CN1724646A - Bacillus subtilis mutant strain, breeding method and application in fermentation method of producing adenosin - Google Patents

Bacillus subtilis mutant strain, breeding method and application in fermentation method of producing adenosin Download PDF

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CN1724646A
CN1724646A CNA2005100139000A CN200510013900A CN1724646A CN 1724646 A CN1724646 A CN 1724646A CN A2005100139000 A CNA2005100139000 A CN A2005100139000A CN 200510013900 A CN200510013900 A CN 200510013900A CN 1724646 A CN1724646 A CN 1724646A
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adenosine
medium
subtilis
strain
isoleucine
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陈宁
黄金
刘淑云
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Tianjin University of Science and Technology
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Tianjin University of Science and Technology
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Abstract

The invention relates to a wilted hay bacilli mutant, the cultivation method and the application in producing adenosine by fermentation method. It uses wilted hay bacilli mutant TX-1 as source strain, after mutagenizing by DES, adds isoleucine and valine into culture medium as supplemental nutrition. It would be gained after cultivating, filtering and resume filtering. Verifying the genetic marker, it can be confirmed to have 6-meraptopurine resistant. The application in producing adenosine is picking a circle of HJ04130 to 25-35mL/500mL cone bottle that contains fermenting culture medium, under the condition of 30-34 degree centigrade and 200-240rpm rotate speed, fermenting for 48-64 hours to gain adenosine. The fungus could improve the capability to produce adenosine. And the manufacture method is low cost, and is easy to be popularized.

Description

Bacillus subtilis mutant strain, selection and the application of this bacterium in producing adenosine through zymotechnics
[technical field]: the present invention relates to a kind of technical field of bioengineering, particularly to produce bacterium be bacillus subtilis mutant strain to adenosine, with and selection and with this bacterium producing adenosine through zymotechnics.
[background technology]: adenosine (Adenosine) belongs to important nucleotide derivative, plays important regulation in physiological and biochemical procedure, has pharmaceutical use widely.In the U.S., adenosine is the line medicine through the treatment PSVT of FDA approval, also be through the FDA approval be used for one of two kinds of medicines of cardiac drug load test, become the routine administration of emergency Treatment tachyarrhythmia and drug load test.Fermentation method scale operation goes out adenosine and still belongs to blank in China, and the production of present domestic adenosine mainly is chemical method and enzyme process, and cost height, seriously polluted applies being subjected to serious restriction.Raw material sources are extensive, cost is low, product purity is high, the fermentation method of reflection mild condition has been the trend of the times of research.Many universities and colleges and research institution all are being engaged in the research in this field.
[summary of the invention]: the objective of the invention is to solve the production cost height, seriously polluted of existing adenosine, apply the problem that is severely limited, provide seed selection that a kind of adenosine produces bacterium and selection thereof and with this bacterium producing adenosine through zymotechnics.
This laboratory (University Of Science and Technology Of Tianjin metabolic control fermentation research department) utilizes subtilis (Bacillussubtilis) ATCC 6872 (being preserved in American Type Culture Collecti) (to be preserved in Chinese industrial microbial strains preservation administrative center through repeatedly improveing acquisition one strain inosine production bacterium TX-1, deposit number CICC-10082), be the auxotroph of VITAMIN B4 (Ade), Histidine (His), xanthine (Xan), promptly need these nutritive substances just can keep growth and accumulate high-caliber inosine.Subtilis TX-1 also has 8-azaguanine (8-AG), Ismipur (6-MP), Sulphaguanidine (SG) resistance simultaneously.By measuring, its adenosine output is almost nil.
The present invention proposes a kind of mutant strain Hj04130 on the basis of subtilis TX-1, be that this bacterial strain is to be starting strain with subtilis TX-1, after ethyl sulfate (DES) mutagenic treatment, in substratum, add Isoleucine and Xie Ansuan nutritive substance as a supplement again, through cultivating screening and multiple sieve acquisition, its genetic marker is verified, determine that it still has the Ismipur resistance, its genetic marker is Ile -+ Val -+ 6-MP r
The present invention is a starting strain with subtilis TX-1, be intended to utilize TX-1 from glucose to basis that the high-throughput of t-inosinic acid metabolism main flow is established the output of further raising adenosine.
