CN103525893A - Method of preparing 7-amino-cephalsporanic acid under catalysis of cephalosporin acylase - Google Patents
Method of preparing 7-amino-cephalsporanic acid under catalysis of cephalosporin acylase Download PDFInfo
- Publication number
- CN103525893A CN103525893A CN201310497063.8A CN201310497063A CN103525893A CN 103525893 A CN103525893 A CN 103525893A CN 201310497063 A CN201310497063 A CN 201310497063A CN 103525893 A CN103525893 A CN 103525893A
- Authority
- CN
- China
- Prior art keywords
- cephalosporin
- reaction
- amino
- acylases
- catalysis
- 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.)
- Pending
Links
Abstract
The invention discloses a method of preparing 7-amino-cephalsporanic acid under catalysis of cephalosporin acylase. The method comprises the steps of: adding a buffer solution with a pH value of 7.5-8.0 into a reaction kettle, regulating the temperature, adding cephalosporin C into the reaction kettle, and then adding immobilized cephalosporin acylase into a reaction system; after the reaction starts, regulating the pH value of the system by virtue of a sodium hydroxide solution; and stopping the reaction when the mass percentage of the cephalosporin C as a raw material is less than 0.2 percent, filtering the immobilized cephalosporin acylase out, and treating a filter liquor to obtain the 7-amino-cephalsporanic acid. Compared with the existing process of synthesizing 7-ACA (7-Amino-Cephalsporanic Acid) by virtue of a multistep enzymatic method, the process has the advantages that the operation is simple, the aftertreatment is simplified, and therefore the cost is saved; and compared with the traditional chemical method, the process has the advantages that the process is simple, the period is short, the reaction conditions are milds, and immobilized enzyme can be reused, is easily separated from the reaction system, is strong in the stability and has certain mechanical strength, so that the catalytic reaction is more easily controlled.
Description
Technical field
The present invention relates to the preparation method of 7-amino-cephalosporanic acid, be specifically related to utilize Cephalosporin Acylases catalysis to prepare the method for 7-amino-cephalosporanic acid.
Background technology
7-amino-cephalosporanic acid (7-ACA) is to prepare the requisite crucial parent nucleus of cephalosporins, and its structure following (I) is shown:
The synthetic method of traditional 7-ACA is mainly chemical method, and the synthetic 7-ACA domestic and foreign literature report of relevant chemical process is a lot.But all there is a common drawback: not only cost is high for the synthetic 7-ACA of chemical method, complex steps, and exist group activation, intermolecular condensation, radical protection and de-protected process; Chemosynthesis process is tediously long and complicated, severe reaction conditions, a large amount of three wastes of generation etc.
Summary of the invention
Technical problem to be solved by this invention is: what provide that a kind of technique is simple, the cycle is short, reaction conditions is gentle prepares the method for 7-amino-cephalosporanic acid with Cephalosporin Acylases catalysis.
For solving the problems of the technologies described above, technical scheme of the present invention is: the method for 7-amino-cephalosporanic acid is prepared in a kind of Cephalosporin Acylases catalysis, the steps include: the buffered soln of pH=7.5～8.0 to add reactor, adjusting temperature is 35～38 ℃, cephalosporin is added in reactor, again immobilization Cephalosporin Acylases is added in reaction system, after reaction starts, with sodium hydroxide solution, regulate and maintain reaction system pH=7.5～8.0, HPLC follows the tracks of detection reaction process, when the quality percentage composition of raw material cephalosporin is less than 0.2%, reaction transforms completely, then stopped reaction, filter out immobilization Cephalosporin Acylases, filtrate is washed by ethyl acetate, water is through nanofiltration, lyophilize obtains product 7-amino-cephalosporanic acid.
