CN105130792A - Method for separating and purifying (R)-(-)-mandelic acid synthesized with biological method - Google Patents
Method for separating and purifying (R)-(-)-mandelic acid synthesized with biological method Download PDFInfo
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Abstract
The invention provides a method for separating and purifying (R)-(-)-mandelic acid synthesized with a biological method. The method comprises steps as follows: a mandelonitrile solution is added to a conversion solution containing E.coli engineering bacteria used for secreting J2315 nitrilase and has a reaction, then a reaction solution is obtained, the reaction solution is sequentially subjected to pre-treatment, acidizing treatment, concentration treatment, crystallization treatment and refining treatment, and the purified (R)-(-)-mandelic acid is obtained. The method for separating and purifying (R)-(-)-mandelic acid has the advantages of simple operation steps, mild reaction conditions, good separation effects, low cost and short period, contributes to large-scale production of (R)-(-)-mandelic acid synthesized with the biological method and has a good industrial application prospect.
Description
Technical field
The invention belongs to technical field of bioengineering, relate to a kind of to synthesized by biological process (R) ?(?) ?amygdalic acid carry out the method for separation and purification.
Background technology
Amygdalic acid is extremely important chirality pharmaceutical intermediate compound, has the dual function of anti-inflammatory and sterilization.At present, in the international market, to the demand of amygdalic acid about with average annual about 10% speed increment, especially (R) ?(?) ?amygdalic acid become the product be badly in need of already both at home and abroad.
(R) ?(?) ?amygdalic acid and derivative thereof be the important drugs intermediate of the vasodilators such as synthesis Cyclelate, Hydrobenzole, pemoline, sterilant, spasmolytic, and there is good Biodegradable, it is the acid optical resolution agent attracted most attention at present, most racemic modification amine and amino acids can be made to carry out optical resolution through diastereomer isomery salt formation method, as antitussive first south different caye the beautiful jade derivative of intermediate octahydro can by (R) ?(?) ?amygdalic acid split.
At present, the common method preparing amygdalic acid opticity monomer roughly has: (1) physico-chemical processes, comprising dissymmetric synthesis, Optical Instruments Industry method and chromatography etc., dissymmetric synthesis can the isomer of direct chemosynthesis amygdalic acid, Split Method first synthesizes racemic modification amygdalic acid, then splits acquisition chiral mandelic acid; (2) biological synthesis process.
Application number be 201510334809.2 patent propose the synthesis of a kind of biological process (R) ?(?) ?the method of amygdalic acid to replace traditional chemical resolution method.The method single batch reaction (R) ?(?) ?the cumulative concentration of amygdalic acid reach 2.9M, ee value is greater than 97%.Produce to make the method (R) ?(?) ?amygdalic acid more easily reach industrialization and produce, be necessary to develop one can easy and at an easy rate will (R) ?(?) ?amygdalic acid carry out being separated and the method for purifying from end product mixture.
Summary of the invention
Main purpose of the present invention be to provide a kind of to synthesized by biological process (R) ?(?) ?amygdalic acid carry out the method for separation and purification, the method has easy and simple to handle, the advantage such as good separating effect, cost are low and the cycle is short.
For achieving the above object, solution of the present invention is:
To synthesized by biological process (R) ?(?) ?amygdalic acid carry out the method for separation and purification, it comprises the steps:
(1), mandelonitrile stream is added in the conversion fluid containing the E.coli engineering bacteria being used for secreting J2315 nitrilase, pH be 6.0 ?9.0 and temperature be 20 ?react under the condition of 40 DEG C 20 ?24h, stop stream adduction continue reaction 2 ?6h, obtain reaction solution;
(2), to step (1) gained reaction solution carry out pre-treatment to remove insolubles, obtain treatment solution;
(3), to step (2) gained treatment solution carry out acidification, can 20 ?collect the crystal of precipitation at the temperature of 30 DEG C and obtain acidizing fluid;
(4), to step (3) gained acidizing fluid carry out concentration and crystallization treatment, collect the crystal of separating out;
(5), combining step (3) and step (4) gained crystal, obtain after carrying out refinement treatment (R) ?(?) ?amygdalic acid.
Wherein, the method involved by step (1) and application number be 201510334809.2 Chinese patent method used identical.
In step (1), by mandelonitrile with first-class acceleration stream add 10 ?12h, within the remaining reaction times, carry out stream with second acceleration add, first-class acceleration is greater than second acceleration, is preferably 2 times that first-class acceleration is second acceleration.
In step (1), first-class acceleration can be 10 ?40gL
?1h
?1, second acceleration can be 5 ?20gL
?1h
?1; Preferably, first-class acceleration can be 15 ?35gL
?1h
?1, second acceleration can be 7.5 ?17.5gL
?1h
?1; More preferably, first-class acceleration can be 20 ?30gL
?1h
?1, second acceleration can be 10 ?15gL
?1h
?1; Most preferably, first-class acceleration is 24gL
?1h
?1, second acceleration is 12gL
?1h
?1.
In step (1), pH can be 7 ?8.5, can also be preferably 7.9 ?8.1; Temperature can be 25 ?37 DEG C, can also be preferably 30 DEG C.
In step (1), the preparation method of conversion fluid comprises the steps:
A, E.coli engineering bacteria is carried out amplification cultivation and abduction delivering in the fermentation medium, centrifugal to obtain resting cell;
B, collect resting cell, and be suspended in pH be 6.0 ?9.0 damping fluid in, obtain conversion fluid.
Wherein, in step a, component and the content of fermention medium are as follows: yeast extract paste 16 ?60gL
?1, peptone 12 ?80gL
?1, glycerine 1 ?500mlL
?1, K
2hPO
424.75gL
?1, KH
2pO
43.47gL
?1, MgSO
41gL
?1.
In the conversion fluid of step b, the concentration of resting cell can be greater than 10gL
?1, be preferably 10 ?100gL
?1, more preferably 10 ?50gL
?1, most preferably be 10 ?20gL
?1.
In stepb, damping fluid can be NaH
2pO
4/ Na
2hPO
4damping fluid, KH
2pO
4/ K
2hPO
4in damping fluid and any one of Tris ?HCl damping fluid.The concentration of damping fluid can be 20 ?200mM.
In stepb, conversion fluid can also contain solubility promoter, solubility promoter to be volume fraction be 1 ?25% methyl alcohol, ethanol, any one or a few in Virahol and normal hexane, be preferably volume fraction be 5 ?10% methyl alcohol, ethanol, any one or a few in Virahol and normal hexane.
In step (2), pre-treatment step can for filtrations, absorption, rotary evaporation, centrifugal and extract in any one or a few.Filtration can adopt diatomite filtration; Absorption can adopt charcoal absorption.
In step (3), the pH value for the treatment of solution can be adjusted to 1 ?7 for using acid by acidification, is preferably adjusted to 1 ?5, is more preferably and is adjusted to 2 ?4.Acid can be any one or a few in hydrochloric acid, phosphoric acid and acetic acid.The interpolation volume of acid can be 5% ?30% with the ratio of the volume for the treatment of solution, is preferably 10% ?15%.
In step (3), the collection mode of crystal is collecting by filtration or collected by centrifugation, is preferably vacuum filtration and collects.
In step (4), concentration be 55 ?adopt at the temperature of 100 DEG C rotary evaporation by the volume concentration to 10 of acidizing fluid ?60%, be preferably concentrated into 20 ?40%.Concentration also can preferably 65 ?carry out under the condition of 85 DEG C, more preferably carry out under the condition of 75 DEG C.The number of times of rotary evaporation can be 1 ?5 times, can also be preferably 2 ?3 times.
In step (4), after concentration, proceed crystallization treatment.Crystallization treatment be the temperature of the acidizing fluid after concentration (i.e. Tc) is down to 0 ?50 DEG C, be preferably down to 10 ?30 DEG C, be more preferably be down to 20 ?30 DEG C.When temperature is down to after below 50 DEG C, the amygdalic acid being in hypersaturated state just can crystallization.
In step (5), refinement treatment comprise the steps: the crystal after by the merging of step (5) gained 50 ?100 DEG C of oven dry, adopt dissolution with solvents, and proceed the concentration of step (4) and crystallization treatment 2 ~ 3 times, collect the crystal of separating out, continue 50 ?dry 12h at 100 DEG C, obtain (R) ?(?) ?amygdalic acid.
In step (5), bake out temperature can be 70 ?80 DEG C.Solvent can be any one in water, methyl alcohol, ethanol, Virahol, ethyl acetate, toluene, methylene dichloride and normal hexane.
Owing to adopting such scheme, the invention has the beneficial effects as follows:
The present invention to synthesized by biological process (R) ?(?) ?amygdalic acid carried out a series of process, thus realize in end product mixture (R) ?(?) ?the separation and purification of amygdalic acid.
First, method of the present invention especially for synthesized by biological process (R) ?(?) ?the separation of amygdalic acid, its operation steps is less, and therefore the cycle that is simple to operate, separation and purification is shorter, and production efficiency is higher.
Secondly, each operation steps of method of the present invention does not need large-scale plant and instrument to complete, reaction conditions is gentleer, energy consumption is lower, drop into if drop into industrial applications without the need to larger cost in early stage, be conducive to by biological process synthesize (R) ?(?) ?the follow-up industrialization of amygdalic acid produce.
Finally, method good separating effect of the present invention.Prove through test, after adopting method of the present invention, the yield of amygdalic acid can reach 96%; Adopt multiple detection method (as alkali formula volumetry, high performance liquid chromatography and nucleus magnetic resonance etc.) to detect to the amygdalic acid of gained, result shows, and the amygdalic acid purity after surperficial separation and purification is higher than 99%.
Embodiment
The invention provides a kind of to synthesized by biological process (R) ?(?) ?amygdalic acid carry out the method for separation and purification, it comprises the steps:
(1), mandelonitrile stream is added in the conversion fluid containing the E.coli engineering bacteria being used for secreting J2315 nitrilase, pH be 6.0 ?9.0 and temperature be 20 ?react under the condition of 40 DEG C 20 ?24h, stop stream adduction continue reaction 2 ?6h, obtain reaction solution;
(2), to step (1) gained reaction solution carry out pre-treatment to remove insolubles, obtain treatment solution;
(3), to step (2) gained treatment solution carry out acidification, 20 ?collect the crystal of precipitation at the temperature of 30 DEG C and obtain acidizing fluid;
(4), to step (3) gained acidizing fluid carry out concentration, 20 ?collect the crystal of precipitation at the temperature of 30 DEG C;
(5), combining step (3) and step (4) gained crystal, obtain after carrying out refinement treatment (R) ?(?) ?amygdalic acid.
Wherein, in step (1), J2315 nitrilase (BCJ2315) is that bulkholderia cepasea J2315 bacterial strain (BurkholderiacenocepaciaJ2315) is secreted.For secrete the E.coli engineering bacteria of J2315 nitrilase be by EscherichiacoliM15, clone BCJ2315 gene and process LAN thus build E.coliM15/BCJ2315 recombinant chou (RecombinantE.coliM15/BCJ2315).About this E.coli engineering bacteria, please refer to the record of the people such as Wang non-patent literature " Discoveryandcharacterizationofahighlyefficientenantiosel ectivemandelonitrilehydrolasefromBurkholderiacenocepacia J2315byphylogeny ?basedenzymaticsubstratespecificityprediction " disclosed in BMCBiotechnology2013,13:14.
In step (1), mandelonitrile can carry out stream with different flow accelerations in the different steps that whole stream adds reaction process and add.Stream add reaction initial 10 ?12h, mandelonitrile can carry out stream with first-class acceleration and add, and adds in the reaction times at remaining stream, and mandelonitrile can carry out stream with second acceleration and add, but first-class acceleration should be greater than second acceleration, and be preferably the twice of second acceleration.The present invention adopts different flow accelerations to carry out in the different steps that whole stream adds reaction process (20 ?24h), and reason that stream adds is as follows: mandelonitrile has certain toxicity to J2315 nitrilase, along with stream adds the carrying out of reaction, the catalytic efficiency of J2315 nitrilase can reduce gradually.First-class acceleration adopts larger numerical value can make full use of the highest catalytic efficiency of J2315 nitrilase, and when the catalytic efficiency of J2315 nitrilase is reduced to a certain degree, then adopts second acceleration thus ensure that mandelonitrile is fully transformed.If the whole process that whole stream adds reaction only adopts first-class acceleration, mandelonitrile transformation efficiency can be caused to reduce, the whole process that whole stream adds reaction all adopts second acceleration then can cause the waste of the catalytic efficiency of J2315 nitrilase.
In step (1), the preparation method of conversion fluid comprises the steps:
A, E.coli engineering bacteria is carried out amplification cultivation and abduction delivering in the fermentation medium, centrifugal to obtain resting cell;
B, collect resting cell, and be suspended in pH be 6.0 ?9.0 damping fluid in, obtain conversion fluid.
Wherein, in step a, component and the content of fermention medium are as follows: yeast extract paste 16 ?60gL
?1, peptone 12 ?80gL
?1, glycerine 1 ?500mlL
?1, K
2hPO
424.75gL
?1, KH
2pO
43.47gL
?1, MgSO
41gL
?1.But, fermentation feed medium can also be added according to practical situation.
In the conversion fluid of step b gained, the concentration of resting cell should be greater than 10gL
?1, be preferably 10 ?100gL
?1, be more preferably 10 ?50gL
?1, most preferably be 10 ?20gL
?1.J2315 nitrilase needed for catalyzed reaction is secreted by resting cell, so the concentration impact of resting cell is the concentration of J2315 nitrilase.The higher then relative catalytic efficiency of concentration of J2315 nitrilase is higher, and namely in the unit time, the inversion quantity of mandelonitrile is higher.In theory along with the raising of the concentration of resting cell, first-class acceleration can proportional increase, namely the concentration of resting cell needed for first-class acceleration equal 15 ?can transform the concentration of the resting cell of 100mM mandelonitrile in 20min completely.
In a preferred embodiment of the invention, first-class acceleration can be 10 ?40gL
?1h
?1, second acceleration can be 5 ?20gL
?1h
?1; In another preferred embodiment of the invention, first-class acceleration also can be 15 ?35gL
?1h
?1, second acceleration also can be 7.5 ?17.5gL
?1h
?1; In another preferred embodiment of the present invention, first-class acceleration can be 20 ?30gL
?1h
?1, second acceleration can be 10 ?15gL
?1h
?1; In most preferred embodiment of the present invention, first-class acceleration can be 24gL
?1h
?1, second acceleration can be 12gL
?1h
?1.
Above-mentioned fed-batch mode is a kind of preferred fed-batch mode just, due in the process of whole reaction, the catalytic efficiency of J2315 nitrilase declines gradually along with the carrying out of reaction, therefore, the different steps that can also add reaction at stream is carried out stream with the flow acceleration reduced gradually and is added, or whole stream is added step of reaction and is divided into several stages, each stage adopts a different flow acceleration, and the flow acceleration of previous stage is greater than the flow acceleration of the latter half.As: whole stream can be added step of reaction and be divided into three phases, each stage corresponds respectively to first-class acceleration, second acceleration and the 3rd flow acceleration, and first-class acceleration is greater than second acceleration, and second acceleration is greater than the 3rd flow acceleration; Whole stream can also be added step of reaction and be divided into four-stage, each stage corresponds respectively to first-class acceleration, second acceleration, the 3rd flow acceleration and the 4th flow acceleration, first-class acceleration is greater than second acceleration, second acceleration is greater than the 3rd flow acceleration, and the 3rd flow acceleration is greater than the 4th flow acceleration; Whole stream can also be added step of reaction and be divided into double teacher, each stage corresponds respectively to first-class acceleration, second acceleration, the 3rd flow acceleration, the 4th flow acceleration and the 5th flow acceleration, first-class acceleration is greater than second acceleration, second acceleration is greater than the 3rd flow acceleration, 3rd flow acceleration is greater than the 4th flow acceleration, 4th flow acceleration is greater than the 5th flow acceleration, by that analogy.
In stepb, damping fluid can be NaH
2pO
4/ Na
2hPO
4damping fluid, KH
2pO
4/ K
2hPO
4in damping fluid, Tris ?any one in HCl damping fluid or other any conventional pH damping fluids.The concentration of this damping fluid can be 20 ?200mM.
In step b except adding damping fluid, the solubility promoter of mandelonitrile can also be added, solubility promoter to be volume fraction be 1 ?25% methyl alcohol, ethanol, any one or a few in Virahol and normal hexane, be preferably volume fraction be 5 ?10% methyl alcohol, ethanol, any one or a few in Virahol and normal hexane.The interpolation volume of solubility promoter can be long-pending 5% ?10% of buffering liquid.
In step (1), pH can also be 7 ?8.5, be preferably 7.5 ?8.3, be more preferably 7.9 ?8.1.Ammoniacal liquor, NaOH solution or Na can be adopted to the adjustment of pH
2cO
3solution.Wherein, the concentration of ammoniacal liquor for 1M is to pure ammoniacal liquor, can be preferably 2 ?10M, be more preferably 4 ?8M, most preferably be 6M.During conversion temperature can also be 25 ?37 DEG C, be preferably 30 DEG C.During conversion, pH and temperature directly have an impact to the vigor of J2315 nitrilase and the catalytic efficiency of J2315 nitrilase.The vigor of enzyme is different under different invert points or different conversion pH conditions.The invert point that the present invention selects is suitable for the performance of the best catalytic efficiency of enzyme with transforming pH.By affecting the catalytic efficiency of enzyme thus affecting the first-class acceleration of mandelonitrile, final impact be namely when having reacted (R) ?(?) ?the ultimate density of amygdalic acid.
In step (1), the reaction solution obtained is the same with the reaction solution that the method that the Chinese patent that application number is 201510334809.2 is recorded obtains.
In step (2), pre-treatment step can for filtrations, absorption, rotary evaporation, centrifugal and extract in any one or a few.Wherein, filtration can preferably adopt diatomite to filter, and absorption can preferably adopt gac to adsorb.The object for the treatment of step is the insolubles such as biological catalyst or substrate for removing in reaction solution.
In a preferred embodiment of the invention, pre-treatment step comprises centrifugation, diatomite filtration and charcoal absorption.Wherein, centrifugation (can be generally 25 DEG C) at normal temperatures, with the centrifugal 60min of 8000rpm; Diatomite filtration can adopt super-cell and adopt Büchner funnel vacuum filtration, loads the object that one deck diatomite retains to reach solid in Büchner funnel; Gac selected by charcoal absorption is preferably columnar activated carbon.
In step (3), acidification be use acid the pH value of step (2) gained treatment solution is adjusted to 1 ?7, be preferably adjusted to 1 ?5, be more preferably be adjusted to 2 ?4.Acid can be any one or a few in hydrochloric acid, phosphoric acid and acetic acid.Acid interpolation volume and the ratio of the volume for the treatment of solution be 5% ?30%, be preferably 10% ?15%.The object of acidifying makes the mandelate in solution be converted into amygdalic acid, and therefore, the too low meeting of the addition as tartaric acid makes mandelate all cannot be converted into amygdalic acid, thus affects quality product; The too high meeting of addition as tartaric acid makes liquor capacity expanded many, produces dilution effect, affect subsequent purification flow process to amygdalic acid.
In step (3), the crystal of separating out should 20 ?collect at the temperature of 30 DEG C, this is because: the crystallization of amygdalic acid is relevant to its solubleness in the solution, amygdalic acid after acidifying in solution is in hypersaturated state and crystallization, to gather in the crops more crystal, then need the solubleness reducing amygdalic acid, because solubleness can increase along with the rising of temperature, therefore too high temperature is unfavorable for the crystallization of amygdalic acid, but low temperature can increase energy consumption again, therefore the temperature of crystallization is arranged on 20 ?30 DEG C for good.
In step (3), be collecting by filtration or collected by centrifugation to the collection mode of the crystal of separating out, be preferably vacuum filtration and collect.The concrete steps that vacuum filtration is collected are as follows: adopt Büchner funnel, spread one deck filter cloth or filter paper, pour the solution after acidifying into funnel in funnel surface, by vacuum pump using circulatory water suction filtration, crystal is separated from the solution after acidifying, after suction filtration terminates, collects the crystal on filter cloth.In production, adopt the mode of centrifuge with cutter discharge of solid collected by centrifugation, rotary drum rotating speed 1000 ± 50rpm.
In step (4), concentration be 55 ?adopt at the temperature of 100 DEG C rotary evaporation by the volume concentration to 10 of acidizing fluid ?60%, be preferably concentrated into 20 ?40%.The temperature (i.e. the temperature of rotary evaporation) of concentration can be preferably 65 ?85 DEG C, 75 DEG C can also be more preferably.The number of times of rotary evaporation be 1 ?5 times, be preferably 2 ?3 times.In step (3), due to quite a few amygdalic acid existing crystallization being collected from supersaturated solution (i.e. the treatment solution of step (2) gained), now, the amygdalic acid in this solution has reached non-hypersaturated state and cannot crystallization.Therefore need, by concentrating with remove portion water, to make the amygdalic acid in this solution again reach hypersaturated state.By the volume concentration of acidizing fluid to original volume 10% ?60% be to make amygdalic acid again reach hypersaturated state.
In step (4), after concentration, proceed crystallization treatment.Crystallization treatment be the temperature of the acidizing fluid after concentration (i.e. Tc) is down to 0 ?50 DEG C, be preferably down to 10 ?30 DEG C, be more preferably 20 ?30 DEG C.When temperature is down to after below 50 DEG C, the amygdalic acid being in hypersaturated state just can crystallization.
In step (5), refinement treatment comprise the steps: the crystal after by the merging of step (5) gained 50 ?100 DEG C of oven dry, adopt dissolution with solvents, and carry out the concentration of step (4) and crystallization treatment 2 ~ 3 times, collect the crystal of separating out, continue 50 ?dry 12h at 100 DEG C, obtain (R) ?(?) ?amygdalic acid.Bake out temperature during refinement treatment be preferably 70 ?80 DEG C, solvent be preferably in water, methyl alcohol, ethanol, Virahol, ethyl acetate, toluene, methylene dichloride and normal hexane any one.
Embodiment
The present embodiment relate to a kind of to synthesized by biological process (R) ?(?) ?amygdalic acid carry out the method for separation and purification, it comprises the steps:
(1), obtain containing (R) ?(?) ?the reaction solution 10L of amygdalic acid, this reaction solution is the reaction solution of application number described in the patent of invention of 201510334809.2;
(2), to step (1) gained reaction solution carry out centrifugation, get supernatant liquor after discarding precipitation, at 60 DEG C, rotary evaporation is to remove methyl alcohol, collects remaining solution, obtains treatment solution after adopting diatomite filtration and charcoal absorption successively;
(3) treatment solution of step (2) gained, is got, slowly add the hydrochloric acid (namely the volume of hydrochloric acid is 11% with the ratio of the volume for the treatment of solution) of 11% (v/v) and constantly stir with the speed of 300rpm, make pH value reach 2 ?3, temperature is adjusted to 25 DEG C, vacuum filtration is collected the crystal of separating out and is continued to employ, and remaining liquid is as acidizing fluid;
(4) step (3) gained acidizing fluid, is got, rotary evaporation is carried out at 75 DEG C, by the volume concentration of acidizing fluid to 30% of original volume, and then temperature is adjusted to 25 DEG C, vacuum filtration is collected the crystal of separating out and is continued to employ, continue to repeat above-mentioned rotary evaporation and vacuum filtration to remaining solution, the crystal again collecting precipitation is continued to employ;
(5), combining step (3) and step (4) gained crystal, drying and processing 6h at 75 DEG C, after crystal drying, ethanol is used again to dissolve crystal, rotary evaporations are adopted by the volume concentration of the ethanolic soln that dissolves crystal to 30% of original volume the temperature of 50 DEG C, then temperature value 25 DEG C is regulated, vacuum filtration is collected the crystal of separating out and is continued to employ, remaining liquid continues to adopt rotary evaporation to be concentrated into 30% of original volume at the temperature of 50 DEG C, vacuum filtration is collected the crystal of separating out and is continued to employ, the crystal of twice vacuum filtration collection is merged, 12h is dried at 75 DEG C, obtain after purifying (R) ?(?) ?amygdalic acid.
Above-mentioned is can understand for ease of those skilled in the art and use the present invention to the description of embodiment.Person skilled in the art obviously easily can make various amendment to these embodiments, and General Principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (10)
1. (R) ?(?) the ?amygdalic acid synthesized by biological process is carried out to a method for separation and purification, it is characterized in that: comprise the steps:
(1), mandelonitrile stream is added in the conversion fluid containing the E.coli engineering bacteria being used for secreting J2315 nitrilase, pH be 6.0 ?9.0 and temperature be 20 ?react under the condition of 40 DEG C 20 ?24h, stop stream adduction continue reaction 2 ?6h, obtain reaction solution;
(2), to step (1) gained reaction solution carry out pre-treatment to remove insolubles, obtain treatment solution;
(3), to step (2) gained treatment solution carry out acidification, collect the crystal of precipitation and obtain acidizing fluid;
(4), to step (3) gained acidizing fluid carry out concentration and crystallization treatment, collect the crystal of separating out;
(5), combining step (3) and step (4) gained crystal, obtain after carrying out refinement treatment (R) ?(?) ?amygdalic acid.
2. method according to claim 1, it is characterized in that: in step (1), by described mandelonitrile with first-class acceleration stream add 10 ?12h, within the remaining reaction times, carry out stream with second acceleration add, described first-class acceleration is greater than described second acceleration; Or, described pH be 7 ?8.5; Or, described temperature be 25 ?37 DEG C;
Preferably, described first-class acceleration be 10 ?40gL
?1h
?1, described second acceleration be 5 ?20gL
?1h
?1; Or, described pH be 7.9 ?8.1; Or described temperature is 30 DEG C;
Further preferably, described first-class acceleration be 15 ?35gL
?1h
?1, described second acceleration be 7.5 ?17.5gL
?1h
?1;
Still more preferably, described first-class acceleration be 20 ?30gL
?1h
?1, described second acceleration be 10 ?15gL
?1h
?1;
Most preferably, described first-class acceleration is 24gL
?1h
?1, described second acceleration is 12gL
?1h
?1.
3. method according to claim 1, is characterized in that: in step (1), the preparation method of described conversion fluid comprises the steps:
A, described E.coli engineering bacteria is carried out amplification cultivation and abduction delivering in the fermentation medium, centrifugal to obtain resting cell;
B, collect described resting cell, and be suspended in pH be 6.0 ?9.0 damping fluid in, obtain described conversion fluid.
4. method according to claim 3, is characterized in that: in step a, and component and the content of described fermention medium are as follows: yeast extract paste 16 ?60gL
?1, peptone 12 ?80gL
?1, glycerine 1 ?500mlL
?1, K
2hPO
424.75gL
?1, KH
2pO
43.47gL
?1, MgSO
41gL
?1.
5. method according to claim 3, is characterized in that: in the conversion fluid of step b, and the concentration of described resting cell is greater than 10gL
?1; Or the damping fluid in step b is NaH
2pO
4/ Na
2hPO
4damping fluid, KH
2pO
4/ K
2hPO
4in damping fluid and any one of Tris ?HCl damping fluid; Or, also containing solubility promoter in the conversion fluid of step b, described solubility promoter to be volume fraction be 1 ?25% methyl alcohol, ethanol, any one or a few in Virahol and normal hexane;
Preferably, in the conversion fluid of step b, the concentration of described resting cell be 10 ?100gL
?1; Or, the concentration of the damping fluid in step b be 20 ?200mM; Or, described solubility promoter to be volume fraction be 5 ?10% methyl alcohol, ethanol, any one or a few in Virahol and normal hexane;
More preferably, in the conversion fluid of step b, the concentration of described resting cell be 10 ?50gL
?1;
Most preferably, in the conversion fluid of step b, the concentration of described resting cell be 10 ?20gL
?1.
6. according to described method arbitrary in claim 1 to 5, it is characterized in that: the pre-treatment step in step (2) is filtrations, absorption, rotary evaporation, centrifugal and any one or a few in extracting;
Preferably, in step (2), described in be filtered into employing diatomite filtration; Or, described in be adsorbed as employing charcoal absorption.
7., according to described method arbitrary in claim 1 to 5, it is characterized in that: the acidification in step (3) be use acid the pH value of described treatment solution is adjusted to 1 ?7; Or the collection mode of the crystal in step (3) is collecting by filtration or collected by centrifugation;
Preferably, described acid is any one or a few in hydrochloric acid, phosphoric acid and acetic acid; Or, the pH value of described treatment solution be adjusted to 1 ?5; Or, the interpolation volume of described acid and the ratio of the volume of described treatment solution be 5% ?30%; ; Or described collecting by filtration is that vacuum filtration is collected;
Most preferably, the pH value of described treatment solution be adjusted to 2 ?4; Or, the interpolation volume of described acid and the ratio of the volume of described treatment solution be 10% ?15%.
8. according to described method arbitrary in claim 1 to 5, it is characterized in that: the concentration in step (4) be 55 ?adopt at the temperature of 100 DEG C rotary evaporation by the volume concentration to 10 of described acidizing fluid ?60%, the crystallization treatment in step (4) be the temperature of the acidizing fluid through concentration is down to 0 ?50 DEG C;
Preferably, described acidizing fluid volume concentration to 20 ?40%; Or, the temperature of described concentration be 65 ?85 DEG C; Or, the number of times of described rotary evaporation be 1 ?5 times; Or, through the temperature of the acidizing fluid of concentration be down to 10 ?30 DEG C;
More preferably, or, the number of times of described rotary evaporation be 2 ?3 times.
9. according to described method arbitrary in claim 1 to 5, it is characterized in that: described refinement treatment comprise the steps: by step (5) gained crystal 50 ?100 DEG C of oven dry, adopt dissolution with solvents, and carry out the concentration of step (4) and crystallization treatment 2 ~ 3 times, collect the crystal of separating out, continue 50 ?dry 12h at 100 DEG C, obtain described (R) ?(?) ?amygdalic acid.
10. method according to claim 9, is characterized in that: the bake out temperature of described refinement treatment be 70 ?80 DEG C; Or described solvent is any one in water, methyl alcohol, ethanol, Virahol, ethyl acetate, toluene, methylene dichloride and normal hexane.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114410699A (en) * | 2021-12-27 | 2022-04-29 | 安徽泰格生物科技有限公司 | Method for producing R-mandelic acid by biological catalysis |
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|---|---|---|---|---|
| CN1753990A (en) * | 2003-02-27 | 2006-03-29 | 巴斯福股份公司 | Modified nitrilases and their use in methods for producing carboxylic acids |
| CN102559781A (en) * | 2011-12-15 | 2012-07-11 | 重庆大学 | Method for preparing R-mandelic acid |
| CN103772188A (en) * | 2014-02-25 | 2014-05-07 | 成都百事兴科技实业有限公司 | Preparation method of R-(+)-alpha-cyclohexyl mandelic acid |
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| CN102559781A (en) * | 2011-12-15 | 2012-07-11 | 重庆大学 | Method for preparing R-mandelic acid |
| CN103772188A (en) * | 2014-02-25 | 2014-05-07 | 成都百事兴科技实业有限公司 | Preparation method of R-(+)-alpha-cyclohexyl mandelic acid |
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