The selection of subtilis HJ04130 comprises:
1) starting strain is chosen:
Choose subtilis (Bacillus subtilis) TX-1, be the auxotroph of VITAMIN B4 (Ade), Histidine (His), xanthine (Xan), TX-1 also has 8-azaguanine (8-AG), Ismipur (6-MP), Sulphaguanidine (SG) resistance simultaneously;
2) culture medium preparation:
---slant medium: extractum carnis 8-20g, peptone 2-5g, yeast extract powder 3-7g, NaCl1.0-5.0g, agar 15-30g, tap water is settled to 1L, pH7.0-7.2;
-seed culture medium: glucose 30-60g, KH 2PO 42.0-5.0g, MgSO 47H 2O 0.5-0.8g, NH 4Cl 3.0-6.0g, FeSO 47H 2O 0.005-0.02g, MnSO 47H 2O 0.005-0.02g, soya-bean cake hydrolyzed solution 30-50ml, yeast extract powder 0.1-0.7, urea 1.0-4.0, guanine 0.05-0.2g, tap water is settled to 1L, pH7.0-7.2;
---fermention medium: glucose 70-100g, KH 2PO 40.5-3.0g, NHCl 10-25g, MgSO 47H 2O 0.2-0.5g, soya-bean cake hydrolyzed solution 30-50ml, FeSO 47H 2O 0.005-0.02g, MnSO 4H 2O 0.005-0.02g, guanine 0.05-0.2g, CaCl 23-8g, CaCO 320-35g adds when shaking bottle; Isoleucine and Xie Ansuan addition separately is 0-0.04g, and tap water is settled to 1L, pH7.0-7.2;
---minimum medium (MM): glucose l0-30g, NH 4Cl 3-8g, KH 2PO 40.5-2g, MgSO 40.2-0.5g, Trisodium Citrate 0.2-1.0g, Sodium Glutamate 0.5-2.0g, FeSO 47H 2O 0.005-0.02g, MnSO 4H 2O 0.005-0.02g, pH7.0-7.2;
---screening culture medium: on the basis of MM substratum, add 0.015-0.040g Isoleucine and 0.015-0.040g Xie Ansuan;
---perfect medium (CM): glucose 0.5-5g, peptone 5-15g, extractum carnis 5-15g, yeast extract paste 3-10g, NaCl 5-20g, pH7.0-7.2;
---minimum medium, screening culture medium, perfect medium must add the agar of 1.5-3.0% when preparation solid plate substratum;
3) mutagenesis of mutant strain and separation screening:
---connect ring subtilis (Bacillus subtilis) TX-1 bacterial classification from fresh inclined-plane and go into seed culture medium 25-35mL/500mL Erlenmeyer flask, under 30-34 ℃, 160-200r/min, cultivate 12-16h;
---centrifugal collecting cell, wash with stroke-physiological saline solution.Then, after 0.1mol/L, the washing once of pH7.0-7.2 phosphoric acid buffer, the cell suspension of breaing up is suspended in 20-30ml contains among the 0.1mol/L, pH7.0-7.2 phosphoric acid buffer liquor of 1.5-2.5ml ethyl sulfate (DES) 30-34 ℃ of following oscillation treatment 0.25-1h;
---reaction 10 times of termination reactions of hypo solution dilution of 10-15mL, centrifugal collecting cell is with will doing middle the cultivation 24 hours in its access seed culture medium after the stroke-physiological saline solution washing;
---dilution is coated with the CM flat board then, cultivates 48h down for 30-34 ℃;
---choose the big bacterium colony of bacterium colony circle on the CM flat board, corresponding dibbling is dull and stereotyped and screening flat board at CM, MM, dull and stereotyped bacterium colony of going up growth and not growing on the MM flat board of picking CM and screening or the fainter bacterium colony of growth are inoculated in slant medium, cultivate 12-48h in 30-34 ℃;
---then, every strain connects a ring and goes into fermention medium, determine to sieve again the bacterial strain of usefulness according to the height of adenosine output, before the multiple sieve, will confirm with the amino acid defective type genetic marker of bacterial strain multiple sieve, when sieving again, with the continuous passage ten times on the inclined-plane of every strain bacterium, every strain connects 3 bottles of fermention mediums, investigates every monobasic adenosine throughput, screen definite bacterial strain according to adenosine output height and product glycosides stability, obtain HJ04130 Isoleucine and Xie Ansuan defective adenosine and produce bacterium.
---with the bacterial strain of subtilis HJ04130, meet a ring HJ04130 and fill in the Erlenmeyer flask of fermention medium, 30-34 ℃, under the rotating speed 200-240rpm, fermentation culture 48-64 hour, make adenosine in 25-35mL/500mL as producing adenosine through zymotechnics.
Advantage of the present invention and positively effect: from the Isoleucine and the Xie Ansuan deficient strain HJ04130 of subtilis (Bacilus subtilis) TX-1 seed selection, show the ability of accumulation higher level adenosine, this be since with respect to starting strain its IMP dehydrogenase activity be lowered manyly, in substratum, add micro-Isoleucine and Xie Ansuan and activated the IMP desaturase.The test of the producing adenosine through zymotechnics by shaking a bottle level, 5L fermentor tank level with this bacterium has certain industrial applicibility and is worth.Adenosine production method raw material sources provided by the invention are extensive, cost is low, product purity is high, the reflection mild condition.
[embodiment]:
Embodiment 1: the selection of subtilis (Bacillus subtilis) HJ04130:
1) starting strain is chosen:
Choose subtilis (Bacillus subtilis) TX-1;
2) culture medium preparation:
---slant medium: extractum carnis 10g, peptone 4g, yeast extract powder 5g, NaCl 2.5g, agar 20g, tap water is settled to 1L, pH7.0;
---seed culture medium: glucose 40g, KH 2PO 43.0g, MgSO 47H 2O 0.6g, NH 4Cl5.0g, FeSO 47H 2O 0.01g, MnSO 47H 2O 0.01g, soya-bean cake hydrolyzed solution 40ml, yeast extract powder 0.5, urea 2.0, guanine 0.1g, tap water is settled to 1L, pH7.0;
---fermention medium: glucose 80g, KH 2PO 41.0g, NH 4Cl 15g, MgSO 47H 2O 0.4g, soya-bean cake hydrolyzed solution 40ml, FeSO 47H 2O 0.01g, MnSO 4H 2O 0.01g, guanine 0.1g, CaCl 25g, CaCO 330g adds when shaking bottle; Isoleucine and Xie Ansuan optimum addition separately is 0.015g, and the blending ratio of the two is 1: 1, and tap water is settled to 1L, pH7.0-7.2;
---minimum medium (MM): glucose 20g, NH 4Cl 5g, KH 2PO 41g, MgSO 40.4g, Trisodium Citrate 0.5g, Sodium Glutamate 0.5g, FeSO 47H 2O 0.01g, MnSO 4H 2O 0.01g, pH7.0-7.2;
---screening culture medium: on the basis of MM substratum, add 0.02g Isoleucine and 0.02g Xie Ansuan;
---perfect medium (CM): glucose 1g, peptone 10g, extractum carnis 10g, yeast extract paste 5g, NaCl 5g, pH7.0-7.2;
-minimum medium, screening culture medium, perfect medium must add 2.0% agar when preparation solid plate substratum;
3) mutagenesis of mutant strain and separation screening:
---connect ring subtilis (Bacillus subtilis) TX-1 bacterial classification from fresh inclined-plane and go into seed culture medium 30mL/500mL Erlenmeyer flask, under 34 ℃, 160r/min, cultivate 12h;
---centrifugal collecting cell, wash three times then with stroke-physiological saline solution, after 0.1mol/L, the washing once of pH7.0-7.2 phosphoric acid buffer, the cell suspension of breaing up is suspended in 30ml contains among the 0.1mol/L, pH7.0-7.2 phosphoric acid buffer liquor of 2.0ml ethyl sulfate (DES) 32 ℃ of following oscillation treatment 0.25-1h;
---reaction 10 times of termination reactions of hypo solution dilution of 10mL, centrifugal collecting cell is with will doing middle the cultivation 24 hours in its access seed culture medium after the stroke-physiological saline solution washing;
---dilution is coated with the CM flat board then, cultivates 48h down for 32 ℃;
---choose the big bacterium colony of bacterium colony circle on the CM flat board, corresponding dibbling is dull and stereotyped and screening flat board at CM, MM, and the dull and stereotyped bacterium colony or the fainter bacterium colony of growth of growing and not growing on the MM flat board of going up of picking CM and screening is inoculated in slant medium, cultivates 48h in 34 ℃;
---then, every strain connects a ring and goes into fermention medium, determine to sieve again the bacterial strain of usefulness according to the height of adenosine output, before the multiple sieve, to confirm with the amino acid defective type genetic marker of bacterial strain multiple sieve, when sieving again, with the continuous passage ten times on the inclined-plane of every strain bacterium, every strain connects 3 bottles of fermention mediums, investigate every monobasic adenosine throughput, screen definite bacterial strain according to adenosine output height and legacy stability (with output 〉=5g/L, 10 genetic markers of continuous passage are not lost and are standard), obtain HJ04130 Isoleucine and Xie Ansuan defective adenosine and produce bacterium.
By repeating mutagenic treatment, can obtain HJ04130 Isoleucine and Xie Ansuan defective adenosine and produce bacterium.The test of the producing adenosine through zymotechnics by shaking a bottle level, 5L fermentor tank level with this bacterium has industrial applicibility and is worth.
Analytical procedure
The plastic centrifuge tube that will contain the 8mL fermented liquid places table model high speed centrifuge (TGL-16G), centrifugal 5min under 8000r/min, and it is standby to get supernatant liquor.
Adenosine concentration and glucose concn adopt high performance liquid chromatography and SBA-40C multifunctional bio sensing assays instrument to measure respectively.
Cell concentration OD 620Measure.
Shaking the adenosine leavening property of investigating original seed and mutant strain on bottle level, the results are shown in table 1.As seen from Table 1, the adenosine output of mutant strain is high, and productive rate is also high.To the mutant strain experiment of going down to posterity, it is strong and stable to find that mutant strain HJ04130 adenosine in the process of going down to posterity produces ability.
The performance of table 1 original seed and mutant strain fermentation adenosine relatively
Bacterium adenosine output (g/L) glucosides transformation efficiency (g/g)
Starting strain 00
HJ04130 6.5 0.08
The experiment of going down to posterity of mutant strain HJ04130
HJ04130 goes down to posterity repeatedly to mutant strain, shaking the ability of measuring its accumulation adenosine on bottle level, found that as shown in table 2.The heredity of HJ04130, leavening property are stablized, and are bacterium of adenosine generation preferably.
Generation 123456789 10
Genetic marker++++++++++
Output (g/L) 6.52 6.58 6.50 6.50 6.53 6.58 6.52 6.51 6.50 6.54
The seed selection of embodiment 2, HJ04130:
Choose subtilis (Bacillus subtilis) TX-1, the seed selection process is with example 1.By mutagenic treatment repeatedly, obtain Ile -+ Val -+ 6-MP rOn the basis of MM substratum, add 0.02g Isoleucine and 0.02g Xie Ansuan;
Connect a ring HJ04130 bacterial classification from fresh slant medium and go into fermention medium (30ml/500ml Erlenmeyer flask), fermention medium is formed:
Glucose 80g, KH 2PO 41.0g, NH 4Cl 15g, MgSO 47H 2O 0.4g, soya-bean cake hydrolyzed solution 40ml, FeSO 47H 2O 0.01g, MnSO 4H 2O 0.01g, guanine 0.1g, CaCl 25g, CaCO 330g, tap water is settled to 1L, pH7.0-7.2;
Shake-flask culture: the fermention medium liquid amount is 30ml in the 500ml Erlenmeyer flask, and leavening temperature is 34 ℃, and rotating speed is 220rpm, and fermentation time is 48h, and the adenosine production peak is 6-52g/L, is 0.08g/g to the transformation efficiency of glucose.