Under the condition of pH=7.5～8.0, Cephalosporin Acylases is activity stabilized, and substrate conversion efficiency is high; Under the condition of 35～38 ℃ of temperature, Cephalosporin Acylases activity is high, and substrate conversion efficiency is high.When pH and temperature are set in respectively 8.0 and 35 ℃, reaction obtains optimum conventionally.
The concentration of described cephalosporin can change, but concentration is 1mM～1M conventionally, preferably 100mM～500mM.
The buffered soln of described pH=7.5-8.0 is: potassium phosphate buffer, sodium-acetate buffer or three (methylol) aminomethane-hydrochloric acid (Tris-HCl) damping fluid.
The mass ratio of described cephalosporin and immobilization Cephalosporin Acylases is 10:1～12:1, is preferably 10:1; Enzyme dosage speed of response of many times is fast, effective, yet corresponding cost can be high, the few long reaction time of consumption, and reaction is not exclusively.
In described immobilization Cephalosporin Acylases, Cephalosporin Acylases is genetic modification enzyme, chooses certainly: Shanghai Shang Ke biological medicine company limited; The preparation method of described immobilization Cephalosporin Acylases is: polyvinyl alcohol, linking agent are configured to mixed aqueous solution; acylase is joined in the mixed aqueous solution of above-mentioned polyvinyl alcohol and linking agent and stir; the quality percentage composition acylase polyvinyl alcohol gel aqueous solution that is 0.1%-1%, through shaping curing process, process and obtain polyvinyl alcohol immobilized acylase.
Described cephalosporin becomes the method for 7-ACA by enzymatic conversion method, in preparation process, Study of operational conditions is as follows:
1) enzymic activity
Cephalosporin Acylases catalysis cephalosporin under sodium acetate buffer system is converted into 7-ACA:
Carry out as follows the enzyme analytical test alive of standard: by cumulative volume 1mL primitive reaction mixture (200 μ L1M AcOH-AcONa pH of buffer=8.0,100 μ L0.50M cephalosporins, 600 μ L pure water) adding 1mg immobilization cephalosporinase (is immobilized in cephalosporinase in resin, so that can recycled for multiple times enzyme), and 37 ℃ of reactions.One unit enzyme activity is defined as per minute and generates the enzyme amount that 1mM cynnematin consumes.
Detection method: get 1mL reactant, by reactant UV spectrophotometer measuring, by with standard substance to recently determining the concentration of resultant 7-ACA.
2) optimal temperature
By using and 1) identical enzyme analytical test is described at the vigor of 37 ℃ of measurement enzymes in enzymic activity:
Use cephalosporin as substrate.As summarized in Table 1, the optimal temperature of enzyme activity is 35-38 ℃.
Table 1
| Temperature (℃) | Relative activity (%) |
| 15 | 30 |
| 20 | 50 |
| 25 | 60 |
| 30 | 95 |
| 35 | 98 |
| 38 | 98 |
| 40 | 90 |
| 42 | 80 |
| 45 | 15 |
3) optimum pH
By using and 1) the identical enzyme analytical test measurement vigor of enzyme and the mutual relationship between the pH value of reaction mixture described in enzymic activity:
Use various pH and damping fluid, and use cephalosporin as substrate.Optimum pH=7.5～8.0 of enzyme reaction, as shown in table 2:
Table 2
4) impact of concentration of substrate
By using and 1) describe identical enzyme analytical test in enzymic activity and study the impact of concentration of substrate on enzyme activity:
Use cephalosporin as substrate, different concentration of substrate results are as shown in table 3:
Table 3
| Concentration of substrate (mM) | Relative enzyme (%) alive |
| 1 | 100 |
| 10 | 102 |
| 50 | 101 |
| 100 | 110 |
| 200 | 115 |
| 300 | 102 |
| 500 | 105 |
| 800 | 95 |
| 1000 | 90 |
[0033]beneficial effect of the present invention: first, that the present invention adopts is the synthetic 7-ACA of a step enzymatic method, and this technique is compared with the synthetic 7-ACA of using multi-step enzyme method in the past, simple to operate, has simplified aftertreatment, thereby has saved cost; Secondly, compare with traditional chemical method, not only technique is simple, cycle is short, and reaction conditions is gentle, and reaction is without organic solvent, the region and the stereoselectivity that have height more, without protection and deprotection, have avoided chemical process to synthesize the required poisonous and harmful chemical feedstocks of using simultaneously; Again, immobilized enzyme can be reused, and easily separated with reaction system, the stability of immobilized enzyme is high, has certain physical strength, makes catalyzed reaction more easy to control.
