CN105154489A - Method for preparing cephalosporin intermediates by aid of enzymatic processes - Google Patents
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Abstract
The invention discloses a method for preparing cephalosporin intermediates by the aid of enzymatic processes. Penicillin G potassium salt and expandase are subjected to catalytic reaction to prepare 7-ADCA (azodicarbonamide), the penicillin G potassium salt is used as a substrate, the expandase is used as a catalyst, and the 7-ADCA is used as a main raw material for producing antibiotics such as cefalexin, cefradine and cefaclor. Intermediate substrates are added in catalytic procedures by the aid of feed-batch processes and include alpha-ketoglutaric acid and vitamin C. The method includes particular steps of adding parts of the intermediate substrates into a reactor for the first time, adding water into the reactor to regulate pH (potential of hydrogen) of the intermediate substrates until the pH of the intermediate substrates reaches 6-6.5, adding the penicillin G potassium salt and solution with Fe<2+> into the reactor, adding enzyme powder of the expandase into the reactor under ventilation conditions, adding the residual intermediate substrates into the reactor in a batch manner, carrying out stirring reaction until the monitored penicillin G potassium salt is completely converted and then stopping reaction; sequentially carrying out side-chain phenylacetic group removal, denatured protein removal, impurity removal, crystallization and suction filtration on obtained reaction liquid so as to obtain the pure 7-ADCA. The method has the advantages of integral pollution-free preparation procedures, high substrate concentration, reaction speed and conversion rate and low production cost.
Description
Technical field
The present invention relates to bio-pharmaceuticals and technical field of biochemical industry, particularly relate to a kind of method that enzyme process prepares cephalosporin intermediate.
Background technology
7-aminodesacetoxycephalosporanic acid (7-ADCA) (structural formula is as Fig. 1) is the important intermediate of oral cephalosporins medicine, be mainly used in Cephalexin Monohydrate Micro/Compacted, S 578, the antibiotic synthesis such as Cephradine, that China imports and exports one of most active medical material, initial by 7-amino-cephalosporanic acid (7-ACA) through reducing obtained (reaction scheme is shown in Fig. 2), but because the fermentation level of cephalosporin is far below penicillin (Penicillin), and hydrogenation catalyst platinum is expensive, productive rate is low again, therefore seldom use, the method preparing 7-ADCA conventional at present has chemical method, biological fermentation process, enzyme process.
20 century 70s start; there is chemical method; its preparation is raw material with penicillin, is first converted into penicillin sulfoxide through oxidation, is 7-Cephalosporanic acid, finally sloughs phenylacetyl three-step reaction obtained (reaction process is shown in Fig. 3) with chemistry or enzymatic method again through expansion.But suppress owing to there is substrate, the concentration of substrate that this method can realize is on the low side, as in the research of the people such as Liu Xinghui, the substrate 7-Cephalosporanic acid concentration that product yield reaches under 95% condition is only 6%, and reaction process is complicated, oxidation products sulfoxide need can carry out subsequent reactions after recrystallization extracts, and each step all needs strict Controlling Technology parameter just can ensure high-quality height yield, cost is high, causes severe contamination to environment simultaneously.
20th century the mid-80, occur manufacturing the report of 7-ADCA about fermentation method.This method utilizes the technological transformation Penicllium chrysogenums such as recombinant DNA, makes it to have in vivo penicillin and derivative ring expansion to be produced the 7-ADCA product that 7 β bit strips have various side chain, then obtains after suitable acyltransferase removes side chain.Conder etc. as Merck company prepares in the method for 7-ADCA at biology the Penicllium chrysogenum using the culture medium culturing containing hexanodioic acid raw material to transform; thus produce Ad-6-APA (Ad-6-APA); on the spot Ad-6-APA ring expansion is become adipyl-7-ADCA with the above-mentioned Penicllium chrysogenum of ring enlargement enzyme (expandase) gene transformation from clavuligerus, then obtain 7-ADCA with the cracking of adipyl transferring enzyme.But find in research; the gene transformation of ring enlargement enzyme is expressed to Penicllium chrysogenum in clavuligerus; the activity of enzyme is very low and affect the synthesis of penicillin; therefore the Crawford etc. of Panlabs company proposes clavuligerus cefE or top spore cefEF gene to proceed in Penicllium chrysogenum to express; or add acetyltransferase cefG gene again to express simultaneously simultaneously; in the substratum of transformant, add hexanodioic acid side chain precursor, the cynnematin of band adipyl side chain can be made efficiently to produce.
And in recent years, enzyme process prepares 7-ADCA becomes new study hotspot with the feature that environmental protection catalytic efficiency is high.Mainly through the technique means such as rite-directed mutagenesis, random mutation directional transformation ring enlargement enzyme, the enzyme catalysed in vitro potassium penicillin G one step ring expansion after expression generates Cephalosporanic acid, then obtains 7-ADCA (Fig. 4) through penicillin acylase hydrolysis side-chain radical.Although the method prepares 7-ADCA become main trend, but along with the increase of concentration of substrate, the relative reactivity of enzyme reduces, transformation efficiency also can reduce, reaction times extends, and current domestic 7-ADCA overall throughput has reached about 1000 tons/year, therefore, those skilled in the art are badly in need of overcoming above-mentioned defect, to improve transformation efficiency to meet the need of market.
Summary of the invention
The object of this invention is to provide a kind of method that enzyme process prepares cephalosporin intermediate, overcome in prior art by this method the pollution problem that adopts and produce in chemical method preparation process and adopt in biological fermentation process preparation process that concentration of substrate is low, fermentation time long, easily produce the problem of Cephalosporins derivative.
For achieving the above object, the technical solution used in the present invention is: a kind of enzyme process prepares the method for cephalosporin intermediate, described method is take penicilline g potassium as substrate, ring enlargement enzyme prepares cephalosporin intermediate 7-ADCA for catalyzer carries out catalyzed reaction, the method that have employed batch feeding in catalytic process carries out adding of middle substrate, described middle substrate comprises α-ketoglutaric acid and vitamins C, and concrete steps are as follows:
1) add the described middle substrate of part in the reactor first, adding water and being stirred to pH is 6 ~ 6.5, then adds described substrate penicilline g potassium and contains Fe
2+solution, under aeration condition, adding ring enlargement enzyme enzyme powder, then add remaining described middle substrate, stirring reaction in batches, transforming stopped reaction completely when monitoring penicilline g potassium;
Wherein: the final concentration of α-ketoglutaric acid is 20 ~ 35g/L, and ascorbic final concentration is 1.6 ~ 2.3g/L, the starting point concentration of penicilline g potassium is 10 ~ 50g/L, Fe
2+starting point concentration be 0.1 ~ 0.5g/L, by mass, the add-on of ring enlargement enzyme enzyme powder is 37% ~ 50% of penicilline g potassium;
In whole reaction process, the pH of reaction system controls all the time between 6 ~ 6.5, and the dissolved oxygen amount of reaction solution maintains between 25% ~ 35%;
2) to step 1) obtained by reaction solution carry out successively acylation reaction except side chain phenylacetyl, except after metaprotein, removal of impurities, crystallization, suction filtration, obtain sterling 7-ADCA.
The preparation method of the ring enlargement enzyme enzyme powder adopted in the present invention is as follows: the recombination bacillus coli bacterial classification of construction expression ring enlargement enzyme being inoculated in the LB liquid medium containing Kan resistance activates, when cell optical density(OD) reaches 0.6 ~ 0.8, seed liquor after activation is forwarded in the TB substratum containing Kan resistance and continues activation, wherein, by volume, seed liquor during switching/TB substratum=0.5% ~ 1%, when cell optical density(OD) reaches 0.6 ~ 0.8 again, inductor is added to seed liquor, continue activation 18 ~ 20h, centrifugal collecting precipitation; In precipitation, add the damping fluid of pH6 ~ 6.5, collect and suspend; Then in ice-water bath, ultrasonication is dissolved to cell protein, centrifuging and taking supernatant liquor, and described supernatant liquor is the crude enzyme liquid of ring enlargement enzyme, and namely freeze-drying obtains ring enlargement enzyme enzyme powder;
Wherein, described LB liquid medium comprises peptone 10g/L, yeast powder 10g/L and NaCl5g/L;
Described TB substratum comprises peptone 12g/L, yeast powder 24g/L, glycerine 5g/L, dipotassium hydrogen phosphate 16g/L, potassium primary phosphate 2.3g/L;
Before adding inductor, activation temperature controls at 37 DEG C; After adding inductor, activation temperature controls at 25 DEG C; The final concentration of inductor is 0.4mM.
Here the LB liquid medium containing Kan resistance is the liquid Escherichia coli culture medium containing kalamycin resistance, and the TB substratum containing Kan resistance is the inducible protein substratum containing kalamycin resistance.
The structure of the recombination bacillus coli in the present invention conveniently molecular biosciences has operated [seeing " molecular cloning " (Science Press, the second edition, 2002)].
Adopt the ring enlargement enzyme enzyme powder that aforesaid method is obtained, effectively can improve the speed of reaction of preparation 7-ADCA, improve the reaction density of substrate penicilline g potassium, improve transformation efficiency, the reaction times shortens.
If without specified otherwise, the cell optical density(OD) described in the present invention all refers to OD
600value.OD
600it is the standard method of following the trail of microorganism growth in liquid culture.OD is the abbreviation of opticaldensity (optical density(OD)), and representing the optical density(OD) that detected material sponges, is the proper noun in detection method.Light is by detected material, and namely the capacity volume variance of front and back is the energy that detected material sponges, and under specific wavelength, the concentration of same detected material becomes quantitative relationship with absorbed energy.OD
600what refer to is exactly the light absorption value of certain solution at 600nm wavelength place.Light absorption value is proportional to the concentration of the extinction material in solution, is correspondingly inversely proportional to the transmitance of sample.
Preferably, described inductor is isopropyl-beta D-thio galactopyranoside (i.e. inductor IPTG).
Preferably, in 7-ADCA preparation process of the present invention, the dosage first of described middle substrate is 1/3 of overall dosage.
Further preferably, step 1) in, after adding ring enlargement enzyme enzyme powder, add remaining described middle substrate at least in two batches, the dosage often criticized is all equal.
Preferably, the final concentration of described α-ketoglutaric acid is 28 ~ 32g/L.
Further preferably, described ascorbic final concentration is 1.9 ~ 2.3g/L.
Further preferably, the starting point concentration of described penicilline g potassium is 30 ~ 50g/L.
Due to the utilization of technique scheme, the present invention compared with prior art has following advantages:
1) solve employing chemical method and prepare in 7-ADCA process the problem occurring to pollute;
2) solving employing biological fermentation process prepares in 7-ADCA process, and concentration of substrate is low, fermentation time is long, easily produce the problems such as cynnematin analog derivative;
3) problems such as cost in production process is high, yield is low are solved.
Accompanying drawing explanation
Accompanying drawing 1 is the structural formula of 7-aminodesacetoxycephalosporanic acid;
Accompanying drawing 2 is adopt 7-amino-cephalosporanic acid (7-ACA) through reducing the reaction scheme figure of obtained 7-ADCA;
Accompanying drawing 3 is the reaction scheme figure adopting chemical method to prepare 7-ADCA;
Accompanying drawing 4 is the reaction scheme figure adopting enzyme process to prepare 7-ADCA;
Accompanying drawing 5 is the transformation efficiency situation map using in embodiment 1 substrate after obtained ring enlargement enzyme catalyzed reaction 2h in embodiment 2;
Accompanying drawing 6 is the substrate conversion efficiency comparison diagram adopted in embodiment 3 under different feed profile;
Accompanying drawing 7 is adopt the comparison diagram of ventilating with substrate conversion efficiency under stuffiness condition in embodiment 4;
Accompanying drawing 8 is the transformation efficiency comparison diagram of substrate under different concns condition in embodiment 5;
Accompanying drawing 9 is the substrate conversion efficiency comparison diagram in embodiment 6 under different ring enlargement enzyme concentration conditions;
Accompanying drawing 10 is the substrate conversion efficiency comparison diagram whether adopted in embodiment 7 under buffer conditions;
Accompanying drawing 11 is the comparison diagram adopting substrate conversion efficiency under differential responses volume in embodiment 8;
Accompanying drawing 12 is the substrate conversion efficiency comparison diagram whether adopted in embodiment 9 under DTT (dithiothreitol (DTT)) condition.
Embodiment
Below in conjunction with specific embodiment, the present invention will be further described in detail, but the present invention is not limited to following examples.The implementation condition adopted in embodiment can require to do further adjustment according to the concrete difference used, and not marked implementation condition is the condition in normal experiment.
Embodiment 1
The present embodiment provides a kind of preparation method of ring enlargement enzyme enzyme powder, and concrete steps are as follows:
The recombination bacillus coli bacterial classification of construction expression ring enlargement enzyme being inoculated in the LB liquid medium containing Kan resistance activates, and temperature controls, at 37 DEG C, to work as OD
600when value reaches 0.6 ~ 0.8, the seed liquor after activation is forwarded in the TB substratum containing Kan resistance, wherein, by volume, seed liquor/TB substratum=1% during switching; Work as OD
600when value reaches 0.6 ~ 0.8, add the inductor IPTG that final concentration is 0.4mM, continue under 25 DEG C of conditions to cultivate 20h, centrifugal collecting precipitation, adds the Tris-Hcl damping fluid (being butylamine triol damping fluid) of pH6.5, collects and suspends, in ice-water bath, ultrasonication 10min dissolves to cell protein, centrifuging and taking supernatant liquor, this supernatant liquor is the crude enzyme liquid of ring enlargement enzyme, and freeze-drying obtains ring enlargement enzyme enzyme powder.
Here, each substratum is composed as follows:
LB liquid medium: peptone 10g/L, yeast powder 10g/L and NaCl5g/L, high-temperature sterilization;
TB substratum: peptone 12g/L, yeast powder 24g/L, glycerine 5g/L, dipotassium hydrogen phosphate 16g/L, potassium primary phosphate 2.3g/L, high-temperature sterilization.
Embodiment 2
The present embodiment verifies the catalyzed reaction of the obtained ring enlargement enzyme of embodiment 1, is dissolved in the 9ml aqueous solution, after regulating pH to 6.5, adds the Fe of 0.5g substrate penicilline g potassium, 1mg successively by the α-ketoglutaric acid of 0.3g, the vitamins C of 21mg
2+be mixed with mix-solution (mixture), then add the crude enzyme liquid of ring enlargement enzyme obtained in the embodiment 1 of 1ml, stirring reaction 2h under normal temperature, it is 20.7% (shown in Figure 5 that HPLC monitors substrate conversion efficiency, wherein, retention time be the crest at 1.566min place represent what be that the crest at product 7-ADCA, 2.482min place represents is substrate penicilline g potassium).
Prepare 7-ADCA under the different feed profile of embodiment 3, the transformation efficiency of different time sections substrate compares
In 8ml, pH6.5 aqueous solution, adopt different dosing methods according to table 1, carry out material and add, the feed supplement time is 2h, 4h, 6h, and under normal temperature, stirring reaction 7h, HPLC detect the turnover ratio of substrate under each condition each time period, as shown in Figure 6.In table, PenicillinG is substrate penicilline g potassium; α-OG is α-ketoglutaric acid; Vc is vitamins C.
Table 1
From Fig. 6, we can find out, adopt mode 2, namely only carry out batch feeding to middle substrate, and the transformation efficiency of substrate penicilline g potassium at any time in section is all high compared with other three kinds of modes, and transformation efficiency is steady.When employing mode 2 carries out middle substrate batch feeding, the final concentration of α-OG is 30g/L, Vc final concentration is 2.1g/L.
Embodiment 4 reaction system is ventilated with under stuffiness condition, the comparison of substrate conversion efficiency
Middle substrate α-OG0.1g, Vc7mg are dissolved in the 6mL aqueous solution, regulate pH to 6.5, add substrate PenicillinG0.4g successively, Fe
2+1mg is made into mix-solution, add 4mL ring enlargement enzyme crude enzyme liquid, add α-OG, Vc in batches, scheme of adding is with mode 2 in embodiment 3, react respectively with ventilation, stuffiness, under stirring at normal temperature reaction 7h, HPLC detection each time period, the transformation efficiency of substrate is as Fig. 7, and the transformation efficiency of the substrate as can be seen from Figure under aeration condition is higher.
The comparison of transformation efficiency under the different concentration of substrate of embodiment 5
Middle substrate α-OG0.1g, Vc7mg are dissolved in the 6mL aqueous solution, after regulating pH to 6.5, add the PenicillinG of different concns successively, under being namely respectively 30g/L, 40g/L, 50g/L concentration, add Fe
2+1mg is made into mix-solution, add 4mL crude enzyme liquid again, add α-OG, Vc in batches, scheme of adding is with mode 2 in embodiment 3, ventilatory response, the transformation efficiency under stirring at normal temperature reaction 7h, HPLC detection each time period is as Fig. 8, can find out that substrate is under reaction density 40g/L, substrate conversion efficiency is more steady.
The comparison of substrate conversion efficiency under the different enzyme concn of embodiment 6
Middle substrate α-OG0.1g, Vc7mg is dissolved in the 6mL aqueous solution, after regulating pH to 6.5, adds substrate PenicillinG0.4g successively, Fe
2+1mg is made into mix-solution, after adding the lyophozyme powder of 5g/L, 10g/L, 15g/L, 20g/L respectively, add α-OG, Vc in batches, scheme of adding is with mode 2 in embodiment 3, ventilatory response, transformation efficiency under stirring at normal temperature reaction 7h, HPLC detection each time period is as Fig. 9, and final selection enzyme optimum response concentration is 20g/L.
Embodiment 7 adopts or does not adopt the transformation efficiency of substrate under buffer conditions to compare
Mix-solution is respectively with Tris-Hcl damping fluid and water preparation (pH6.5), substrate PenicillinG40g/L, add 20g/L lyophozyme powder, add α-OG, Vc in batches, scheme of adding is with mode 2 in embodiment 3, ventilatory response, transformation efficiency under stirring at normal temperature reaction 7h, HPLC detection each time period is as Figure 10, and water reaction system transformation efficiency and Tris-Hcl system transformation efficiency are more or less the same, for reducing production cost, final water of selecting is as enzymic catalytic reaction system.
Under embodiment 8 iodine system, the transformation efficiency of substrate compares
Mix-solution is with water preparation (pH6.5), respectively in the reaction system of 10ml, 100ml, 2L, control substrate PenicillinG concentration 40g/L, add 20g/L lyophozyme powder, add α-OG, Vc in batches, scheme of adding is with mode 2 in embodiment 3, ventilatory response, transformation efficiency under stirring at normal temperature reaction 7h, HPLC detection each time period, as Figure 11, shows that the reaction conditions after optimizing does iodine and still can reach higher transformation efficiency.
Embodiment 9 is added and is not added the transformation efficiency of substrate under dithiothreitol (DTT) (DTT) condition and compare
Mix-solution compares to add DTT and not add DTT, solution (pH6.5), substrate PenicillinG final concentration 40g/L, add 20g/L lyophozyme powder, when adding DTT, according to mode 2 in embodiment 3, add α-OG in batches, Vc, add DTT by the method for 3mg+3*2mg simultaneously in batches, when not adding DTT, then only need add α-OG in batches, Vc, scheme of adding is with mode 2 in embodiment 3, ventilatory response, stirring at normal temperature reaction 7h, transformation efficiency under HPLC detection each time period is as Figure 12, though do not add DTT system transformation efficiency have reduction, but impact is little, from the viewpoint of cost-saving, reaction system can be selected not add DTT.
Embodiment 107-ADCA gram of level preparation method
Prepare mix-solution by table 2, substrate PenicillinG concentration is 40g/L, adds 20g/L lyophozyme powder, and in reaction system, dissolved oxygen amount is 35%, stopped reaction after normal temperature (25 DEG C) stirring reaction 7h, HPLC detection substrate transformation efficiency reaches 91%; And in backward reaction solution, add final concentration 30g/L acylase, 30 DEG C are stirred hydrolysis and slough phenylacetyl, and then control pH is at 7.8-8.0, in the reaction solution after hydrolysis, add a certain amount of CH
2cl
2with rare H
2sO
4, to pH<1, use separating funnel to remove metaprotein, backward reaction solution in add a certain amount of charcoal absorption impurity, suction filtration is to vacuum except gac, and then regulate about pH to 2.0 crystallization with ammoniacal liquor, namely suction filtration obtains sterling 7-ADCA5g.
| Material | Dosage |
| Penicillin G | 80g |
| α-OG | 20g+3*13.35g |
| Vc | 1.4g+3*935mg |
| Fe 2+ | 200mg |
| Water | 2L(pH6.5) |
Table 2
Above-described embodiment is only for illustrating technical conceive of the present invention and feature; its object is to person skilled in the art can be understood content of the present invention and be implemented; can not limit the scope of the invention with this; all equivalences done according to spirit of the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (8)
1. an enzyme process prepares the method for cephalosporin intermediate, it is characterized in that, described method is take penicilline g potassium as substrate, ring enlargement enzyme prepares cephalosporin intermediate 7-ADCA for catalyzer carries out catalyzed reaction, the method that have employed batch feeding in catalytic process carries out adding of middle substrate, oxygen concn in control reactor is in fixing scope, described middle substrate comprises α-ketoglutaric acid and vitamins C, and concrete steps are as follows:
1) add the described middle substrate of part in the reactor first, add water and regulate pH to 6 ~ 6.5, then add penicilline g potassium and contain Fe
2+solution, under aeration condition, adding ring enlargement enzyme enzyme powder, then add remaining described middle substrate, stirring reaction in batches, transforming stopped reaction completely when monitoring penicilline g potassium;
Wherein: the final concentration of α-ketoglutaric acid is 20 ~ 35g/L, and ascorbic final concentration is 1.6 ~ 2.3g/L, the starting point concentration of penicilline g potassium is 10 ~ 50g/L, Fe
2+starting point concentration be 0.1 ~ 0.5g/L, by mass, the add-on of ring enlargement enzyme enzyme powder is 37% ~ 50% of penicilline g potassium;
In whole reaction process, the pH of reaction system controls all the time between 6 ~ 6.5, and the oxyty of reaction solution maintains between 25% ~ 35%;
2) carry out acylation reaction successively to the reaction solution obtained by step 1) to remove side chain phenylacetyl, except after metaprotein, removal of impurities, crystallization, suction filtration, obtain sterling 7-ADCA.
