and reaches as high as 20.3g/l under best shake flask fermentation processing condition.
Embodiment
Embodiment one:
Preservation subtilis (Bacillus substilis) the XG15 CCTCC M 205040 inclined-plane kinds [composition of preservation slant medium (g/l): peptone 4, extractum carnis 14, yeast extract paste 7, guanine 0.05, agar 20, pH7.0 with preservation in 4 ℃ of refrigerators.], being transferred on the activated inclined plane substratum of new configuration, (g/l) is as follows for the composition of activated inclined plane substratum: glucose 20, urea 4.0, corn steep liquor 6, peptone 10, yeast extract paste 10, guanine 0.025, agar 20, pH7.0., connect one and encircle in the liquid activation medium after 20 hours through 32 ℃ of constant temperature culture, (g/l) is as follows for the composition of liquid activation medium: glucose 20, urea 4, corn steep liquor 6, peptone 10, yeast extract paste 10, guanine 0.025, pH7.0.The liquid activation culture was based on 32 ℃ of shaking baths (stroke 70mm, rotating speed 96r/m) shaking culture 15 hours, at this moment, (20 times of bacterium liquid dilutions are with 721 spectrophotometer 650nm wavelength, 1cm cuvette for the general 0D of bacterial concentration, aquae destillata is made reference, measures absorbance value) reach more than 0.7.Get 1ml bacterium liquid (5% inoculum size), insert in the fermention medium, (g/l) is as follows for the composition of fermention medium: glucose 110, corn steep liquor 12, yeast powder 18, ammonium sulfate 10, potassium primary phosphate 1, sal epsom 1, urea 5 (separately sterilization), CaCO
320 (being weighed into earlier in the sky triangular flask) MgSO
47H
2O1, pH7.0.
Fermention medium is loaded in the 500ml triangular flask, loading amount 20ml, and in 76mm stroke, the reciprocating type shaking table shaking culture of rotating speed 120r/m, temperature control mode is as follows: 0～12 hour, 32 ℃; 12～24 hours, 33 ℃; 24～36 hours, 34 ℃; 36～48 hours, 35 ℃; 48～61 hours, 36 ℃.After shake flask fermentation finished, fermented liquid was measured AICAR nucleosides content with Paper Chromatography.Adopt this method shake flask fermentation 72h to produce the AICAR nucleosides and can reach 20.3g/l.
Embodiment two:
With preservation subtilis (Bacillus substilis) the XG15 CCTCC M 205040 inclined-plane kinds of preservation in 4 ℃ of refrigerators [composition of preservation slant medium (g/l): peptone 3, extractum carnis 10, yeast extract paste 5, guanine 0.03, agar 20, pH 6.7.], being transferred on the activated inclined plane substratum of new configuration, (g/l) is as follows for the composition of activated inclined plane substratum: glucose 10, urea 3.0, corn steep liquor 4, peptone 5, yeast extract paste 5, guanine 0.001, agar 20, pH6.7., connect one and encircle in the liquid activation medium after 14 hours through 32 ℃ of constant temperature culture, (g/l) is as follows for the composition of liquid activation medium: glucose 10, urea 3, corn steep liquor 4, peptone 5, yeast extract paste 5, guanine 0.001, pH6.7.The liquid activation culture was based on 30 ℃ of shaking baths (stroke 65mm, rotating speed 80r/m) shaking culture 12 hours, after the cultivation, get 1ml bacterium liquid (5% inoculum size), insert in the fermention medium, (g/l) is as follows for the composition of fermention medium: glucose 100, corn steep liquor 10, yeast powder 15, ammonium sulfate 8, potassium primary phosphate 1, sal epsom 0.5, urea 2 (separately sterilization), CaCO
310 (being weighed into earlier in the sky triangular flask) MgSO
47H
2O 0.5, pH6.5.
Fermention medium is loaded in the 500ml triangular flask, and loading amount 20ml is in 65mm stroke, the reciprocating type shaking table shaking culture of rotating speed 90r/m, 32 ℃ of temperature control modes.After shake flask fermentation finished, fermented liquid was measured AICAR nucleosides content with Paper Chromatography.Adopt this method shake flask fermentation 60h to produce AICAR nucleosides 8.5g/l.
Embodiment three:
With preservation subtilis (Bacillus substilis) the XG15 CCTCC M 205040 inclined-plane kinds of preservation in 4 ℃ of refrigerators [composition of preservation slant medium (g/l): peptone 6, extractum carnis 18, yeast extract paste 10, agar 25, pH 7.5.], being transferred on the activated inclined plane substratum of new configuration, (g/l) is as follows for the composition of activated inclined plane substratum: glucose 30, urea 6.0, corn steep liquor 10, peptone 15, yeast extract paste 15, guanine 0.04, agar 25, pH7.5., connect one and encircle in the liquid activation medium after 24 hours through 34 ℃ of constant temperature culture, (g/l) is as follows for the composition of liquid activation medium: glucose 30, urea 6.0, corn steep liquor 10, peptone 15, yeast extract paste 15, guanine 0.04, pH7.5.The liquid activation culture was based on 34 ℃ of shaking baths (stroke 75mm, rotating speed 110r/m) shaking culture 18 hours, after the cultivation, get 1ml bacterium liquid (5% inoculum size), insert in the fermention medium, (g/l) is as follows for the composition of fermention medium: glucose 140, corn steep liquor 20, yeast powder 20, ammonium sulfate 15, potassium primary phosphate 3, sal epsom 1.5, urea 6 (separately sterilization), CaCO
330 (being weighed into earlier in the sky triangular flask) MgSO
47H
2O 1.5, pH7.5.