Embodiment
Embodiment 1
The preparation of immobilization Cephalosporin Acylases: preparation is containing 5g PVA (polyvinyl alcohol, the polymerization degree 2000), 5g HPMC (Vltra tears, viscosity is 5), the aqueous solution of 100g deionized water, add 10mL cephalosporin C acrylase (crude enzyme liquid of content 25mg/mL), acylase concentration reaches 2.4g/L, at room temperature stir 1 hour, then cephalosporin C acrylase-PVA mixed aqueous solution of preparation is added drop-wise in 2% glutaraldehyde water solution of continuous stirring, dropwise, filtering also water rinses to noresidue glutaraldehyde, transfer to subsequently in (w/w) potassium dihydrogen phosphate aqueous solution of 10% dipping sclerosis 1 hour, filter standby.
To injecting the 40L potassium phosphate buffer of the pH=7.5 preparing in 50L reactor, opening, stir and start heating, temperature of reaction is set 37 ℃.In question response still, temperature-stable has after 37 ℃; join in reactor load weighted cephalosporin (8.0Kg) is disposable; accurately take 500g immobilization Cephalosporin Acylases again and add in reaction system, the sodium hydroxide solution adjusting with 2M after reaction starts maintains reaction system pH=8.0.In whole reaction process, pay close attention to temperature of reaction system, pH value, and note following the tracks of and detect.To substrate conversion completely after, reaction stops, and filters out immobilized enzyme, filtrate is by 10L ethyl acetate washed twice, water after sodium filter again lyophilize obtain product.
Embodiment 2
To injecting the 80L sodium-acetate buffer of the pH=7.8 preparing in 100L reactor, opening, stir and start heating, temperature of reaction is set 37 ℃.In question response still, temperature-stable has after 37 ℃; join in reactor load weighted cephalosporin (16.0Kg) is disposable; accurately take 1000g immobilization Cephalosporin Acylases again and add in reaction system, the sodium hydroxide solution with 3M after reaction starts regulates maintenance system pH=8.0.In whole reaction process, pay close attention to temperature of reaction system, pH value, and note following the tracks of and detect.To substrate conversion completely after, reaction stops, and filters out immobilized enzyme, filtrate is by 20L ethyl acetate washed twice, water after sodium filter again lyophilize obtain product.
Embodiment 3
To injecting the Tris-HCl damping fluid of the 160L of the pH=8.0 preparing in 200L reactor, opening, stir and start heating, temperature of reaction is set 37 ℃.In question response still, temperature-stable has after 37 ℃; join in reactor load weighted cephalosporin (32.0Kg) is disposable; accurately take 2000g immobilization Cephalosporin Acylases again and add in reaction system, the sodium hydroxide solution adjusting with 3M after reaction starts maintains reaction system pH=8.0.In whole reaction process, pay close attention to temperature of reaction system, pH value, and note following the tracks of and detect.To substrate conversion completely after, reaction stops, and filters out immobilized enzyme, filtrate is by 40L ethyl acetate washed twice, water after sodium filter again lyophilize obtain product.
The evaluation of product: by HPLC, analyze, with standard substance Identification product be 7-ACA.
