CN1155692C - Aspergillus niger strain and its culture method and application - Google Patents
Aspergillus niger strain and its culture method and application Download PDFInfo
- Publication number
- CN1155692C CN1155692C CNB001274511A CN00127451A CN1155692C CN 1155692 C CN1155692 C CN 1155692C CN B001274511 A CNB001274511 A CN B001274511A CN 00127451 A CN00127451 A CN 00127451A CN 1155692 C CN1155692 C CN 1155692C
- Authority
- CN
- China
- Prior art keywords
- aspergillus niger
- nutrient solution
- thalline
- cultural method
- value
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to an asperill us niger 5450 bacterial strain which has the registration number which is CGMCCNO. 0496 and is preserved in the common micro-organism center of the CCCCM. A pure bacterial strain is obtained by sieving large quantity of bacterial strains in soil, and well grows in a culture medium and culture fluid. After the bacterial strain grows for 2 to 3 days in the culture fluid, the bacterial strain has high reaction activity and epoxide hydrolase with enantioselectivity. The present invention can be used for the asymmetric hydrolyzation of a racemic 1, 2-epoxide. Then, S-type epoxide and R-shaped 1, 2-diol can be obtained.
Description
Technical field
The present invention relates to a kind of microbial catalyst and cultural method thereof
Background technology
Have optically actively 1,2-epoxide and two corresponding alcoholate thereof have high using value at aspects such as agricultural chemicals, medicine and fine chemistry industries.As: calcium ion channel blocker (a class cardiovascular agent), according to Press release, Zeneca, 1996 infer: the global market share reaches 4,000,000,000 dollars, American market accounts for 20 hundred million, and beta-blocker wherein surpasses 3.5 hundred million dollars of 22%[Pharmaprojects CD v2.1 that account for antianginal market, the whole America, copyright 1999 PJB Publication Ltd.Richmond in the sales volume in market every year of the U.S., Surrey, UK.].And have an optically active epoxy and glycol key intermediate of synthetic-this class cardiovascular agent of receptor-blocking agent just.
Obtain at present optically actively 1,2-epoxy and 1,2-glycol mainly are to use chemical process.For example the Sharpless asymmetric dihydroxylation prepares chirality 1, the 2-glycol, the mixture that uses salen-Co (II) is to 1, the 2-epoxy carries out asymmetric hydrolysis, they to substrate all have certain restriction [1) .Johnson, R.A.; Sharpless, K.B.in:Catalytic Asymmetric Synthesis, Ed.:Ojima, I., Verlag Chemie, New York, 1993, p.103. 2) .Kolb, H.C., van Nieuwenhze, M.S.; Sharpless, K.B.Chem.Rev.1994,94,2483. 3) .Katsuki, T.J.Synth.Org.Chem.Jpn.1995,53,940. 4) .Jacobsen, E.S.; Kakiuchi, F.; Fonsler, R.G.; Larrow J.F.; Tokunaga, M.Tetrahedron Lett.1997,38,773. 5) .Larrow, J.F.; Schauss, S.E.; Jacobsen, E.N.J.Am.Chem.Soc.1996,50,249.] ￡ in addition because this class cardiovascular agent of beta-blocker often needs to take for a long time, this just need not use harmful reagent as heavy metal ion etc. in synthesizing as far as possible.And use chemical catalysis must use the chiral catalyst that contains heavy metal ion.
Use microbe transformation method catalysis asymmetric hydrolysis 1, the report of 2-epoxy is few at present.Be mainly France R.Furstoss group and K.Farber group [1) .Chen, X.J.; Archelas, A.; Furstoss, R.J.Org.Chem.1993,58,5528. 2) .Mischitz, M.; Kroutil, W.; Wandel, U.; Faber, K.Tetrahedron:Asymmetry, 1995,6 (6), 1261.] and found have the high enantioselectivity catalysis of a high vigor asymmetric hydrolysis 1, the microorganism of 2-epoxy compounds ability is also very rare.So be necessary to seek the bacterial strain of the high vigor highly selective of having of other epoxide hydrolase.
Summary of the invention
The object of the invention provides the Aspergillus niger strain that a brace has high vigor highly selective epoxide hydrolase.
The object of the invention also provides a kind of method of cultivating above-mentioned bacterial strains.
