CA2075699A1 - Process for increasing the enantio-selectivity of a candida lipase in the esterification of chiral alcohols, and an immobilized candida lipase - Google Patents
Process for increasing the enantio-selectivity of a candida lipase in the esterification of chiral alcohols, and an immobilized candida lipaseInfo
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- CA2075699A1 CA2075699A1 CA002075699A CA2075699A CA2075699A1 CA 2075699 A1 CA2075699 A1 CA 2075699A1 CA 002075699 A CA002075699 A CA 002075699A CA 2075699 A CA2075699 A CA 2075699A CA 2075699 A1 CA2075699 A1 CA 2075699A1
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
- C12N11/082—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer obtained by reactions only involving carbon-to-carbon unsaturated bonds
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- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
- 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
Abstract Immobilized lipase from a microorganism of the genus Candida which is covalently bonded to epsilon-amino groups of the lysine by N-alkylation with opened epoxide groups of an epoxide-activated macroscopic carrier, a process for its preparation, its use in a process for the enantioselective esterification of enantiomer mixtures of an alcohol which contains at least one asymmetric center in the molecule with the aid of an enol ester, and a process for the enantioselective transesterification of an enantiomer mixture of an alcohol which contains at least one asymmetric center in the molecule, with the aid of the immobilized lipase in the presence of an enol ester.
Description
~ c~9 Process for increasing the enantioselectivity of a Candida lipase in the esterification of chiral alcohols, and an immobilized Carldida lip~se.
The invention relates to a process for increasing the enantioselectivity, activity and stability of a lipase from a microorganism of the genus Candida in an enzymatic transesterification process in which ~ chiral alcohol which contains at least one asymmetric center in the molecule is esterified enantioselectively with the aid of an enol ester, to an immobilized Candida lipase, and to a process for its preparation.
Chiral, enantiomerically pure alcohols are important for a wide range of uses, for example for the synthesis of pharmaceutical active substances or agrochemicals~
They can be prepared, for example, by enantioselective transesterification, an enantiomer mixture of a chiral alcohol being reacted in the presence of a car~oxylate with the aid of a hydrolase. Since hydrolases generally catalyze both the forward reaction and the reverse reaction, the desired end product of such reactions is frequently only foxmed very slowly and in insufficient optical purity. M. Degueil-Castaing et al., Tetrahedron Letters, Vol 28, 9 (1987), 953-954, therefore propose to employ an enol ester as carboxylate since a reverse reaction can no longer take place. However, as can be seen from Zakrzewska et al., Acta Med. Pol., 29, (1988), 1-2, page 44, the aldehydes which are foxmed from the enol ester during the transesterification may deactivate the enzyme by reacting with terminal functional groups of amino acids of the enzyme. US-PS-4,963,492 describes a process for the enzymatic racemate resolution of enantiomer mixtuxes of a racemic alcohol with, or in, a vinyl e~ter with the aid of a lipase, claiming that the liberated aldehyde does not deactivate the lipase.
~owever, it has emerged that repeated use of a Candida lipase in such a process causes a very rapid decline in both its activity and its selectivity.
~-? J~ 9 C.J. Gr~y et al., Enzyme Microb. Technol., Vol .
12 (1~90), pages 800 to 807, disclose that the stability of a Candida cylindracea lipase can be increased for repeated use by a range of immobilization techniques.
However, this publication doe!s not mention trans-esterifications using enol esters.
Surprisingly, it has now been found that the stability to aldehyde as well as the activity and, exceptionally, also the selectivity of a Candida lipase are increased when the lipase i5 immobilized before use in a transesterification process using enol esters by reacting epsilon~amino groups of the lysine of the lipase with epoxide groups of an epoxide-activated macroporous carrier, as a result of which N-alkylation takes place.
This immobilization increase~ both the resi~tance of the lipase to aldehydes and its activity and substrate selectivity compared with a non-immobilized lipase, and the substrate selectivity does not decline on repPated use, but remains entirely constant.
The invention therefore relates to an ~bilized lipase from a microor~ism of the genus Candida in which epRilon-amino groupR of the lysines in the lipase are bonded covalently by N-alkylation via opened epoxide groups of an epoxide-activated macroporous carrier.
Lipase from a microorganism of the genus Candida is understood as meaning unpurified microorgani~m suspensions of the genufi Candida as well as purified enzyme fractions. Specie~ from the genu~ Candida (C.) are, for example, C.antarctica. C. ruqo~a C.
cylindracea. A lipase from C.cylindracea i8 preferably employed. Lipases from the genu~ Candida are commercially available in various degrees of purity.
An epoxide-activated macroporous carrier is employed for immobilizing the lipase. It is prepared, for example, by suspension polymerization of vinyl acetate and a monomer which is copolymerizable with vinyl acetate, for example N,N'-divinylethyleneurea, in water, _ 3 _ 2~?~r ~9 partial hydrolysis of the acetate groups to give hydroxyl groups, followed by a reaction with epichlorohydrin which reacts with free hydroxyl groupspreserving the epoxide ring.
This results in the formation of spacer groups with reactive epoxide gro~lps in the polymerizate which are suitable for linking the carrier with a wide range of substrates. Such carriers and their preparation are described, for example, in K.Burg et al., Angew.
Makromol. Chem. 157, ~1988), pages 105 to 121. They are commercially available. Particularly preferably used carriers are Eupergit C (Rohm Pharma, Germany) or VA-epoxy-biosynth, Riedel de Haen (Germany).
A.N. Glazer, "The proteins", ed. H. Neurath and R.L.
~ill, Academic press London, 1976, Volume II, pages 1-109, discloses that in this type of enzyme immobilizationit is mainly the epsilon-amino groups of the lysine which react with epoxide groups of the carrier, amino groups of the lysine being alkylated.
~ his means that the lipaRe ac~ording to the invention is alkylated on epsilon-amino groups of the lysine of the lipase via opened epoxide groups of the carrier, i.e. it is bonded covalently with the carrier.
~ he invention also relate~ to a process for the preparation of the immobilized lipase according to the invention. To this end, an epoxide-activated macroporous carrier i8 brought into contact with an aqueous enzyme solution of a Candida lipase at temperatures from 15C up to the deactivation temperature of the lipase, preferably from 18 to 30C, for example by moving in a shaker flask.
The ratio of carrier and lipase to each other must at least be such that one epoxide group of the carrier is available in the reaction mixture per free amino group of the lysine in the lipase. It i8 preferred to use approx.
0.05 to 0.1 g of lipase per 1 g of carrier. The lipase can be dissolved in pure water, or in a buffer or salt solution. In this process, epsilon-amino groups of the lysine in the lipase react with epoxide groups of the carrier, an N-alkylation, iOe. a covalent bond between .. ¢~ 9 lipase and carrier, being formed. When the reaction is complete, salts, for example NaCl, are optionally added to the reaction mixture, and the Lmmobilized lipase is then filtered off and washed with buffer solution. The immobilized lipase can be stored in the buffer or under humid or, alternatively, dry conditions until use.
The lipase which has been prepared in this manner is employed for the enantioselective esterification of enantiomer mixtures of chiral alcohols by transesterific-ation using enol esters.
The invention then also relates to a proce~s forthe enantioselective esterification of an alcohol which contains at least one asymmetric center in the molecule, compris~ng esterify~g the alcohol in the presence of an enol ester and of a lipase from a microorganism of the genus Candida, which is covalently bonded to the epsilon-amino groups of the lysine in the lipase by N-alkylation by opened epoxide groups of an epoxide-activated macroscopic carrier and isolat~g the resulting ester of the chiral alcohol and/or the unreacted alcohol from the reaction mixture.
The alcohol employed has at least one asymmetric center and is therefore optically active. It can exist in the form of racemic enantiomer mixtures or those mixtures in which one or the other enantiomer i3 enriched.
The immobilized lipase according to the invention can react both primary and secondary alcohols. The alcohol can al~o be a dialcohol having two asymmetric centexs in the molecule, mixtures of R,S-alcohols, S,R-alcohols, R,R-alcohols or S,S-alcohols being possible. It is preferred to employ secondary alcohols having one asymmetric center in the molecule as substrate.
Enol esters which can be employed are, for example, enol esters disclosed in US-PS-4,963,492 Prefer-ably employed esters are vinyl ester~ of lower organic acids, particularly preferably vinyl acetate, vinyl propionate and ~inyl butyrate.
_ 5 ~`r`'~ s$ ~
-To carry out the r~action, 2 to 30 g, preferably 6 to 10 g, of Lmmobilized lipase and at least 5 equivalent~ of enol ester, if appropriate mixed with a diluent which is inert under the reaction conditions, are reacted per gram of the enantiomer mixture of the chiral alcohol, with stirring or shaking, at temperatures from 15C up to the deactivation temperature of the lipase, preferably at room temperature.
The reaction can be carried out in diluents which are inert under the reaction conditions, or the enol ester itself which is used for the transesterification is also employed as diluent in great excess. The reaction is prefer~bly not carried out in a diluent which is inert under the reaction conditions, but in the enol ester which is used for the reaction.
The reaction is expediently carried out at temperatures at which the activity of the lipase is at its highest.
This temperature i8 indicated by the manufacturer or can be determined by sLmple tests. Depending on the alcohol mixture, enol ester and specificity of the lipase employ-ed, the R ester, or the S ester, or the R,S ester, or the S,R est~r of the alcohol employed is chiefly formed, while none, or only a small amount, of the corresponding S or R alcohol, or S,R, R,S, R,R or S,S alcohol, is reacted.
The procedure of the reaction is monitored by suitable methods known in enzyme chemistry, for example with the aid of gas-chromatographic methods.
When the desired enantiomeric purity of the product is reached, the reaction is stopped, and the reaction mixture i8 worked up. To this end, the immobilized lipase is filtered off, and the mother liquor is subjected to a suitable separating operation where the desired products are isolated. Separation of the desired products from the 3S reaction mixture can be carried out by customary methods, for example with the aid of extraction, distillation or chromatography.
6 ~ ~ ~J - ~ ~r ~?9 In a preferred embodiment, a racemic mixture of an alcohol is stirred or shaken at room temperature with Candida cylindracea lipasel Lmmobilized with the aid of VA-epoxy-biosynth, Riedel-de-Haen, Germany, in vinyl acetate. When the desired enarltiomeric excess of the resulting R ester or S ester, or R, S or S,R ester, is reached, the enzyme is filtered off, and the desired products are resolved by distillation or by chromato-graphy.
It has emerged that the resistance of-the lipase against acetaldehyde as well as its activity and selec-tivity, increased in the reaction according to the invention compared with a non-immobilized lipase. The more than 5-fold increase in selectivity, which remained entirely constant on repeated use of the immobilized lipase, was particularly surprising.
The Lmmobilized lipase and its use in the enantioselective esterification according to the inven tion therefore represent an enrichment of the art.
Example 1 10.O g of VA-epoxy-biosynth (Riedel-de-Haen, Germany) were suspended in 70 ml of 0.1 N phosphate buffer pH 7.00, 300 mg of lipase of Candida cylindracea lipa~e (AY-30, Amano Pharm. Co., Japan) were added, and the mixture was then shaken for 3 days at 27C and 80 rp~. 70 ml of sodium chloride solution were added, and the solid was filtered off, wafihed twice with 30 ml portions of 0.05 N phosphate buffer pH 7 and dried. The specific activity of the immobilized lipase prepared in this manner was determined by titrating the resulting acetic acid with 0.1 N sodium hydroxide solution during the hydrolysis of triacetin in 0.1 N phosphate buffer pH
7.00 and was 5070 ~mol min~' g~'. The specific activity of the non-immobilized lipase under the same condi~ions was 13 ~mol min~l g~1~
~xam~le 2 1 g of racemic endo-noxhorn-5-en-2-ol (9 mmol) was mixed with 200 mg of Candida cylindracea lipase (Ay-30, Amano Pharm. Co., Japan) and the mixture was shaken in 10 ml of vinyl acetate at 20C and 200 rpm. The pxocedure of the reaction was monitored by gas chromato-graphy. After 4 hours, the reaction was stopped. The lipase was filtered off, and the mother liquor was evaporated in vacuo. Conventional column chromatography of the residue on silica gel gave (+)-endo-norborn-5-en~
The invention relates to a process for increasing the enantioselectivity, activity and stability of a lipase from a microorganism of the genus Candida in an enzymatic transesterification process in which ~ chiral alcohol which contains at least one asymmetric center in the molecule is esterified enantioselectively with the aid of an enol ester, to an immobilized Candida lipase, and to a process for its preparation.
Chiral, enantiomerically pure alcohols are important for a wide range of uses, for example for the synthesis of pharmaceutical active substances or agrochemicals~
They can be prepared, for example, by enantioselective transesterification, an enantiomer mixture of a chiral alcohol being reacted in the presence of a car~oxylate with the aid of a hydrolase. Since hydrolases generally catalyze both the forward reaction and the reverse reaction, the desired end product of such reactions is frequently only foxmed very slowly and in insufficient optical purity. M. Degueil-Castaing et al., Tetrahedron Letters, Vol 28, 9 (1987), 953-954, therefore propose to employ an enol ester as carboxylate since a reverse reaction can no longer take place. However, as can be seen from Zakrzewska et al., Acta Med. Pol., 29, (1988), 1-2, page 44, the aldehydes which are foxmed from the enol ester during the transesterification may deactivate the enzyme by reacting with terminal functional groups of amino acids of the enzyme. US-PS-4,963,492 describes a process for the enzymatic racemate resolution of enantiomer mixtuxes of a racemic alcohol with, or in, a vinyl e~ter with the aid of a lipase, claiming that the liberated aldehyde does not deactivate the lipase.
~owever, it has emerged that repeated use of a Candida lipase in such a process causes a very rapid decline in both its activity and its selectivity.
~-? J~ 9 C.J. Gr~y et al., Enzyme Microb. Technol., Vol .
12 (1~90), pages 800 to 807, disclose that the stability of a Candida cylindracea lipase can be increased for repeated use by a range of immobilization techniques.
However, this publication doe!s not mention trans-esterifications using enol esters.
Surprisingly, it has now been found that the stability to aldehyde as well as the activity and, exceptionally, also the selectivity of a Candida lipase are increased when the lipase i5 immobilized before use in a transesterification process using enol esters by reacting epsilon~amino groups of the lysine of the lipase with epoxide groups of an epoxide-activated macroporous carrier, as a result of which N-alkylation takes place.
This immobilization increase~ both the resi~tance of the lipase to aldehydes and its activity and substrate selectivity compared with a non-immobilized lipase, and the substrate selectivity does not decline on repPated use, but remains entirely constant.
The invention therefore relates to an ~bilized lipase from a microor~ism of the genus Candida in which epRilon-amino groupR of the lysines in the lipase are bonded covalently by N-alkylation via opened epoxide groups of an epoxide-activated macroporous carrier.
Lipase from a microorganism of the genus Candida is understood as meaning unpurified microorgani~m suspensions of the genufi Candida as well as purified enzyme fractions. Specie~ from the genu~ Candida (C.) are, for example, C.antarctica. C. ruqo~a C.
cylindracea. A lipase from C.cylindracea i8 preferably employed. Lipases from the genu~ Candida are commercially available in various degrees of purity.
An epoxide-activated macroporous carrier is employed for immobilizing the lipase. It is prepared, for example, by suspension polymerization of vinyl acetate and a monomer which is copolymerizable with vinyl acetate, for example N,N'-divinylethyleneurea, in water, _ 3 _ 2~?~r ~9 partial hydrolysis of the acetate groups to give hydroxyl groups, followed by a reaction with epichlorohydrin which reacts with free hydroxyl groupspreserving the epoxide ring.
This results in the formation of spacer groups with reactive epoxide gro~lps in the polymerizate which are suitable for linking the carrier with a wide range of substrates. Such carriers and their preparation are described, for example, in K.Burg et al., Angew.
Makromol. Chem. 157, ~1988), pages 105 to 121. They are commercially available. Particularly preferably used carriers are Eupergit C (Rohm Pharma, Germany) or VA-epoxy-biosynth, Riedel de Haen (Germany).
A.N. Glazer, "The proteins", ed. H. Neurath and R.L.
~ill, Academic press London, 1976, Volume II, pages 1-109, discloses that in this type of enzyme immobilizationit is mainly the epsilon-amino groups of the lysine which react with epoxide groups of the carrier, amino groups of the lysine being alkylated.
~ his means that the lipaRe ac~ording to the invention is alkylated on epsilon-amino groups of the lysine of the lipase via opened epoxide groups of the carrier, i.e. it is bonded covalently with the carrier.
~ he invention also relate~ to a process for the preparation of the immobilized lipase according to the invention. To this end, an epoxide-activated macroporous carrier i8 brought into contact with an aqueous enzyme solution of a Candida lipase at temperatures from 15C up to the deactivation temperature of the lipase, preferably from 18 to 30C, for example by moving in a shaker flask.
The ratio of carrier and lipase to each other must at least be such that one epoxide group of the carrier is available in the reaction mixture per free amino group of the lysine in the lipase. It i8 preferred to use approx.
0.05 to 0.1 g of lipase per 1 g of carrier. The lipase can be dissolved in pure water, or in a buffer or salt solution. In this process, epsilon-amino groups of the lysine in the lipase react with epoxide groups of the carrier, an N-alkylation, iOe. a covalent bond between .. ¢~ 9 lipase and carrier, being formed. When the reaction is complete, salts, for example NaCl, are optionally added to the reaction mixture, and the Lmmobilized lipase is then filtered off and washed with buffer solution. The immobilized lipase can be stored in the buffer or under humid or, alternatively, dry conditions until use.
The lipase which has been prepared in this manner is employed for the enantioselective esterification of enantiomer mixtures of chiral alcohols by transesterific-ation using enol esters.
The invention then also relates to a proce~s forthe enantioselective esterification of an alcohol which contains at least one asymmetric center in the molecule, compris~ng esterify~g the alcohol in the presence of an enol ester and of a lipase from a microorganism of the genus Candida, which is covalently bonded to the epsilon-amino groups of the lysine in the lipase by N-alkylation by opened epoxide groups of an epoxide-activated macroscopic carrier and isolat~g the resulting ester of the chiral alcohol and/or the unreacted alcohol from the reaction mixture.
The alcohol employed has at least one asymmetric center and is therefore optically active. It can exist in the form of racemic enantiomer mixtures or those mixtures in which one or the other enantiomer i3 enriched.
The immobilized lipase according to the invention can react both primary and secondary alcohols. The alcohol can al~o be a dialcohol having two asymmetric centexs in the molecule, mixtures of R,S-alcohols, S,R-alcohols, R,R-alcohols or S,S-alcohols being possible. It is preferred to employ secondary alcohols having one asymmetric center in the molecule as substrate.
Enol esters which can be employed are, for example, enol esters disclosed in US-PS-4,963,492 Prefer-ably employed esters are vinyl ester~ of lower organic acids, particularly preferably vinyl acetate, vinyl propionate and ~inyl butyrate.
_ 5 ~`r`'~ s$ ~
-To carry out the r~action, 2 to 30 g, preferably 6 to 10 g, of Lmmobilized lipase and at least 5 equivalent~ of enol ester, if appropriate mixed with a diluent which is inert under the reaction conditions, are reacted per gram of the enantiomer mixture of the chiral alcohol, with stirring or shaking, at temperatures from 15C up to the deactivation temperature of the lipase, preferably at room temperature.
The reaction can be carried out in diluents which are inert under the reaction conditions, or the enol ester itself which is used for the transesterification is also employed as diluent in great excess. The reaction is prefer~bly not carried out in a diluent which is inert under the reaction conditions, but in the enol ester which is used for the reaction.
The reaction is expediently carried out at temperatures at which the activity of the lipase is at its highest.
This temperature i8 indicated by the manufacturer or can be determined by sLmple tests. Depending on the alcohol mixture, enol ester and specificity of the lipase employ-ed, the R ester, or the S ester, or the R,S ester, or the S,R est~r of the alcohol employed is chiefly formed, while none, or only a small amount, of the corresponding S or R alcohol, or S,R, R,S, R,R or S,S alcohol, is reacted.
The procedure of the reaction is monitored by suitable methods known in enzyme chemistry, for example with the aid of gas-chromatographic methods.
When the desired enantiomeric purity of the product is reached, the reaction is stopped, and the reaction mixture i8 worked up. To this end, the immobilized lipase is filtered off, and the mother liquor is subjected to a suitable separating operation where the desired products are isolated. Separation of the desired products from the 3S reaction mixture can be carried out by customary methods, for example with the aid of extraction, distillation or chromatography.
6 ~ ~ ~J - ~ ~r ~?9 In a preferred embodiment, a racemic mixture of an alcohol is stirred or shaken at room temperature with Candida cylindracea lipasel Lmmobilized with the aid of VA-epoxy-biosynth, Riedel-de-Haen, Germany, in vinyl acetate. When the desired enarltiomeric excess of the resulting R ester or S ester, or R, S or S,R ester, is reached, the enzyme is filtered off, and the desired products are resolved by distillation or by chromato-graphy.
It has emerged that the resistance of-the lipase against acetaldehyde as well as its activity and selec-tivity, increased in the reaction according to the invention compared with a non-immobilized lipase. The more than 5-fold increase in selectivity, which remained entirely constant on repeated use of the immobilized lipase, was particularly surprising.
The Lmmobilized lipase and its use in the enantioselective esterification according to the inven tion therefore represent an enrichment of the art.
Example 1 10.O g of VA-epoxy-biosynth (Riedel-de-Haen, Germany) were suspended in 70 ml of 0.1 N phosphate buffer pH 7.00, 300 mg of lipase of Candida cylindracea lipa~e (AY-30, Amano Pharm. Co., Japan) were added, and the mixture was then shaken for 3 days at 27C and 80 rp~. 70 ml of sodium chloride solution were added, and the solid was filtered off, wafihed twice with 30 ml portions of 0.05 N phosphate buffer pH 7 and dried. The specific activity of the immobilized lipase prepared in this manner was determined by titrating the resulting acetic acid with 0.1 N sodium hydroxide solution during the hydrolysis of triacetin in 0.1 N phosphate buffer pH
7.00 and was 5070 ~mol min~' g~'. The specific activity of the non-immobilized lipase under the same condi~ions was 13 ~mol min~l g~1~
~xam~le 2 1 g of racemic endo-noxhorn-5-en-2-ol (9 mmol) was mixed with 200 mg of Candida cylindracea lipase (Ay-30, Amano Pharm. Co., Japan) and the mixture was shaken in 10 ml of vinyl acetate at 20C and 200 rpm. The pxocedure of the reaction was monitored by gas chromato-graphy. After 4 hours, the reaction was stopped. The lipase was filtered off, and the mother liquor was evaporated in vacuo. Conventional column chromatography of the residue on silica gel gave (+)-endo-norborn-5-en~
2-yl acetate and (-)-endo-norborn-5-en-2-ol.
The test results regarding lipase activity, conversion rate and enantiomeric ratios are summarized in Table 1 Example 3 was carried out as Example 2, with the difference that the lipase which had been immobilized as in Example 1 was employed. The test results regarding lipase activity, conversion rate and enantiomeric ratios are summarized in Table 1.
20 Table 1 U Lipase Act. % ee ~ ee S
(-) Alc. (+)Est 1. C 39 51.4 66.5 8 2. C 1 11.0 54.0 4 25 3. C
1. I 100 62.2 91.3 42 2. I 50 66.0 91.7 42
The test results regarding lipase activity, conversion rate and enantiomeric ratios are summarized in Table 1 Example 3 was carried out as Example 2, with the difference that the lipase which had been immobilized as in Example 1 was employed. The test results regarding lipase activity, conversion rate and enantiomeric ratios are summarized in Table 1.
20 Table 1 U Lipase Act. % ee ~ ee S
(-) Alc. (+)Est 1. C 39 51.4 66.5 8 2. C 1 11.0 54.0 4 25 3. C
1. I 100 62.2 91.3 42 2. I 50 66.0 91.7 42
3. I 23 59.1 91.8 42 The symbols in the table have the following meanings:
30 U: Number of uses C Compari~on I: immobilized lipase Act: Relative activity determined by comparing the increase in the level of r~action over the first 20% of the reaction (tangent method) ~ee (-)Alc: Enantiomeric excess of the unreacted (-)-endo-norborn-5-en-2-ol %ee (+)Es~: Enantiomeric excess of the res~lting (+)-endo-norborn-5-en-2-yl acetate The enantiomeri.c excess was in each case deter-mined with the aid of gas chromatographi.c separation of the corresponding menthyl chloroformate, which was prepared by derivatization with(-)-menthyl chloroformate by the method of H. Berger et al., Tetrahedron:
Asymmetry, 1 (1990), pages 541-546.
S: Enantioselectivity of the reaction Determined by the method of Chen et al., J.Am.Chem. Soc, 104 (1982), pages 7294-7299~
30 U: Number of uses C Compari~on I: immobilized lipase Act: Relative activity determined by comparing the increase in the level of r~action over the first 20% of the reaction (tangent method) ~ee (-)Alc: Enantiomeric excess of the unreacted (-)-endo-norborn-5-en-2-ol %ee (+)Es~: Enantiomeric excess of the res~lting (+)-endo-norborn-5-en-2-yl acetate The enantiomeri.c excess was in each case deter-mined with the aid of gas chromatographi.c separation of the corresponding menthyl chloroformate, which was prepared by derivatization with(-)-menthyl chloroformate by the method of H. Berger et al., Tetrahedron:
Asymmetry, 1 (1990), pages 541-546.
S: Enantioselectivity of the reaction Determined by the method of Chen et al., J.Am.Chem. Soc, 104 (1982), pages 7294-7299~
Claims (10)
1.Imobilized lipase from a microorganism of the genus Candida, in which epsilon-amino groups of the lysine in the lipase are bonded covalently by N-alkylation via opened epoxide groups of an epoxide-activated macroporous carrier.
2. Immobilized lipase according to Claim 1, wherein the macroscopic carrier is prepared by suspension polymerization of vinyl acetate and a monomer which is copolymerizable with vinyl acetate, in water, partial hydrolysis of the acetate groups to give hydroxyl groups, followed by reaction with epichlorohydrin which reacts with free hydroxyl groups preserving the epoxide ring.
3. Process for immobilizing a lipase from a micro-organism of the genus Candida, comprising reacting an epoxide-activated macroporous carrier with a solution of Candida lipase in water, a buffer solution or a salt solution at temperatures of 15°C up to the deactivation temperature of the lipase, resulting in the formation of covalent bonds between epsilon-amino groups of the lysine in the lipase with opened epoxide groups of the epoxide-activated macroporous carrier by N-alkylation.
4. Process as claimed in Claim 3, comprising employing 0.05 to 0.1 g of lipase per g of the macroporous carrier.
Use of an immobilized lipase according to Claim 1 for the enantioselective esterification of a chiral alcohol with the aid of an enol ester.
6. Process for the enantioselective esterification of a chiral alcohol, comprising esterifying the alcohole in the presence of an enol ester and a lipase from a microorganism of the genus Candida which is covalently bonded via epsilon-amino groups of the lysine by N-alkylation with epoxide groups of an epoxide-activated macroporous carrier and isolating the resulting ester of the chiral alcohol and/or the unreacted alcohol from the reaction mixture.
7. Process as claimed in Claim 6, comprising employing at least 5 equivalents of enol ester and 2 to 30 grams of immobilized lipase per gram of the chiral alcohol.
8. Process according to Claim 6, comprising employing the enol ester which is used as reactant for esterification also as diluent.
9. Process according to Claim 6 comprising employing vinyl acetate, vinyl propionate or vinyl butyrate as enol ester.
10. Process according to Claim 6 comprising employing a lipase from a microorganism of the species Candida cylindracea.
O.Z.981 25.6.1992
O.Z.981 25.6.1992
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AT0171591A AT398310B (en) | 1991-08-30 | 1991-08-30 | IMMOBILIZED LIPASE, METHOD FOR THE PRODUCTION THEREOF AND METHOD FOR INCREASING THE ENANTIOSELECTIVITY OF A CANDIDA LIPASE IN THE Esterification of CHIRAL ALCOHOLS |
| ATA1715/91 | 1991-08-30 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2075699A1 true CA2075699A1 (en) | 1993-03-01 |
Family
ID=3519425
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002075699A Abandoned CA2075699A1 (en) | 1991-08-30 | 1992-08-12 | Process for increasing the enantio-selectivity of a candida lipase in the esterification of chiral alcohols, and an immobilized candida lipase |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP0529424A3 (en) |
| JP (1) | JPH05207881A (en) |
| KR (1) | KR930004462A (en) |
| AT (1) | AT398310B (en) |
| AU (1) | AU649347B2 (en) |
| CA (1) | CA2075699A1 (en) |
| CZ (1) | CZ252792A3 (en) |
| HU (1) | HUT63658A (en) |
| NZ (1) | NZ243708A (en) |
| SI (1) | SI9200192A (en) |
| SK (1) | SK252792A3 (en) |
| YU (1) | YU76692A (en) |
| ZA (1) | ZA926022B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3431204B2 (en) * | 1993-04-22 | 2003-07-28 | 塩野義製薬株式会社 | Norbornane type ester hydrolase |
| EP0893422A1 (en) * | 1997-07-18 | 1999-01-27 | Mitsubishi Gas Chemical Company, Inc. | Optically active alcohol and process for the production thereof |
| IL152290A0 (en) * | 2002-10-14 | 2003-05-29 | Enzymotec Ltd | Immobilization of compounds on polymeric matrix |
| ITMI20070435A1 (en) | 2007-03-05 | 2008-09-06 | Innovate Biotechnology Srl | 2 ', 3'-DI-O-acyl-5-FLUORONUCLEOSIDI |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3743824C2 (en) * | 1987-12-23 | 1997-03-06 | Hoechst Ag | Process for the enzymatic resolution of racemic alcohols with / in vinyl esters by transesterification |
| DE3819467A1 (en) * | 1988-06-08 | 1989-12-14 | Basf Ag | METHOD FOR PRODUCING A BIO CATALYST AND ITS USE FOR RAZEMATE CUTTING |
| US5108916A (en) * | 1989-06-05 | 1992-04-28 | Rhone-Poulenc Rorer, S.A. | Process for stereoselectively hydrolyzing, transesterifying or esterifying with immobilized isozyme of lipase from candida rugosa |
| JPH03183480A (en) * | 1989-12-13 | 1991-08-09 | Ajinomoto Co Inc | Immobilized lipase and ester exchange reaction of fat or oil with the same |
| DE4131546A1 (en) * | 1991-09-21 | 1993-03-25 | Chemie Linz Deutschland | Candida lipase for high activity and aldehyde resistance - comprises covalent bonding to epoxy activated macroporous carrier for immobilisation and enantioselective esterification of chiral alcohol with enol ester |
-
1991
- 1991-08-30 AT AT0171591A patent/AT398310B/en not_active IP Right Cessation
-
1992
- 1992-07-24 NZ NZ243708A patent/NZ243708A/en unknown
- 1992-08-05 AU AU20844/92A patent/AU649347B2/en not_active Ceased
- 1992-08-11 ZA ZA926022A patent/ZA926022B/en unknown
- 1992-08-12 CA CA002075699A patent/CA2075699A1/en not_active Abandoned
- 1992-08-13 EP EP19920113794 patent/EP0529424A3/en not_active Withdrawn
- 1992-08-14 YU YU76692A patent/YU76692A/en unknown
- 1992-08-17 SK SK2527-92A patent/SK252792A3/en unknown
- 1992-08-17 CZ CS922527A patent/CZ252792A3/en unknown
- 1992-08-27 JP JP4228986A patent/JPH05207881A/en not_active Withdrawn
- 1992-08-27 SI SI19929200192A patent/SI9200192A/en unknown
- 1992-08-28 KR KR1019920015541A patent/KR930004462A/en not_active Application Discontinuation
- 1992-08-28 HU HU9202785A patent/HUT63658A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| AU2084492A (en) | 1993-03-04 |
| JPH05207881A (en) | 1993-08-20 |
| AT398310B (en) | 1994-11-25 |
| EP0529424A3 (en) | 1993-11-10 |
| NZ243708A (en) | 1993-10-26 |
| SK252792A3 (en) | 1995-11-08 |
| AU649347B2 (en) | 1994-05-19 |
| CZ252792A3 (en) | 1993-03-17 |
| EP0529424A2 (en) | 1993-03-03 |
| YU76692A (en) | 1995-03-27 |
| HUT63658A (en) | 1993-09-28 |
| ZA926022B (en) | 1993-03-10 |
| SI9200192A (en) | 1993-03-31 |
| KR930004462A (en) | 1993-03-22 |
| ATA171591A (en) | 1994-03-15 |
| HU9202785D0 (en) | 1992-12-28 |
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