HPLC analysis condition:
Chromatographic column: Hypersil ODS C18 post (250mm * 4.6mm10 μ m);
Moving phase: A is 0.05mol/L, pH=6.0 phosphate buffered saline buffer, and B is acetonitrile, gradient elution; Flow velocity: 2.0mL/min; Column temperature: 30 ℃ of sample sizes: 5 μ L; DAD detects wavelength: 220nm;
Product appearance time: 11.5min.
Claims (5)
1. the method for 7-amino-cephalosporanic acid is prepared in a Cephalosporin Acylases catalysis, the steps include: the buffered soln of pH=7.5～8.0 to add reactor, adjust temperature to 35～38 ℃, cephalosporin is added in reactor, again immobilization Cephalosporin Acylases is added in reaction system, the sodium hydroxide solution of take after reaction starts regulates and maintains reaction system pH as 7.5～8.0, HPLC follows the tracks of detection reaction process, when the quality percentage composition of raw material cephalosporin is less than 0.2%, stopped reaction, then filter out immobilization Cephalosporin Acylases, filtrate is washed by ethyl acetate, water is through nanofiltration, lyophilize, obtain 7-amino-cephalosporanic acid.
2. the method for 7-amino-cephalosporanic acid is prepared in a kind of Cephalosporin Acylases catalysis according to claim 1, it is characterized in that: the concentration of described cephalosporin is 1mM～1M.
3. the method for 7-amino-cephalosporanic acid is prepared in a kind of Cephalosporin Acylases catalysis according to claim 2, it is characterized in that: the concentration of described cephalosporin is 100mM～500mM.
4. according to a kind of Cephalosporin Acylases catalysis described in claim 1,2 or 3, prepare the method for 7-amino-cephalosporanic acid, it is characterized in that: the mass ratio of described cephalosporin and immobilization Cephalosporin Acylases is 10:1～12:1.
5. according to a kind of Cephalosporin Acylases catalysis described in claim 1,2 or 3, prepare the method for 7-amino-cephalosporanic acid, it is characterized in that: the buffered soln of described pH=7.5～8.0 is: potassium phosphate buffer, sodium-acetate buffer or three (methylol) aminomethane-hydrochloride buffer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310497063.8A CN103525893A (en) | 2013-10-18 | 2013-10-18 | Method of preparing 7-amino-cephalsporanic acid under catalysis of cephalosporin acylase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310497063.8A CN103525893A (en) | 2013-10-18 | 2013-10-18 | Method of preparing 7-amino-cephalsporanic acid under catalysis of cephalosporin acylase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103525893A true CN103525893A (en) | 2014-01-22 |
Family
ID=49928240
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310497063.8A Pending CN103525893A (en) | 2013-10-18 | 2013-10-18 | Method of preparing 7-amino-cephalsporanic acid under catalysis of cephalosporin acylase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103525893A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105506050A (en) * | 2016-01-21 | 2016-04-20 | 湖北凌晟药业有限公司 | Cephalosporin parent nucleus enzymolysis reaction catalysis method |
| CN105624257A (en) * | 2014-11-04 | 2016-06-01 | 北京科技大学 | 7-aminocephalosporanic acid preparation method and use |
| CN111549089A (en) * | 2020-04-23 | 2020-08-18 | 山东立海润生物技术有限公司 | Method for producing 7-aminocephalosporanic acid by enzyme method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557962A (en) * | 2004-02-10 | 2004-12-29 | 清华大学 | Production method of 7-amino cephalosporanic acid |
| CN102154429A (en) * | 2010-12-28 | 2011-08-17 | 哈药集团制药总厂 | One-step enzymatic method for preparing 7-aminocephalosporanic acid |
| CN103014114A (en) * | 2012-12-27 | 2013-04-03 | 华北制药河北华民药业有限责任公司 | Method for preparing 7-aminocephalosporanic acid via enzymic method |
-
2013
