CN103221370A - Preparation of isomerically pure substituted cyclohexanols - Google Patents
Preparation of isomerically pure substituted cyclohexanols Download PDFInfo
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- C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
- C12P41/004—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of alcohol- or thiol groups in the enantiomers or the inverse reaction
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Abstract
Disclosed is a method for preparing isomerically pure substituted cyclohexanols starting from a mixture of cis/trans substituted cyclohexanols, which comprises reacting the cis/trans mixture of a substituted cyclohexanol with a dicarboxylic acid anhydride in the presence of a lipase, to give the trans semi-ester which is separated from the unreacted substituted cyclohexanol cis isomer.
Description
The present invention relates to the mixture of the hexalin that is substituted from cis/trans, prepare the method for the pure hexalin that is substituted of isometry basically.
Can be by using multiple technologies well known by persons skilled in the art, for example, distillation, chromatogram, crystallization realize separating the mixture of cis/trans isomer.
WO 2005/073215 has described the method for the alkamine of production enantiomer-pure.It discloses existing under the condition of lipase and with succinyl oxide racemic alcohol has been carried out the enantioselectivity acylations and produce succinic acid half-ester, described succinic acid half-ester can with unreacted stage enantiomer separation.
EP 1069183 A2 instructed immobilized from false pseudomonas bacillus (Pseudomonas burkholderia) lipase of uncle Ke Shi in the presence of with succinyl oxide to racemic trans-2-methoxyl group hexalin carries out the enantioselectivity acylations.
Usually, fractionation by distillation is used to separate the cis/trans isomer.Yet under the situation of the hexalin that is substituted, because the distillation feature of the hexalin that is substituted, this method is not satisfied.Therefore, technical problem to be solved is to find to be used to separate the method for the mixture of the hexalin that cis/trans is substituted.
The present invention has solved this problem by the method that is provided for separating the hexalin that is substituted that is in the pure form of basic isometry, and described method comprises:
(i) lipase of the protein sequence of in existence has as SEQ ID No:2, being showed, perhaps have the protein sequence identical and have under the condition of lipase of at least 50% enzymatic activity of SEQ ID No:2 with shown whole aminoacid sequence at least 75% among the SEQ ID No:2, cis/trans mixture and dicarboxylic acids anhydride reactant with the hexalin that is substituted, produce trans half ester
(ii) from the unreacted hexalin syn-isomerism body that is substituted, separate trans half ester,
(iii) separate pure cis that is substituted of isometry or trans hexalin.
Surprisingly, the lipase that uses in inventive method causes the acylations of dicarboxylic acid anhydride to the hexalin that is substituted of trans forms with highly selective.The result of the inventive method is cis or the anti-type isomerism body that acquisition is in the hexalin that is substituted of the pure form of basic isometry.
" basic isometry pure " means cis or trans product substantially not by another kind of isometry body pollution.Therefore, " basic isometry pure " means acquisition at least 80%, and preferably at least 90%, more preferably at least 95%, at least 96,97,98,99% isomers especially.
The first step of inventive method:
The lipase of the protein sequence of in existence has as SEQ ID No:2, being showed, perhaps have the protein sequence identical and have under the condition of lipase of at least 50% enzymatic activity of SEQ ID No:2 with shown whole aminoacid sequence at least 75% among the SEQ ID No:2, cis/trans mixture (following formula A1 to A3) and dicarboxylic acids anhydride reactant with the hexalin that is substituted produce half ester (following formula C1 to C3) and unreacted isomers (following formula B1 to B3).It should be noted that by using optionally acylations anti-type isomerism body of described lipase.
In preferred embodiments, initial substance is the cis/trans mixture (following formula A3) of the hexalin that is substituted of 4-, the lipase of the protein sequence that it is showed in existence has as SEQ ID No:2, perhaps have the protein sequence identical and have under the condition of lipase of at least 50% enzymatic activity of SEQ ID No:2 with shown whole aminoacid sequence at least 75% among the SEQ ID No:2, with the dicarboxylic acids anhydride reactant, produce half ester (following formula C3) and unreacted isomers (following formula B3).
The hexalin that is substituted according to formula A1 to A3 that uses in the inventive method is replaced by R1.R1 can be any substituting group of inert under reaction conditions.
For example, R1 can be the C that is substituted or be unsubstituted
1-C
10Alkyl, the C that is substituted or be unsubstituted
3-C
8-cycloalkyl, the C that is substituted or be unsubstituted
2-C
10-alkenyl or alkynyl, the heterocycle that is substituted or be unsubstituted, the aryl that is substituted or be unsubstituted.
" the C that is unsubstituted
1-C
10" refer to have the saturated alkyl of the straight or branched of 1 to 10 carbon atom, for example methyl, ethyl, propyl group, 1-methyl-ethyl, 1-methyl-propyl, 2-methyl-propyl and 1-1 dimethyl ethyl etc.These are to be called the substituting group that is unsubstituted in the context of the invention.
Term " the C that is unsubstituted
3-C
8-cycloalkyl " mean monocyclic saturated hydrocarbon group base, for example cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, suberyl or ring octyl group with 3 to 8 carbocyclic ring members.
" the C that is unsubstituted
2-C
10Thiazolinyl " refer to have 2 to 10 carbon atoms and be in the unsaturated alkyl of straight or branched of two keys of any position, for example vinyl, 1-propenyl, 2-propenyl (allyl group), 1-methyl ethylene, 1-butylene base, crotyl, 3-butenyl, 1-methyl isophthalic acid-propenyl, 2-methyl isophthalic acid-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl etc.For purposes of the present invention, also comprise ring unsaturated alkyl in this definition, for example cyclopentadienyl, cyclohexadienyl, cycloheptadiene base, cyclooctadiene base with 5 to 8 carbocyclic ring members.
Term " C
2-C
10-alkynyl " mean the unsaturated alkyl that has 2 to 10 carbon atoms and contain at least one triple-linked straight or branched, for example ethynyl, 1-proyl, 2-propynyl (propargyl), ethyl acetylene base, 2-butyne base, 3-butynyl, 1-methyl-2-propynyl etc.
The term heterocycle for example means, " 5-; 6-, or 7 element heterocycles " wherein heterocyclic ring members atom comprises and is interpreted as 1,2,3 or 4 heteroatoms that is selected from N, O and S except carbon atom to mean the saturated undersaturated and heteroaromatic (being heteroaryl) with part.Example comprises:
◇ saturated with the undersaturated 5-of part, 6-or 7 element heterocycles, wherein heterocyclic ring members atom comprise except carbon atom to be selected from N, O and S 1,2 or 3 heteroatomic, and its be saturated or part undersaturated, for example: tetramethyleneimine-2-base, tetramethyleneimine-3 base, tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base, tetramethylene sulfide-2-base, tetramethylene sulfide-3-base, 1,3-dioxolane-4-base, different
Azoles alkane-3-base, different
Azoles alkane-4-base, different
Azoles alkane-5-base, isothiazolidine 3-base, isothiazolidine-4-base, isothiazolidine-5-base, pyrazolidine-3-base, pyrazolidine-4-base, pyrazolidine-5-base,
Azoles alkane-2-base,
Azoles alkane-4-base,
Azoles alkane-5-base, thiazolidine-2-Ji, thiazolidine-4-base, thiazolidine-5-base, imidazolidine-2-base, imidazolidine-4-base, 2-pyrroline-2-base, 2-pyrroline-3-base, 3-pyrroline-2-base, 3-pyrroline-3-base, piperidines-2-base, piperidines-3-base, piperidin-4-yl, 1,3-two
Alkane-5-base, tetrahydropyrans-2-base, tetrahydropyran-4-base, tetramethylene sulfide-2-base, hexahydro-pyridazine-3-base, hexahydro-pyridazine-4-base, hexahydropyrimidine-2-base, hexahydropyrimidine-4-base, 5-hexahydropyrimidine base and piperazine-2-base;
5 Yuans heteroaryls of ◇ (heteroaromatic base), wherein the ring members atom of heteroaryl comprises 1,2 or 3 heteroatoms that is selected from N, O and S except carbon atom, for example, pyrroles-1-base, pyrroles-2-base, pyrroles-3-base, thiophene-2-base, thiene-3-yl-, furans-2-base, furans-3-base, pyrazol-1-yl, pyrazole-3-yl, pyrazoles-4-base, pyrazoles-5-base, imidazoles-1-base, imidazoles-2-base, imidazol-4 yl, imidazoles-5-base
Azoles-2-base,
Azoles-4-base,
Azoles-5-base, different
Azoles-3-base, different
Azoles-4-base, different
Azoles-5-base, thiazol-2-yl, thiazole-4-base, thiazole-5-base, isothiazole-3-base, isothiazole-4-base, isothiazole-5-base, 1,2,4-triazolyl-1-base, 1,2,4-triazole-3-base 1,2,4-triazole-5-base, 1,2,4-
Diazole-3-base, 1,2,4-
Diazole-5-base and 1,2,4-thiadiazoles-3-base, 1,2,4-thiadiazoles-5-base;
6 Yuans heteroaryls of ◇ (heteroaromatic base), wherein the ring members atom of heteroaryl comprises 1,2 or 3 heteroatoms that is selected from N, O and S except carbon atom, for example pyridine-2-base, pyridin-3-yl, pyridin-4-yl, pyridazine-3-base, pyridazine-4-base, pyrimidine-2-base, pyrimidine-4-base, pyrimidine-5-base, pyrazine-2-base and 1,3,5-triazine-2-base.
" aryl that is unsubstituted " be phenyl, naphthyl, anthryl or phenanthryl in particular.
In the context of the present invention, " being substituted " means, and by comparing with the corresponding substituting group that is unsubstituted, one or more H atoms are by other atoms of inert in process of the present invention or molecular radical (alkyl for example, N (alkyl)
2, O-alkyl, S-alkyl, CN, NO
2, I, Cl, Br, F, carbonyl, carboxyl, R3 are COOR3, the 5-of alkyl, 6-, or 7 element heterocycles, aryl-back both as defined above) replace.In the context of inert substituent, " alkyl " means the C for the straight or branched saturated hydrocarbyl with 1 to 10 carbon atom
1-C
10-alkyl, for example methyl, ethyl, propyl group, sec.-propyl, butyl, isobutyl-etc.
Lipase-catalyzed acylation reaction of the present invention need use dicarboxylic acid anhydride.Each dicarboxylic acid anhydride sees that for example formula D can be used for this purpose in principle.
Preferably, R2 is C
2-C
10Alkyl.The example that is ready to use in the dicarboxylic acid of method of the present invention is: propanedioic acid (being daucic acid) acid anhydrides, Succinic Acid (being succsinic acid) acid anhydrides, pentanedioic acid (being glue acid) acid anhydrides, hexanodioic acid (being adipic acid) acid anhydrides, pimelic acid (pimelic acid) acid anhydrides, suberic acid (being cork acid) acid anhydrides, nonane diacid (being lepargylic acid) acid anhydrides, sebacic acid (being sebacic acid) acid anhydrides, undecane diacid acid anhydrides, dodecanedioic acid acid anhydrides.Especially preferably use C
3-C
8Dicarboxylic acid anhydride, for example succinyl oxide.
Preferably with equimolar amount, more preferably with employed dicarboxylic acid anhydride at least 10% excessive use the inventive method, to allow the complete acylations of anti-type isomerism body.The composition of initial substance, i.e. the per-cent of anti-type isomerism body in the mixture of the hexalin that cis/trans is substituted is for the decisive role that measures of the dicarboxylic acid anhydride of using in decision the inventive method.For example, for 30: 70 mixtures of cis/trans isomers, use 0.7 equivalent or 0.8 excessive normal dicarboxylic acid anhydride should play the almost completely purpose of the trans hexalin that is substituted of acylations.
The lipase that uses in the inventive method is selected from such lipase, described lipase have according to the aminoacid sequence of SEQ ID No:2 or be derived from showed as SEQ ID No:2, show by disappearance, replace, insert or its combination has changed at the most 25%, preferably at the most 20%, more preferably at the most 15%, the sequence of 10,9,8,7,6,5,4,3,2,1% amino-acid residue at the most especially.This means, the lipase that uses in the inventive method have with SEQ ID No:2 in the whole aminoacid sequence at least 75% showed, preferably at least 80%, more preferably at least 85%, at least 90,91,92,93,94,95,96,97,98,99% identical sequence especially.Per-cent sequence identity between two sequences is the function (being per-cent sequence identity=identical positional number/total number of positions x100) of the total same position number of sequence.
The lipase that uses in the inventive method can be expressed in the biology of producing lipase.The biology of producing lipase means can be natural or any biology by genetic modification (for example by in the genome that lipase gene is inserted into biology) yielding lipase in next life, described lipase have according to the aminoacid sequence of SEQ ID No:2 or with SEQ ID No:2 in the whole aminoacid sequence at least 75% showed, preferably at least 80%, more preferably at least 85%, at least 90,91,92,93,94,95,96,97,98,99% identical sequence especially.The example of producing the biology of lipase is Aspergillus (Aspergillus), genus arthrobacter (Arthrobacter), Alkaligenes (Alcaligenes), bacillus (Bacillus), brevibacterium sp (Brevibacterium), Rhodopseudomonas (Pseudomonas), chromobacterium (Chromobacterium), Candida (Candida), Fusarium (Fusarium), geotrichum (Geotrichum), Humicola (Humicola), mucor (Mucor), Pichia (Pichia), Penicillium (Penicillium), Rhizomucor (Rhizomucor), the microorganism of Rhizopus (Rhizopus) or Thermus (Thermus).Preferably in uncle's false pseudomonas bacillus of Ke Shi (being plant Burkholderia (Burkholderia plantarii)), express.
The preferred lipase gene that is inserted in the biology of producing lipase is a) as defined polynucleotide among the SEQ ID No:1, b) on the total length in the sequence encoding district of SEQ ID No:1 with the sequence of SEQ ID No:1 at least about 50%, preferably at least about 60%, more preferably at least about 70%, 75%, 80%, 85% or 90% and even more preferably at least about 95%, 96%, 97%, 98%, 99% or higher identical polynucleotide.Per-cent sequence identity between two sequences is the function (being per-cent sequence identity=identical positional number/total number of positions x100) of the total same position number of sequence.
Can cultivate the biology of producing lipase in a manner known way, for example by fermenting in nutritional medium, described substratum contains except nutrition, trace element with when suitable the microbiotic, for example, and the buffer system of stabilizing protein and enzyme.For example,, particularly described in embodiment 1 paragraph 1.1 and cultivated the biology of producing lipase, wherein used the plant Burkholderia as an example at US 6596520 B1.
Compare with SEQ ID No:2, the deutero-aminoacid sequence that uses in the inventive method should have at least 50%, and preferably 65%, more preferably 80%, especially more than the enzymatic activity of 90% SEQ ID No:2.In this context, the enzymatic activity of SEQ ID No:2 means the ability that causes the hexalin that trans selective acylation is substituted.Trans selectivity is at least 95%, more preferably at least 98%, even more preferably 99%.Can use 4-tertiary butyl hexalin as reference substance.
Can measure lipase activity (people .Review:Lipase assays for conventional and molecular screening:an overview such as Gupta itself by currently known methods, Biotechnol.Appl.Biochem. (2003) 37,63-71).In aqueous matrix, preferably measure catalytic activity by using tributyrin to test.In organic system, can use the phenylethyl alcohol test.
Fail to be folded into 3 of expection and tie up protein or the enzyme that shapes are produced non-activity usually.Fold usually and under the supervision of the molecule that is called the folding proteic specialization of assistant, carry out.In a preferred embodiment
◇ have according to the aminoacid sequence of SEQ ID No:2 or with SEQ ID No:2 in the whole aminoacid sequence at least 75% showed, preferably at least 80%, more preferably at least 85%, especially the lipase of at least 90,91,92,93,94,95,96,97,98,99% identical sequence by
Defined polynucleotide among ◇ such as the SEQ ID No:1, or on the total length in the sequence encoding district of SEQ ID No:1 with the sequence of SEQ ID No:1 at least about 50%, preferably at least about 60%, more preferably at least about 70%, 75%, 80%, 85% or 90%, with in addition more preferably at least about 95%, 96%, 97%, 98%, 99% or higher identical polynucleotide coded, described polynucleotide exist
Express in the ◇ plant Burkholderia.
Therefore, folding assistant's albumen and lipase coexpression in the plant Burkholderia, described folding assistant's albumen have according to the aminoacid sequence of SEQ ID No:3 or be derived from showed as SEQ ID No:3, with SEQ ID No:3 in the whole aminoacid sequence at least 85% showed, at least 90,91,92,93,94,95,96,97,98,99% identical sequence especially.
Can be used as the coarse extract of full cell and use the lipase that uses in the method for the present invention with height to the preparation (for example not celliferous extract) of the different purity of height purified form.The preferred lipase that uses the form that is in partial purification or highly purified protein soln.The plant Burkholderia cell of Shi Yonging is little in preferred embodiments, has and the comparable specific density of the specific density of substratum.Preferably, by using the suitable screen plate well known by persons skilled in the art and the microfiltration of strong pump to come to separate described cell from supernatant liquor.For example concentrate by using ultrafiltration as is known to persons skilled in the art to reach further.
Distinguish lipase that is used for conversion of substrate that uses in the solution and the stability that has increase usually and can be used for carrying out continuously or the immobilized lipase of batch reactions.Immobilized meaning uses method known to those skilled in the art to be combined in supporting agent on the solid support usually, is used for the method according to this invention (seeing below) then.When implementation method continuously, using immobilized lipase is embodiment preferred especially.For this purpose, can for example advantageously use lipase simultaneously it to be retained in pillar or the tubular reactor.
Lipase in multiple fixing of possibility the inventive method is arranged.Can be according to for example, as people .Biotechnology Letters 2000,22 (19): 1571-1575 such as Persson; US6,596, the method that 520B1-particularly describes among the embodiment 1, the coarse extract of immobilized whole-cell suspension or the supernatant liquor of cell culture, and purified protein soln.
The substratum (or fermented liquid) itself of producing the biology of lipase also can be at 50-150 ℃, and preferably 70-100 ℃, more preferably 75-85 ℃ and even 80-85 ℃ temperature (temperature out of spray-drier) spraying drying more preferably.Also can under the situation that has the supporting agent material, carry out spraying drying.Must select supporting agent in the mode that can carry out method of the present invention.Preferably, polysaccharide Star Dri 5 or mineral compound Na for example for example
2SO
4Be used as supporting agent.Based on the solids content of fermented liquid, the weight of supporting agent is by weight 5 to 200%, 10-200% by weight preferably, more preferably 20-150% and particularly preferably 50-100% by weight by weight.Also can use the purified lipase solution of this class methods immobilization.Based on solid matter, residual moisture is less than 10%, and preferably it is less than 7%, especially preferably is less than 5% residual moisture content.
In the method for the invention, use 0.5-1 more preferably, (immobilized) lipase of 1% amount by weight especially for initial substance (cis/trans mixture) 0.5-10%, 0.5-5% by weight preferably by weight.
Acylation reaction of the present invention can carry out not having or exist under the condition of solvent.Preferably it is at organic solvent, for example hydrocarbon, ether, or carry out in the alcohol.The solvent that is particularly suitable for reacting is:
The ◇ fat hydrocarbon is hexane, heptane or octane or its mixture, particularly sherwood oil for example, or
◇ is aromatic hydrocarbon based as benzene, toluene, and dimethylbenzene, or
The ◇ ethers is methyl tert-butyl ether (MTBE), tetrahydrofuran (THF) (THF), 1 for example, and 4-two
Alkane, or
The ◇ alicyclic ring same clan is pentamethylene, hexanaphthene for example, or
The ◇ tertiary alcohols is the trimethyl carbinol for example, tertiary amyl alcohol.
If the use solvent, with organic solvent diluting initial substance (cis/trans mixture), the feasible 0.2-5 mole that obtains initial substance (cis/trans mixture), preferred 0.5-2 mole, the solution of preferred 0.6-1.2 mole.
Can react continuously or in batches.For the enforcement on the technical scale, recommend to synthesize continuously, particularly use the lipase that support is arranged.
Second step of inventive method:
The mixture of half ester and unreacted isomers need separate half ester from unreacted isomers.This can finish easily by water extraction (for example half ester salt, the particularly water extraction of its basic metal or alkaline earth salt).Embodiment preferred is the water extraction in the presence of alkali (for example yellow soda ash or sodium hydroxide).Reason for this reason preferably, pH should be at 7.5-10,8-10 preferably, 8-9.5 more preferably is especially in the scope of 9-9.5.
The 3rd step of inventive method:
Which kind of isomers that depends on alcohol is needed, can check the organic phase that contains the syn-isomerism body, or contain the water of the anti-type isomerism body that is in the half ester form.The anti-type isomerism body of the alcohol that can use usual method for hydrolysis that half ester is cut into corresponding acid and want is for example by with alkali (NaOH for example, KOH, Na
2CO
3) or acid (H for example
2SO
4, HCl) handle.
Hereinafter, further describe the present invention by the example that provides.This explanation is not intended to limit the present invention.
Embodiment 1: a bottle pre-culture is shaken in preparation
With the Erlenmeyer flask of two 1000ml of velveteen plug sealing, with the aluminium foil covering and at 134 ℃ of sterilization 30min.With the scale glass cylinder of foil sealing 250ml and equally at 134 ℃ of sterilization 30min.
Use following component to prepare micro-salts solution: two liters are removed mineral water fully, 77.2g monohydrate potassium, 22.6g Zinc vitriol, 17.3g six ferrous sulfate hydrate ammoniums, 5.7g Manganous sulfate monohydrate, 1.2g Salzburg vitriol, 0.5g Cobalt monosulfate heptahydrate and 3.0g two hydration calcium chloride.
Make the 500ml substratum that comprises following component: 3.8g dry yeast extract powder, 0.5g potassium primary phosphate, 1.5g Secondary ammonium phosphate, 0.5g bitter salt, 5g trace elements salts solution/500g water.Use phosphoric acid that pH is transferred to 6.5.The substratum that filtration sterilization (0.22 μ m) is finished.Be transferred in each of two Erlenmeyer flasks the 200ml substratum is aseptic, use 1ml plant Burkholderia (LU8093) liquid storage flasks then in each case.
(30 ℃ and 200rpm were hatched the Erlenmeyer flask culture 12 hours in the vibrator orbit radius=25mm) at the vibrator incubator then.
Embodiment 2: the pre-culture of preparation fermentor tank
In stainless steel cask, make among 10 liters of embodiment 1 indicated substratum and use phosphoric acid that pH is transferred to 6.5.Then with media transfer to 21 liters of fermentor tanks being furnished with three traditional paddle stirrers.Then in 121 ℃ of sterilization fermentation jars 60 minutes and be cooled to 30 ℃.
The pre-culture of the Erlenmeyer flask that will grow fully is aseptic then is transferred in the fermentor tank, and moved fermentor tank under the following conditions 8 hours: the air of Ventilation Rate 0.5vvm compression, 30 ℃ of steady temperatures, overburden pressure=0.1bar, rotating speed=1000rpm regulates pH6.5 with the sodium hydroxide solution of 25% intensity and the phosphoric acid of 20% intensity.
Embodiment 3: produce lipase in fermentor tank
Loading cubic capacity continuously with following initial substance is 300 liters fermentor tank:
150 liters are removed mineral water fully, 1207g dry yeast extract powder, 160g potassium primary phosphate, 480g Secondary ammonium phosphate, 432g bitter salt, 1600g trace elements salts solution (embodiment's 1), 30ml
3062 (based on the foam reducing composition of polysiloxane).With the phosphoric acid of 20% intensity the pH of substratum is adjusted to 4.5.
In 121 ℃ of sterilization fermentation jars 60 minutes and be cooled to 30 ℃.Use the pre-culture aseptic inoculation fermentor tank of embodiment 2 then, and move fermentor tank under the following conditions: the air of Ventilation Rate 0.4vvm compression, 30 ℃ of steady temperatures, overburden pressure=0.3bar, rotating speed=550rpm regulates pH6.5 with the sodium hydroxide solution of 25% intensity and the phosphoric acid of 20% intensity.
After through four hours fermentation time, the filtration sterilization unit by hole size 0.2 μ m pumps into rapeseed oil.In first charging stage, carry out feed supplement according to following formula:
Feeding rate [g/l]=19.7*e
(0.11*t)
Wherein t represent in hour fermentation time
Stopped for first charging stage after 17 hours.Thereafter and then, pump into more rapeseed oils according to following formula:
Feeding rate [g/l]=168.6*e
(0.0069*t)
After 96 hours, stop the oil subsidy material, and continue the oil consumption of operation fermentor tank in substratum to the greatest extent.After this, fermentor tank is cooled to 4 ℃.And then, sample thief and measure the total dry matter (DM) of liquid and enzymatic activity (unit/ml).By infrared moisture analysis-e/or determining dry matter content.Use tributyrin as substrate titration determination enzymatic activity.Per minute is defined as an enzymatic unit from the amount that tributyrin discharges the butyro-liquid of 1 μ mol.
In 300 liters fermentor tank, measured 7.56% dry matter content, enzymatic activity is 10256U/ml.Fermentor tank weight is 186.8kg.Total dry matter reaches 14.1kg.Total enzymatic activity reaches 1916MU.1MU=1000000 unit.
Embodiment 4: produce immobilized lipase
To be present in lipase immobilization in the fermented liquid on sodium sulfate by spraying drying.
For this purpose, be added into 14.1kg sodium sulfate in the fermentor tank inclusion and in 1 hour with the dissolving of the minimum speed of fermentor tank.After this, with whole fermentor tank inclusion spraying dryings.Use 250m
3/ h nitrogen operation spray-drier.Temperature in is 180 ℃.Temperature out is 75 ℃.By two substance nozzle (two-substance nozzle) with in the fermentor tank inclusion nitrogen injection stream.Adjust the feasible ideal temperature out (about 15kg/h) that reaches of pump speed.Discharge from cyclonic separator by cyclonic separator separation dry powder and by the cell hub brake.
Obtain to amount to the dry powder of 25.6kg.The residual moisture content of powder is 1.8%.With the sample dissolution of dry powder in water and use tributyrin to measure enzymatic activity.The activity of powder is 67360 units/gDM.
The following transesterification of immobilized enzyme catalysis in organic medium:
Following test fixture is used for the suitability of the conversion of organic system: react in test reactor, described test reactor is made up of the 500ml jacketed vessel of being furnished with the propeller type stirrer of being made by glass.Agitator is driven by magnetic coupling by the engine (RZR2051 type) from Heidolph.By thermostatted (Huber Ministat) heating unit.
At 50ml
The about 0.5g fixture of weighing and write down accurate weight in the pipe.
It is 350rpm that thermostatted is set to 22 ℃ and agitator speed.
By glass funnel, load the exsiccant reactor with the 1-phenylethyl alcohol of 50.0g and the MTBE of 95.0g.Avoid any pollution of water because too much water resistance ends reaction.
Add through the preweighted fixture that contains lipase by glass funnel.
By adding the propionate initial action of 20.4g.
Add propionate after 60 minutes, sampling (about 1ml) and filtering immediately pass through 0.2 μ m syringe filter (
30/02RC, Schleicher ﹠ Schuell).
Filtered sample of 100 μ l and 900 μ l HPLC elutriants (acetonitrile 20%, methyl alcohol 40%, trifluoroacetic acid 1% and water 39%) are placed 2ml
In the container.With 100 these solution of μ l then place the HPLC pipe and similarly replenish 900 μ l HPLC elutriants, wherein pipe seals.By the HPLC analytic sample.
Lipase activity unit in the organic system is PEU (a phenylethyl alcohol unit).1PEU is under above-mentioned test condition, and per minute forms the amount of the lipase of 1 μ mol phenylethyl propionate (PEP) from phenylethyl alcohol catalysis.
Calculate parameters needed:
Phenylethyl propionate concentration (PEP) [mmol/l]
Reaction volume [l]
Time (reaction times) [min]
Weight (amount of the enzyme of use) [g]
Calculate enzymatic activity
Calculate specific activity
The fixture that obtains among the embodiment has the specific activity of 842PEU/g fixture.
Embodiment 5: preparation cis-4-tertiary butyl hexalin
(726mol 1eq) introduces and is equipped with in the reaction vessel of 453l MTBE (1.6mol) with cis/trans (30: the 70) mixture of 113.4kg4-tertiary butyl hexalin.Add 58.1kg succinyl oxide (580mol; 0.8eq) and 1.1kg be fixed on Na
2SO
4On the lipase from the plant Burkholderia (by weight 1%; See above example).At 20 ℃ of stirred reaction mixtures.Check reaction method by gas-chromatography.Because 23h after the reaction times, still detects the anti-type isomerism body (table 1, No. 3) of 0.9GC area %, restir reaction mixture 27h.
Table 1: isometry ratio cis/trans
| Numbering | Survey | Cis (%) | Trans (%) |
| 1 | Beginning | 30.6 | 69.4 |
| 2 | Behind the 17h | 94.6 | 5.4 |
| 3 | Behind the 23h | 99.1 | 0.9 |
| 4 | Behind the 50h | 99.9 | 0.1 |
By diatomite (Kieselgur, for example
) filter reaction mixture and with other MTBE rinsing vessel and MTBE.The MTBE that distilled water is added into filtered reaction mixture and rinse step mutually after, add 25%NaOH solution up to reaching pH9.3 at 20 ℃ of substeps.Add other distilled water and be separated.Extract twice water (pH9.3) again with MTBE.Make up organic phase, and remove solvent up to obtaining white suspension by distillation (50mbar, maximum 40 ℃).In rotary evaporator, further concentrate this suspension (109kg).
Table 2: extract back isometry ratio cis/trans
| Numbering | Survey | Cis (%) | Trans (%) |
| 1 | The organic phase of combination | 99.3 | 0.7 |
| 2 | After the distillation | 99.6 | 0.4 |
For distillation, use and to have the post that carries packing material (packed column, i.e. Raschigrings, 8x8mm) and the simple water distilling apparatus of the solid bridge (condenser of quenching) that is heated.Transition temperature is 118 ℃ of 26mbar water spray vacuum.
Generally speaking, obtained to be in 24.8kg cis-4-(1, the 1-the dimethyl ethyl)-hexalin (73% output) of white solid form
Embodiment 6: preparation cis-4-tertiary butyl hexalin
Mix 1g4-tertiary butyl hexalin (cis/trans: 30: 70; Be 1eq) and 10ml toluene (being 0.6mol).Add 0.5g succinyl oxide (being 0.8eq) and 0,1g is fixed on Na
2SO
4On lipase (promptly by weight 10% /). identical among reaction conditions and the embodiment 1.
Also further handle at 20 ℃ of stirred reaction mixture 24h as embodiment 5.Detection after analysis 1 and the 24h churning time:
| Numbering | Reaction times | Cis (%) | Trans (%) |
| 1 | 1h | 73.2 | 26.8 |
| 2 | 24h | 100 | - |
Embodiment 7: but 20 ℃ 24h after different solvent (0.6mol) similar with embodiment 6
| Numbering | Solvent | Cis (%) | Trans (%) |
| 1 | MTBE | 99 | 0.1 |
| 2 | THF | 98 | - |
| 3 | Hexanaphthene | 79 | 19 |
| 4 | Sherwood oil | 81 | 17 |
Embodiment 8: but 20 ℃ 24h after 1.2mol solvent similar to embodiment
| Numbering | Solvent | Cis (%) | Trans (%) |
| 1 | MTBE | 100 | - |
| 2 | Toluene | 100 | - |
Claims (13)
1. prepare the method for the pure hexalin that is substituted of basic isometry, described method comprises:
I) lipase of the protein sequence of in existence has as SEQ ID No:2, being showed, perhaps have the protein sequence identical and have under the condition of lipase of at least 50% enzymatic activity of SEQ ID No:2 with shown whole aminoacid sequence at least 75% among the SEQ ID No:2, cis/trans mixture and dicarboxylic acids anhydride reactant with the hexalin that is substituted, produce trans half ester
Ii) from the unreacted hexalin syn-isomerism body that is substituted, separate trans half ester,
Iii) separate cis or the trans hexalin that is substituted.
2. according to the method for claim 1, wherein the lipase that uses in the step (i) be by a) as defined polynucleotide among the SEQ ID No:1, b) polynucleotide identical with the sequence at least 75% of SEQ ID No:1 are coded on the total length in the sequence encoding district of SEQ ID No:1.
3. according to the method for claim 2, wherein lipase is expressed in plant Burkholderia (Burkholderia plantarii).
4. according to each method in the aforementioned claim, wherein in step (i), use C
2-C
10Dicarboxylic acid anhydride.
5. according to the method for claim 4, wherein use succinyl oxide.
6. according to each method in the aforementioned claim, wherein the lipase in the step (i) is immobilized.
7. according to each method in the aforementioned claim, wherein step (i) be reflected at hydrocarbon, ether as solvent, or carry out in the alcohol.
8. according to the method for claim 7, wherein solvent is selected from toluene, sherwood oil, MTBE, tetrahydrofuran (THF), or the group of hexanaphthene composition.
9. according to each method in the aforementioned claim, wherein the separation of step in (ii) is to extract by the pH at 8-10 to carry out.
10. according to each method in the aforementioned claim, the mixture of the use therein hexalin that is substituted is substituted with respect to 4 of OH-group.
11., wherein use 4-tertiary butyl hexalin according to the method for claim 10.
12. be used for the method for immobilized protein on solid support, wherein by spraying drying immobilization fermentation liquid.
13., wherein use Na according to the method for claim 12
2SO
4As solid support.
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| EP10192721 | 2010-11-26 | ||
| EP10192721.8 | 2010-11-26 | ||
| PCT/IB2011/055199 WO2012069974A1 (en) | 2010-11-26 | 2011-11-21 | Preparation of isomerically pure substituted cyclohexanols |
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| CN103221370A true CN103221370A (en) | 2013-07-24 |
| CN103221370B CN103221370B (en) | 2015-02-11 |
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| Country | Link |
|---|---|
| EP (1) | EP2643281A4 (en) |
| JP (1) | JP2014500020A (en) |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1280189A (en) * | 1999-07-09 | 2001-01-17 | Basf公司 | Immobilized lipase |
| CN1914190A (en) * | 2004-01-29 | 2007-02-14 | 巴斯福股份公司 | Method for producing enantiomer-pure aminoalcohols |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4665028A (en) * | 1982-10-06 | 1987-05-12 | Novo Industri A/S | Method for production of an immobilized enzyme preparation by means of a crosslinking agent |
| DE10151292A1 (en) * | 2001-10-22 | 2003-04-30 | Basf Ag | New bacterial lipase mutants, useful for enantioselective conversion, e.g. acylation of alcohols, have increased specific activity |
| EP2248906A4 (en) * | 2008-01-23 | 2012-07-11 | Ajinomoto Kk | Method of producing l-amino acid |
| KR20110087273A (en) * | 2008-09-29 | 2011-08-02 | 아커민 인코퍼레이티드 | Process for accelerated capture of carbon dioxide |
-
2011
- 2011-11-21 WO PCT/IB2011/055199 patent/WO2012069974A1/en active Application Filing
- 2011-11-21 CN CN201180056293.XA patent/CN103221370B/en not_active Expired - Fee Related
- 2011-11-21 JP JP2013540467A patent/JP2014500020A/en not_active Withdrawn
- 2011-11-21 EP EP11843794.6A patent/EP2643281A4/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1280189A (en) * | 1999-07-09 | 2001-01-17 | Basf公司 | Immobilized lipase |
| CN1914190A (en) * | 2004-01-29 | 2007-02-14 | 巴斯福股份公司 | Method for producing enantiomer-pure aminoalcohols |
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| EP2643281A4 (en) | 2014-06-04 |
| CN103221370B (en) | 2015-02-11 |
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| EP2643281A1 (en) | 2013-10-02 |
| JP2014500020A (en) | 2014-01-09 |
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