The invention provides a kind of epoxide hydrolase bacterial strain aspergillus niger 5450 (Aspergillus niger 5450) with high vigor highly-solid selectively, this bacterial strain carries out preservation at China Committee for Culture Collection of Microorganisms common micro-organisms center, and that registers on the books is numbered CGMCC NO 0496.This bacterial strain is a pure bacterial strain that obtains through a large amount of screenings in the thousands of kinds of bacterial strains that exist from earth, and growth is 2-3 days in slant medium.After cultivating, can be used for organic chemical reactions.Examine under a microscope, this flora is black, and the top capsule is big, and is spherical in shape.The conidium sphere.
The method of cultivation above-mentioned bacterial strains of the present invention, available following method is cultivated aspergillus niger 5450 bacterial strains and is inoculated usually.Receive on the inclined-plane with transfering loop picking aspergillus niger 5450 thalline or its spore, grew 2-3 days in substratum for 10-35 ℃, the well-grown thalline of picking is received the nutrient solution from the inclined-plane then, and 10-35 ℃ of down reciprocal shaking table cultivated 2-3 days.It is centrifugal that (5,000rpm) 30 minutes, thalline washed with 0.8% physiological saline, and centrifugal again back obtains thalline and can be used for reaction.
Described substratum is 10-30g/L carbon source, 6-15g/L nitrogenous source, 1-3g/L agar, 1.0g/L K
2HPO
4H
2O, 0.5g/L KCl, 0.5g/L MgSO
4H
2O, 0.01g/LFeSO
4H
2O forms, pH value 3.0 to 7.0.Described carbon source can be fructose, glucose or sucrose.Described nitrogenous source can be corn steep liquor, yeast extract paste or Semen Maydis powder.Described nutrient solution is 10-30g/L carbon source, 6-15g/L nitrogenous source, 1.0g/L K
2HPO
4H
2O, 0.5g/L KCl, 0.5g/L MgSO
4H
2O, 0.01g/L FeSO
4H
2O forms, pH value 3.0 to 7.0.Described carbon source can be fructose, glucose or sucrose.Described nitrogenous source can be corn steep liquor, yeast extract paste or Semen Maydis powder.
Fructose is recommended to use as carbon source in slant medium described in the present invention and the nutrient solution, and corn steep liquor uses as nitrogenous source.It is 7 that described slant medium and nutrient solution are recommended the pH value.Available mineral alkali is transferred pH value to 7.Described mineral alkali can be the mineral alkali of monovalence or divalent metal.
Aspergillus niger 5450 bacterial strains of the present invention are the bacterial strains with the high enantioselectivity epoxide hydrolase of high vigor, but Stereoselective catalytic hydrolysis racemization 1, and the 2-epoxy obtains having the epoxy of S-configuration of high optical activity and the glycol of R-configuration.Two kinds of above chipal compounds all are to synthesize some to have the intermediate of important physiologically active compound and chiral drug.Produce the introducing that to avoid harmful reagent such as heavy metal ion with present method simultaneously, make medicine safer.
As: can finish following conversion by aspergillus niger 5450 bacterial strains:
Aspergillus niger 5450 bacterial strains of the present invention are bacterial strains that a strain has high vigor and high enantioselectivity epoxide hydrolase, its cultural method is simple, fast growth, be difficult for variation, and can be used as epoxide hydrolase, widely the catalysis asymmetric hydrolysis dissimilar 1,2-epoxy compounds, Stereoselective obtain chiral diol and chiral epoxy product.Especially those are by the light that contains the equiatomic nucleophilicity of nitrogen oxygen functional group that chemical process was difficult to obtain epoxy compounds alive and diol compound.Has the industrial applications prospect.
Embodiment
The present invention will be helped to understand by following embodiment, but content of the present invention can not be limited.
The cultivation (1) of embodiment 1 thalline
Receive on the above-mentioned inclined-plane with transfering loop picking aspergillus niger CGMCC 0496 thalline, growth is 2-3 days in 10-30 ℃ and substratum, has a large amount of black spore populations to occur.The well-grown thalline of picking is received the nutrient solution from the inclined-plane then, and 10-30 ℃ of down reciprocal shaking table cultivated gained thalline centrifugal (5 2-3 days, 000rmp) 30 minutes, thalline is with the washing of 0.8% physiological saline, and is centrifugal again or obtain thalline by filtering, and the gained thalline can be directly used in reaction.The thalline gray is spherical.
Slant medium contains 10-30g/L fructose, glucose or sucrose, 6-15g/L corn steep liquor, yeast extract paste or Semen Maydis powder, 1-3g/L agar, 1.0g/L K
2HPO
4H
2O, 0.5g/L KCl, 0.5g/LMgSO
4H
2O, 0.01g/L FeSO
4H
2O is with mineral alkali adjust pHs to 7 such as NaOH or KOH.