Fermention medium is loaded in the 500ml triangular flask, and loading amount 20ml is in 80mm stroke, the reciprocating type shaking table shaking culture of rotating speed 130r/m, 38 ℃ of temperature control modes.After shake flask fermentation finished, fermented liquid was measured AICAR nucleosides content with Paper Chromatography.Adopt this method shake flask fermentation 80h to produce AICAR nucleosides 18.1g/l.
Claims (7)
1. the generation bacterium of 5-amino-4-carbamyl imidazole nucleoside is characterized in that this microorganism strains is subtilis (Bacillus substilis) the XG15 CCTCC M 205040 through seed selection.
2. the preparation method of 5-amino-4-carbamyl imidazole nucleoside is characterized in that doing fermentation strain with subtilis XG15CCTCCM 205040, utilizes saccharine material to carry out shake flask fermentation.
3. the preparation method of 5-amino according to claim 2-4-carbamyl imidazole nucleoside is characterized in that said saccharine material is glucose or starch double-enzyme hydrolysis sugar, shake flask fermentation 50-80 hour.
4. the preparation method of 5-amino according to claim 2-4-carbamyl imidazole nucleoside is characterized in that the technological process of said shake flask fermentation production 5-amino-4-carbamyl imidazole nucleoside is as follows:
(1) slant strains activation: cultured preservation subtilis XG15 CCTCC M 205040 inclined-plane kinds are lined on the activated inclined plane of new preparation and carry out actication of culture;
(2) liquid of bacterial classification activation: cultured activated inclined plane kind is connect encircle in the liquid activation medium, make the bacterium number of liquid strain reach 2.0 * 10
9Individual/more than the ml;
(3) shake flask fermentation: by volume per-cent is 2～10% inoculum sizes, inserts in the saccharine material fermention medium, in reciprocating type shaking table shaking culture, treats that residual sugar drops to 3g/l when following, stops to ferment;
(4) the shake flask fermentation mash that fermentation is finished is centrifugal, gets supernatant liquor, promptly gets to contain 5-amino-4-carbamyl imidazole nucleoside fermentation liquid.
5. the preparation method of 5-amino according to claim 4-4-carbamyl imidazole nucleoside, when it is characterized in that carrying out the slant strains activation, activated inclined plane consists of: glucose 10～30g/l, urea 3～6g/l, corn steep liquor 4～10g/l, peptone 5～15g/l, yeast extract paste 5～15g/l, guanine 0.010～0.040g/l, agar 20-25g/l, pH6.5-7.5.
6. the preparation method of 5-amino according to claim 4-4-carbamyl imidazole nucleoside, when it is characterized in that carrying out the liquid activation of bacterial classification, the liquid activation medium consists of: glucose 10～30g/l, urea 3～6g/l, corn steep liquor 4～10g/l, peptone 5～15g/l, yeast extract paste 5～15g/l, guanine 0.010～0.040g/l, initial pH6.5-7.5.
7. the preparation method of 5-amino according to claim 4-4-carbamyl imidazole nucleoside, when it is characterized in that carrying out shake flask fermentation, the fermention medium composition calculates by every liter of contained grammes per square metre g/l of substratum, and is composed as follows: sugar 100～140, corn steep liquor 10～20, yeast powder 15～20, ammonium sulfate 8～15, potassium primary phosphate 1～3, lime carbonate 10～30, urea 2～6, sal epsom 0.5～1.5, pH6.5～7.5.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0463393A2 (en) * | 1990-06-22 | 1992-01-02 | ENIRICERCHE S.p.A. | A mutant of bacillus subtiles and a method of producing surfactin with the use of the mutant |
| WO1992006207A1 (en) * | 1990-10-08 | 1992-04-16 | Basf Aktiengesellschaft | Micro-organism and process for obtaining anthranilic acid |
| CN1536071A (en) * | 2003-04-10 | 2004-10-13 | 华中农业大学 | Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid |
| CN1590535A (en) * | 2004-03-27 | 2005-03-09 | 中国科学院等离子体物理研究所 | Bacillus subtilis strain, preparation method, preparation preparing proess and use |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0463393A2 (en) * | 1990-06-22 | 1992-01-02 | ENIRICERCHE S.p.A. | A mutant of bacillus subtiles and a method of producing surfactin with the use of the mutant |
| WO1992006207A1 (en) * | 1990-10-08 | 1992-04-16 | Basf Aktiengesellschaft | Micro-organism and process for obtaining anthranilic acid |
| CN1536071A (en) * | 2003-04-10 | 2004-10-13 | 华中农业大学 | Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid |
| CN1590535A (en) * | 2004-03-27 | 2005-03-09 | 中国科学院等离子体物理研究所 | Bacillus subtilis strain, preparation method, preparation preparing proess and use |
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