- 2013-10-18 CN CN201310497063.8A patent/CN103525893A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1557962A (en) * | 2004-02-10 | 2004-12-29 | 清华大学 | Production method of 7-amino cephalosporanic acid |
| CN102154429A (en) * | 2010-12-28 | 2011-08-17 | 哈药集团制药总厂 | One-step enzymatic method for preparing 7-aminocephalosporanic acid |
| CN103014114A (en) * | 2012-12-27 | 2013-04-03 | 华北制药河北华民药业有限责任公司 | Method for preparing 7-aminocephalosporanic acid via enzymic method |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105624257A (en) * | 2014-11-04 | 2016-06-01 | 北京科技大学 | 7-aminocephalosporanic acid preparation method and use |
| CN105624257B (en) * | 2014-11-04 | 2019-11-08 | 北京科技大学 | The Preparation method and use of 7-amino-cephalosporanic acid |
| CN105506050A (en) * | 2016-01-21 | 2016-04-20 | 湖北凌晟药业有限公司 | Cephalosporin parent nucleus enzymolysis reaction catalysis method |
| CN105506050B (en) * | 2016-01-21 | 2018-12-25 | 湖北凌晟药业有限公司 | A kind of catalysis process of cephalosporin mother nucleus enzyme digestion reaction |
| CN111549089A (en) * | 2020-04-23 | 2020-08-18 | 山东立海润生物技术有限公司 | Method for producing 7-aminocephalosporanic acid by enzyme method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101899484B (en) | Preparation method of genipin | |
| JP2001037475A (en) | Production of immobilized lipase, enzyme catalytic or enantio-selective conversion of such lipase and substrate, production of optically active compound and use of immobilized lipase | |
| CN106086126B (en) | Method for synthesizing glutathione by enzyme catalysis | |
| CN104087646B (en) | Method for preparing glycyrrhetinic acid | |
| CN105647996A (en) | Method for preparing adenosine triphosphate with immobilized enzyme method | |
| CN103343117B (en) | Preparation method of immobilized cephalosporin C acylase | |
| CN104004002A (en) | Method using penicillin fermentation liquor for direct preparation of 6-aminopenicillanicacid | |
| CN101993867A (en) | Immobilization method using chitosan as carrier | |
| CN101376646A (en) | Novel method for extracting L-tryptophan from fermentation liquor | |
| CN103525893A (en) | Method of preparing 7-amino-cephalsporanic acid under catalysis of cephalosporin acylase | |
| Cheng et al. | Development of immobilized cellulase through functionalized gold nano-particles for glucose production by continuous hydrolysis of waste bamboo chopsticks | |
| CN103756992B (en) | A kind of chocolate microbacterium magnetic cell and its preparation method and application | |
| CN109735582A (en) | A kind of method of lipase-catalyzed online synthesizing cyclohexane 1 alcohols beta-alkamine derivative | |
| CN102719500A (en) | Method for producing gamma-amino butyric acid through continuous conversion of immobilized enzyme | |
| CN103695409A (en) | Preparation method of immobilized enzyme and application of immobilized enzyme in geniposide conversion | |
| CN100584955C (en) | Chitosan oligosaccharide/chito-oligomer single enzyme production process | |
| Belleville et al. | Preparation of hybrid membranes for enzymatic reaction | |
| CN100519564C (en) | Cefradine preparing process | |
| CN102286563A (en) | Method for preparing L-ornithine from immobilized enzyme | |
| CN107523558B (en) | Embedding and curing method of D-pantolactone hydrolase | |
| CN104762360A (en) | High-content nicotinamide synthesis induced by new-feature nitrile hydratase | |
| CN105732663B (en) | The preparation method of 6 aminopenicillanic acids | |
| CN101550411A (en) | Immobilized cellulose and a method for hydrolyzing plant material thereby for extracting active ingredients | |
| CN103937776A (en) | Method for modifying gamma-glutamyl transpeptidase immobilized enzyme by using carrier ampholyte | |
| CN103725726B (en) | The preparation method of enzymatic clarification Simvastatin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140122 |