Nutrient solution is 30-10g/L fructose or glucose, 6-15g/L corn steep liquor or yeast extract paste, Semen Maydis powder, 1.0g/L K
2HPO
4H
2O, 0.5g/L KCl, 0.5g/L MgSO
4H
2O, 0.01g/LFeSO
4H
2O is with monovalence or divalent metal mineral alkali adjust pH to 7.
The all sterilizations 20 minutes under 120 ℃ and 1.1 normal atmosphere of above-mentioned substratum and nutrient solution.
The asymmetric fractionation of 2 pairs of chlorine Styryl oxides of embodiment
To be substrate, obtain product (S)-3, productive rate 40% to the chlorine Styryl oxide.Product (R)-4, productive rate 44%.
Product (S)-3: optical purity: 96%[α]
D 25+ 19.2 ° of (c1.0, CHCl
3)
1NMR(CCl
4+TMS，60MHz)δ＝2.6(dd，J
1＝6Hz，J
2＝2Hz，1H)，δ＝3.1(dd，J
1＝6Hz，J
2＝4Hz，1H)，δ＝3.6-3.8(m，1H)，δ＝6.9-7.4(m，4H)
MS?m/z(rel.instensity?％)156(M
++2，2)，155(M
++1，3)，154(M
+，8)，138(3)，125(40)，119(39)，91(29)，89(100)
Product (R)-4: optical purity: 87%[G]
D 25-33.2 ° (c1.0, EtOH)
1NMR(CD
3COCD
3+TMS，300MHz)δ＝3.13(bs，2H)，δ＝3.53(dd，J
1＝10.9Hz，J
2＝7.4Hz，1H)，δ＝3.61(dd，J
1＝11Hz，J
2＝4.6，1H)，δ＝4.71(dd，J
1＝7.3Hz，J
2＝4.5Hz，1H)，δ＝7.12-7.52(m，4H)
MS?m/z(rel.instensity％)172(M
+，2)，155(11)，141(86)，125(8)，113(26)，77(100)，51(21)
The asymmetric fractionation of 3 pairs of bromine Styryl oxides of embodiment
To be substrate, obtain product (S)-5, productive rate 35% to the bromine Styryl oxide.Product (R)-6, productive rate 43%.
Product (S)-5: optical purity: 100%[α]
D 25+ 14.2 ° of (c1.5, CHCl
3)
1NMR(CCl
4+TMS，90MHz)δ＝2.6(dd，J
1＝5Hz，J
2＝2Hz，1H)，δ＝3.0(dd，J
1＝J
2＝4.5Hz，1H)，δ＝3.6-3.7(m，1H)，δ＝7.1-7.4(AB，J＝6Hz，4H)
MS?m/z(rel.instensity％)200(M
++2，3)，199(M
++1，3)，198(M
+，4)，197(M+-1，3)，169(14)，119(41)，89(100)，63(36)
Product (R)-6: optical purity: 87%[α]
D 25-38.4 ° of (c1.0, CHCl
3)
1NMR(CD
3COCD
3+TMS，300MHz)δ＝3.58(bs，2H)，δ＝3.88-3.94(m，1H)，δ＝3.48(m，1H)，δ＝4.71(m，1H)，δ＝7.36-7.48(AB，J＝8.4Hz，4H)
MS?m/z(rel.instensity?％)218，216(M
+，3)，187(71)，185(83)，159(16)，157(19)，77(100)，51(18)
Claims (5)
1. Aspergillus niger strain, it is characterized in that being undertaken the black-koji mould that is numbered CGMCC NO.0496 5450 (Aspergillus niger 5450) of preservation by China Committee for Culture Collection of Microorganisms common micro-organisms center, examine under a microscope, this flora is black, the top capsule is big, spherical in shape, the conidium sphere.
2. the cultural method of an aspergillus niger 5450 as claimed in claim 1, it is characterized in that aspergillus niger 5450 thalline or its spore receive on the inclined-plane, growth is 2-3 days in the substratum on 10-35 ℃ and the inclined-plane, the picking thalline was cultivated 2-3 days in 10-35 ℃ and nutrient solution, and described substratum is 10-30g/L carbon source, 6-15g/L nitrogenous source, 1-3g/L agar, 1.0g/L K
2HPO
4H
2O, 0.5g/LKCl, 0.5g/L MgSO
4H
2O, 0.01g/L FeSO
4H
2O forms, pH value 7.0, and described nutrient solution is 10-30g/L carbon source, 6-15g/L nitrogenous source, 1.0g/L K
2HPO
4H
2O, 0.5g/L KCl, 0.5g/L MgSO
4H
2O, 0.01g/L FeSO
4H
2C forms, and pH value 7.0, described carbon source are fructose, glucose or sucrose, and described nitrogenous source is corn steep liquor, yeast extract paste or Semen Maydis powder.