2. enzyme process according to claim 1 prepares the method for cephalosporin intermediate, it is characterized in that, the preparation method of described ring enlargement enzyme enzyme powder is as follows: the recombination bacillus coli bacterial classification of construction expression ring enlargement enzyme being inoculated in the LB liquid medium containing Kan resistance activates, when cell optical density(OD) reaches 0.6 ~ 0.8, seed liquor after activation is forwarded in the TB substratum containing Kan resistance and continues activation, wherein, by volume, seed liquor during switching/TB substratum=0.5% ~ 1%, when cell optical density(OD) reaches 0.6 ~ 0.8 again, inductor is added to seed liquor, continue activation 18 ~ 20h, centrifugal collecting precipitation, in precipitation, add the damping fluid of pH6 ~ 6.5, collect and suspend, then in ice-water bath, ultrasonication is dissolved to cell protein, centrifuging and taking supernatant liquor, and described supernatant liquor is the crude enzyme liquid of ring enlargement enzyme, and namely freeze-drying obtains ring enlargement enzyme enzyme powder,
Wherein, described LB liquid medium comprises peptone 10g/L, yeast powder 10g/L and NaCl5g/L;
Described TB substratum comprises peptone 12g/L, yeast powder 24g/L, glycerine 5g/L, dipotassium hydrogen phosphate 16g/L, potassium primary phosphate 2.3g/L;
Before adding inductor, activation temperature controls at 37 DEG C; After adding inductor, activation temperature controls at 25 DEG C; The final concentration of inductor is 0.4mM.
3. enzyme process according to claim 2 prepares the method for cephalosporin intermediate, it is characterized in that: described inductor is isopropyl-beta D-thio galactopyranoside.
4. enzyme process according to claim 1 prepares the method for cephalosporin intermediate, it is characterized in that: the dosage first of described middle substrate is 1/3 of overall dosage.
5. the enzyme process according to claim 1 or 4 prepares the method for cephalosporin intermediate, it is characterized in that: in step 1), and after adding ring enlargement enzyme enzyme powder, add remaining described middle substrate at least in two batches, the dosage often criticized is all equal.
6. prepare the method for cephalosporin intermediate according to the arbitrary described enzyme process of Claims 1-4, it is characterized in that: the final concentration of described α-ketoglutaric acid is 28 ~ 32g/L.
7. enzyme process according to claim 6 prepares the method for cephalosporin intermediate, it is characterized in that: described ascorbic final concentration is 1.9 ~ 2.3g/L.
8. enzyme process according to claim 6 prepares the method for cephalosporin intermediate, it is characterized in that: the starting point concentration of described penicilline g potassium is 30 ~ 50g/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852912A (en) * | 2020-04-10 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Method for synthesizing 7-aminodesacetoxycephalosporanic acid |
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| US20010006795A1 (en) * | 1998-04-23 | 2001-07-05 | Arnold L. Demain | Penicillin conversion |
| CN1448506A (en) * | 2002-04-01 | 2003-10-15 | 骏翰生化股份有限公司 | Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same |
| CN101631853A (en) * | 2006-10-05 | 2010-01-20 | 帝斯曼知识产权资产管理有限公司 | Production of beta-lactam antibiotics |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010006795A1 (en) * | 1998-04-23 | 2001-07-05 | Arnold L. Demain | Penicillin conversion |
| CN1448506A (en) * | 2002-04-01 | 2003-10-15 | 骏翰生化股份有限公司 | Mutation penicillin ring enlargement enzyme and process preparing 7-ADCA using same |
| CN101631853A (en) * | 2006-10-05 | 2010-01-20 | 帝斯曼知识产权资产管理有限公司 | Production of beta-lactam antibiotics |
Non-Patent Citations (3)
| Title |
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| KARIN VALEGARD等: "The structural basis of cephalosporin formation in a mononuclear ferrous enzyme", 《NATURE STRUCTURAL & MOLECULAR BIOLOGY》 * |
| 付金衡 等: "全细胞催化生产苯乙酰-7-氨基-3-脱乙酰氧基头孢烷酸的条件优化", 《生物工程学报》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852912A (en) * | 2020-04-10 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Method for synthesizing 7-aminodesacetoxycephalosporanic acid |
| CN112852912B (en) * | 2020-04-10 | 2022-01-11 | 中国科学院天津工业生物技术研究所 | Method for synthesizing 7-aminodesacetoxycephalosporanic acid |
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