Embodiment 3, with the HJ04130 producing adenosine through zymotechnics:
Connect a ring HJ04130 bacterial classification from fresh inclined-plane and go into seed culture medium (30ml/500ml Erlenmeyer flask), at 34 ℃, 160rpm inserts fermention medium with 10% inoculum size (V/V) after cultivating 11h down.Slant medium, seed culture medium and fermention medium are formed:
---slant medium: extractum carnis 10g, peptone 4g, yeast extract powder 5g, NaCl 2.5g, agar 20g, tap water is settled to 1L, pH7.0;
---seed culture medium: glucose 40g, KH 2PO 43.0g, MgSO 47H 2O 0.6g, NH 4Cl5.0g, FeSO 47H 2O 0.01g, MnSO 47H 2O 0.01g, soya-bean cake hydrolyzed solution 40ml, yeast extract powder 0.5, urea 2.0, guanine 0.1g, tap water is settled to 1L, pH7.0;
---fermention medium: glucose 90g, KH 2PO 41.0g, NH 4Cl 15g, MgSO 47H 2O 1.2g, soya-bean cake hydrolyzed solution 35ml, FeSO 47H 2O 0.01g, MnSO 4H 2O 0.01g, guanine 0.1g, CaCl 25g, CaCO 320g adds when shaking bottle; Isoleucine and Xie Ansuan optimum addition separately is 0.015g, and the blending ratio of the two is 1: 1, and tap water is settled to 1L, pH7.0-7.2;
Shake-flask culture: the fermention medium liquid amount is 30ml in the 500ml Erlenmeyer flask, and leavening temperature is 34 ℃, and rotating speed is 220rpm, and fermentation time is 48h, and the adenosine production peak is 7.7g/L, is 0.09g/g to the transformation efficiency of glucose.
Embodiment 4, with HJ04130 at 5L fermentor tank synthesizing adenosine:
Inclined-plane and seed culture medium composition and culture condition thereof are with embodiment 3, and fermention medium is formed:
Glucose 90g, KH 2PO 41.0g, NH 4Cl 7.5g, MgSO 47H 2O 1.2g, soya-bean cake hydrolyzed solution 35ml, FeSO 47H 2O 0.01g, MnSO 4H 2O 0.01g, guanine 0.1g, CaCl 25g, Isoleucine and Xie Ansuan optimum addition separately is 0.015g, and the blending ratio of the two is 1: 1, and tap water is settled to 1L, pH7.0-7.2;
5L fermentation cylinder for fermentation culture volume is 3L.Air flow is 3L/min, and 16h is 350rpm before the mixing speed, increases 50rpm every 4 hours (deciding on the fermentation situation) rotating speeds behind the 16h, increases to till the 700rpm.NaOH or ammoniacal liquor with 6mol/L are controlled pH at 6.7-7.0, and leavening temperature is 32-34 ℃, and fermentation equipment is that the model that Shanghai Baoxing Biology Equipment Engineering Co., Ltd produces is the automatic fermenter of Bio-2002.Fermentation time is 64-72h.The adenosine production peak is 16g/L, is 0.2g/g to the transformation efficiency of glucose.

Claims (5)

1, a kind of subtilis (Bacillus subtilis) mutant strain HJ04130, this bacterial strain is (to be preserved in Chinese industrial microbial strains preservation administrative center with subtilis (Bacillus subtilis) TX-1, deposit number CICC-10082) is starting strain, after ethyl sulfate (DES) mutagenic treatment, in substratum, add Isoleucine and Xie Ansuan nutritive substance as a supplement again, through cultivating screening and multiple sieve acquisition, its genetic marker is verified, determine that it still has the Ismipur resistance, its genetic marker is Il e -+ Val -+ 6-MP r
2, the selection of a kind of subtilis (Bacillus subtilis) HJ04130 is characterized in that comprising:
1) starting strain is chosen:
Choose subtilis (Bacillus subtilis) TX-1 and (be preserved in Chinese industrial microbial strains preservation administrative center, deposit number CICC-10082), be the auxotroph of VITAMIN B4 (Ade), Histidine (His), xanthine (Xan), TX-1 also has 8-azaguanine (8-AG), Ismipur (6-MP), Sulphaguanidine (SG) resistance simultaneously;
2) culture medium preparation:
---slant medium: extractum carnis 8-20g, peptone 2-5g, yeast extract powder 3-7g, NaCl1.0-5.0g, agar 15-30g, tap water is settled to 1L, pH7.0-7.2;
---seed culture medium: glucose 30-60g, KH 2PO 42.0-5.0g, MgSO 47H 2O 0.5-0.8g, NH 4Cl 3.0-6.0g, FeSO 47H 2O 0.005-0.02g, MnSO 47H 2O 0.005-0.02g, soya-bean cake hydrolyzed solution 30-50ml, yeast extract powder 0.1-0.7, urea 1.0-4.0, guanine 0.05-0.2g, tap water is settled to 1L, pH7.0-7.2;
---fermention medium: glucose 70-100g, KH 2PO 40.5-3.0g, NH 4Cl 10-25g, MgSO 47H 2O 0.2-0.5g, soya-bean cake hydrolyzed solution 30-50ml, FeSO 47H 2O 0.005-0.02g, MnSO 4H 2O 0.005-0.02g, guanine 0.05-0.2g, CaCl 23-8g, CaCO 320-35g adds when shaking bottle; Isoleucine and Xie Ansuan addition separately is 0-0.04g, and tap water is settled to 1L, pH7.0-7.2;
---minimum medium (MM): glucose 10-30g, NH 4Cl 3-8g, KH 2PO 40.5-2g, MgSO 40.2-0.5g, Trisodium Citrate 0.2-1.0g, Sodium Glutamate 0.5-2.0g, FeSO 47H 2O0.005-0.02g, MnSO 4H 2O 0.005-0.02g, pH7.0-7.2;
---screening culture medium: on the basis of MM substratum, add 0.015-0.040g Isoleucine and 0.015-0.040g Xie Ansuan;
---perfect medium (CM): glucose 0.5-5g, peptone 5-15g, extractum carnis 5-15g, yeast extract paste 3-10g, NaCl 5-20g, pH7.0-7.2;
---minimum medium, screening culture medium, perfect medium must add the agar of 1.5-3.0% when preparation solid plate substratum;
3) mutagenesis of mutant strain and separation screening:
---connect ring subtilis (Bacillus subtilis) TX-1 bacterial classification from fresh inclined-plane and go into seed culture medium 25-35mL/500mL Erlenmeyer flask, under 30-34 ℃, 160-200r/min, cultivate 12-16h;
---centrifugal collecting cell, wash with stroke-physiological saline solution.Then, after 0.1mol/L, the washing once of pH7.0-7.2 phosphoric acid buffer, the cell suspension of breaing up is suspended in 20-30ml contains among the 0.1mol/L, pH7.0-7.2 phosphoric acid buffer liquor of 1.5-2.5ml ethyl sulfate (DES) 30-34 ℃ of following oscillation treatment 0.25-1h;
---reaction 10 times of termination reactions of hypo solution dilution of 10-15mL, centrifugal collecting cell is with will doing middle the cultivation 24 hours in its access seed culture medium after the stroke-physiological saline solution washing;
---dilution is coated with the CM flat board then, cultivates 48h down for 30-34 ℃;
---choose the big bacterium colony of bacterium colony circle on the CM flat board, corresponding dibbling is dull and stereotyped and screening flat board at CM, MM, dull and stereotyped bacterium colony of going up growth and not growing on the MM flat board of picking CM and screening or the fainter bacterium colony of growth are inoculated in slant medium, cultivate 12-48h in 30-34 ℃;
---then, every strain connects a ring and goes into fermention medium, determine to sieve again the bacterial strain of usefulness according to the height of adenosine output, before the multiple sieve, will confirm with the amino acid defective type genetic marker of bacterial strain multiple sieve, when sieving again, with the continuous passage ten times on the inclined-plane of every strain bacterium, every strain connects 3 bottles of fermention mediums, investigates every monobasic adenosine throughput, screen definite bacterial strain according to adenosine output height and product glycosides stability, obtain HJ04130 Isoleucine and Xie Ansuan defective adenosine and produce bacterium.
3, the selection of subtilis HJ04130 according to claim 2 is characterized in that adding respectively in the fermention medium Isoleucine and Xie Ansuan and respectively is 0.015g.
4, the purposes of subtilis HJ04130, it is characterized in that the bacterial strain as producing adenosine through zymotechnics with HJ04130, fermention medium see claim 2 described fermention mediums, meeting a ring HJ04130 fills in the Erlenmeyer flask of fermention medium in 25-35mL/500mL, 30-34 ℃, under the rotating speed 200-240rpm, fermentation culture 48-64 hour, make adenosine.
5, the purposes of subtilis HJ04130 according to claim 4, it is characterized in that with HJ04130 as the bacterial strain of producing adenosine through zymotechnics, in fermention medium, add the Isoleucine of 0.015g/L and 0.015g/L Xie Ansuan nutritive substance the best as a supplement.
CNA2005100139000A 2005-06-24 2005-06-24 Bacillus subtilis mutant strain, breeding method and application in fermentation method of producing adenosin Pending CN1724646A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101475974B (en) * 2009-01-23 2011-06-08 广东省微生物研究所 Culture medium and method for fermentation production of adenosine using the same
CN102154165A (en) * 2011-01-05 2011-08-17 南京工业大学 Bacillus subtilis capable of producing adenosine at high yield
CN103993059A (en) * 2014-05-28 2014-08-20 河北圣雪大成制药有限责任公司 Method for optimizing proportion of components E1 and E2 in bacillus polymyxa fermentation liquid
CN106635799A (en) * 2016-11-02 2017-05-10 广东肇庆星湖生物科技股份有限公司 Screening method of guanosine production bacterial strains
CN112795607A (en) * 2020-12-31 2021-05-14 河南巨龙生物工程股份有限公司 Method for improving adenosine fermentation yield
CN114807266A (en) * 2022-04-20 2022-07-29 天津科技大学 Method for producing adenine by fermentation

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101475974B (en) * 2009-01-23 2011-06-08 广东省微生物研究所 Culture medium and method for fermentation production of adenosine using the same
CN102154165A (en) * 2011-01-05 2011-08-17 南京工业大学 Bacillus subtilis capable of producing adenosine at high yield
CN103993059A (en) * 2014-05-28 2014-08-20 河北圣雪大成制药有限责任公司 Method for optimizing proportion of components E1 and E2 in bacillus polymyxa fermentation liquid
CN106635799A (en) * 2016-11-02 2017-05-10 广东肇庆星湖生物科技股份有限公司 Screening method of guanosine production bacterial strains
CN112795607A (en) * 2020-12-31 2021-05-14 河南巨龙生物工程股份有限公司 Method for improving adenosine fermentation yield
CN112795607B (en) * 2020-12-31 2023-06-23 河南巨龙生物工程股份有限公司 Method for improving adenosine fermentation yield
CN114807266A (en) * 2022-04-20 2022-07-29 天津科技大学 Method for producing adenine by fermentation

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