3. cultural method as claimed in claim 2 is characterized in that the mineral alkali adjust pH of described substratum or nutrient solution system with monovalence or divalent metal.
4. cultural method as claimed in claim 2, it is characterized in that described aspergillus niger 5450 is cultivated in nutrient solution after, the thalline that contains bacterium liquid is with 0.8% sodium chloride solution washing, and is centrifugal again or obtain thalline by filtering.
5. cultural method as claimed in claim 2, when it is characterized in that described aspergillus niger 5450 is cultivated in nutrient solution, the initial pH value of nutrient solution is 7, controls the pH value during the fermentation between 3.0-7.0.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001274511A CN1155692C (en) | 2000-11-17 | 2000-11-17 | Aspergillus niger strain and its culture method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001274511A CN1155692C (en) | 2000-11-17 | 2000-11-17 | Aspergillus niger strain and its culture method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1291647A CN1291647A (en) | 2001-04-18 |
| CN1155692C true CN1155692C (en) | 2004-06-30 |
Family
ID=4592466
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB001274511A Expired - Fee Related CN1155692C (en) | 2000-11-17 | 2000-11-17 | Aspergillus niger strain and its culture method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1155692C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298837C (en) * | 2005-07-28 | 2007-02-07 | 秘鸣 | Aspergillus niger fungus and its application in production of Pu'er tea |
| CN100413957C (en) * | 2006-09-21 | 2008-08-27 | 江苏省农业科学院 | Apergillus niger strain and application thereof |
-
2000
- 2000-11-17 CN CNB001274511A patent/CN1155692C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1291647A (en) | 2001-04-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101392278B (en) | Method for preparing D-pantolactone by microbe mixed fermentation method | |
| CN110157623B (en) | Fusarium strain and method for producing D-pantolactone hydrolase by fermenting same | |
| CN100345974C (en) | Microbiological preparation method of S-(+)-2,2-dimethyl cyclo propyl formamide | |
| CN1111604C (en) | Process for preparing D-lactone valerate by microbe enzyme method | |
| CN101701246B (en) | Method for screening high-yield D-pantolactone hydrolytic enzyme strain | |
| CN1155692C (en) | Aspergillus niger strain and its culture method and application | |
| JP3951008B2 (en) | Capsaicin decomposing / synthesizing enzyme and production method thereof | |
| CN1869197A (en) | Red yeast cell and method of producing optically pure chiral tertiary alcohol | |
| CN1993476A (en) | Process for the production of (s)-5-chloro-2-isopropylpent-4-enoic acid esters | |
| CN1419596A (en) | Method of culturing microorganism | |
| JPH06209781A (en) | Production of optically active alcohol | |
| CN100334198C (en) | Serration and its use in preparation of chiral precurser for dielzepin | |
| CN101821404A (en) | Microbial kinetic resolution of ethyl-3,4-epoxybutyrate | |
| JPH0630790A (en) | Production of optically active 1,2-propanediol | |
| CN102660471B (en) | Production of chiral alpha-hydroxy acid and its strain by using biological catalysis method | |
| JPH04234991A (en) | New microorganism | |
| JPH06141876A (en) | Production of optically active n-benzyl-3-pyrrolidinol | |
| JPH06141888A (en) | Production of d-mandelic acid | |
| JPS58201992A (en) | Preparation of beta-substituted propionic acid or amide thereof by microorganism | |
| JP2001069975A (en) | Chitosanase | |
| JP2594167B2 (en) | Novel antibiotic SF2698 substance and its production method | |
| KR100657204B1 (en) | The method of making optically active 3-hydroxy-?-butyrolactone by enzymatic method | |
| JPH1042886A (en) | Production of beta-alanine by microorganism | |
| CN105755095A (en) | Method for synthesizing (R)-2-hydroxy acid by biological enzyme method | |
| CN1283777C (en) | Method for screening levo-phosphonomycin bioconversion strain with cis-propene phosphoric acid as substrate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |