AU649347B2 - Process for increasing the enantioselectivity of a candida lipase in the esterification of chiral alcohols, and an immobilized candida lipase - Google Patents
Process for increasing the enantioselectivity of a candida lipase in the esterification of chiral alcohols, and an immobilized candida lipase Download PDFInfo
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- AU649347B2 AU649347B2 AU20844/92A AU2084492A AU649347B2 AU 649347 B2 AU649347 B2 AU 649347B2 AU 20844/92 A AU20844/92 A AU 20844/92A AU 2084492 A AU2084492 A AU 2084492A AU 649347 B2 AU649347 B2 AU 649347B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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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Description
jog 4 3 7S F Ref: 216757
AUSTRALIA
PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT
ORIGINAL
I Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: r Chemie Linz Gesellschaft m.b.H.
St. Peter-Strasse A-4021 Linz
AUSTRIA
Kurt Faber and Brigitte Berger Spruson Ferguson, Patent Attorneys Level 33 St Martins Tower, 31 Market Street Sydney, New South Wales, 2000, Australia Process for Increasing the Enantloselectivlty of a Candida Lipase in the Esterification of Chlral Alcohols, and an Immobilized Candida Lipase The following statement Is a full description of this invention, including the best method of performing it known to me/us:- 5845/6 Process for increasing the enantioselectivity of a Candida lipase in the esterification of chiral alcohols, and an immobilized Candida lipase.
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 a 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 carboxylate 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 formed 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 25 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 formed 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 mixtures of a racemic alcohol with, or in, a vinyl ester with the aid of a lipase, claiming that the liberated aldehyde does not deactivate the lipase.
However, 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.
2 C.J. Gray et al., Enzyme Microb. Technol., Vol.
12 (1990), 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 does not mention transesterifications 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 is 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 increases both the resistance 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 repeated use, but remains entirely constant.
The invention therefore relates to an ilmmbilized lipase frmn a microorganism of the genus Candida in which epsilon-amino groups of the lysines in the lipase are bonded covalently by N-alkylation via opened epoxide groups of an epoxide-activated macroporous 25 carrier.
Lipase from a microorganism of the genus Candida is understood as meaning unpurified microorganism suspensions of the genus Candida as well as purified enzyme fractions. Species from the genus Candida are, for example, C.antarctica, C. ruqosa, C.
cvlindracea. A lipase from C.cylindracea is preferably employed. Lipases from the genus Candida are commercially ava;iahle in various degrees of purity.
An epoxide-actirated 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 partial hydrolysis of the acetate groups to give hydroxyl groups, followed by a reaction with epichlorohydrin which reacts with free hydroxyl groups preserving the r-oxide ring.
This results in the formation of spacer groups with reactive epoxide groups 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 (R8hm Pharma, Germany) or VAepoxy-biosynth, Riedel de Haen (Germany).
A.N. Glazer, "The proteins", ed. H. Neurath and R.L.
Hill, Academic press London, 1976, Volume II, pages 1- 109, discloses that in this type of enzyme immobilization it is mainly the epsilon-amino groups of the lysine which react with epoxide groups of the carrier, amino groups of the lysine being alkylated.
This means that the lipase according 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.
The invention also relates to a process for the preparation of the immobilized lipase according to the 25 invention. To this end, an epoxide-activated macroporous carrier is brought into contact with an aqueous enzyme solution of a Candida lipase at temperatures from 15°C up to the deactivation temperature of the lipase, preferably from 18 to 30°C, 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 is 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, i.e. a covalent bond between 4 lipase and carrier, being formed. When the reaction is complete, salts, for example NaCI, are optionally added to the reaction mixture, and the immobilized 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 th, s manner is employed for the enantioselective esterification of enantiomer mixtures of chiral alcohols by transesterification using enol esters.
The invention then also relates to a process for the enantioselective esterification of an alcohol which contains at least one asymmetric center in the molecule, comprising esterifying 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 isolating 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 25 form of racemic enantiomer mixtures or those mixtures in which one or the other enantiomer is enriched.
The immobilized lipase according to the invention can react both primary and secondary alcohols. The alcohol can also be a dialcohol having two asymmetric centers in the molecule, mixtures of R,S-alcohols, S,Ralcohols, 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 -S-PS-4,963,492 Preferably employed esters are vinyl esters of lower organic acids, particularly preferably vinyl acetate, vinyl propionate and vinyl butyrate.
5 To carry out the reaction, 2 to 30 g, preferably 6 to 10 g, of immobilized lipase and at least equivalents 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 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 preferably 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 is indicated by the manufacturer or can be determined by simple tests. Depending on the alcohol mixture, enol ester and specificity of the lipase employed, the R ester, or the S ester, or the R,S ester, or the S,R ester of the alcohol employed is chiefly formed, while none, or only a small amount, of the corresponding S or R alcohol, or S,R, Rj,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 is worked up. To this end, the immob' i.x.z lipase is filtered off, and the mother liqi~i is subjected to a suitable separating operation where the desired products are isolated. Separation of the desired products from the reaction mixture can be carried out by customary methods, for example with the aid of extraction, distillation or chromatography.
6 In a preferred embodiment, a racemic mixture of an alcohol is stirred or shaken at room temperature with Candida cylindracea lipase, immobilized with the aid of VA-epoxy-biosynth, Riedel-de-Haen, Germany, in vinyl acetate. When the desired enantiomeric 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 chromatography.
It has emerged that the resistance of the lipase against acetaldehyde as well as its activity and selectivity, 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 immobilized lipase and its use in the enantioselective esterification according to the invention therefore represent an enrichment of the art.
Example 1 10.0 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 lipase (AY-30, Amano Pharm. Co., Japan) were added, and 25 the mixture was then shaken for 3 days at 27"C and rpm. 70 ml of sodium chloride solution were added, and the solid was filtered off, washed 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 5.70 pmol min 1 g-1. The specific activity of the non-immobilized lipase under the same conditions was 13 pmol min" 1 g-.
7 Example 2 3 g of racemic endo-norborn-5-en-2-ol (9 mmol) was mixed with 200 mg of Candida cylindracea lipase (Ay- Amano Pharm. Co., Japan) and the mixture was shaken in 10 ml of vinyl acetate at 20°C and 20u rpm. The procedure of the reaction was monitored by gas chromatography. 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 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.
r
V
o r o r r 20 Table 1
U
1.
2.
3.
1.
2.
3.
Lipase Act.
39 1 100 50 23 ee Alc.
51.4 11.0 62.2 66.0 59.1 ee (+)Est 66.5 54.0 91.3 91.7 91.8 The
U:
C
I:
Act: symbols in the table have the following meanings: Number of uses Comparison immobilized lipase Relative activity determined by comparing the increase in the level of reaction over the first 20% of the reaction (tangent -8 method) %ee (-)Al1c: Enantiomaric excess of the unreacted endo-norborn-5-en-2 -ol %ee Enantiomeric excess of the resulting endo-norborn-5-en-2-yl acetate The enantiomeric excess wad~ in each case determined with the aid of gas chromatographic separation of the corresponding menthyl chioroformate, which was prepared by derivatizatiin with (.-)-menthyl chioroformate by the method of B. Berger et al., Tetrahedron: Asymmetry, 1 (1990), pages 541-546.
S: Enantioselectivity of the reaction Determined by the meth~ic of Chen et al., J.Am.Chem. Soc, 104 (1982), pages 7294-7299
Claims (8)
1. Irmnbilized 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. Imrobilized ipase 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- 15 organism of the genus Candida, coprising 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. Process as claimed in Claim 3, comprising employing 25 .0.05 to 0.1 g of lipase per g of the macroporous carrier. Use of an imnobilized 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 macroporr.s carrier and isolating the resulting ester of the chiral alcohol and/or the 10 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 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 eploying vinyl acetate, vinyl propionate or vinyl butyrate as enol ester. Process according to Claim 6 comprising employing a lipase from a microorganism of the species Candida cylindracea. 15 11. Immobilized lipase from a microorganism of the genus Candida substantially as hereinbefore described with reference to any one of the Examples. S12. Process for immobilizing a lipase from a microorganism of the genus Candida substantially as hereinbefore described with reference to any one of the Examples.
13. Process for the enantioselective esterification of a chiral alcohol substantially as hereinbefore described with reference to any one of the Examples. 25 14. The product of the process of any one of claims 6 to 10 or 13. DATED this TWENTY-NINTH day of JULY 1992 Chemie Linz Gesellschaft m.b.H. Patent Attorneys for the Applicant SPRUSON FERGUSON Process for Increasing the Enantioselectivity of a Candida Lipase in the Esterification of Chiral Alcohols, and an Immobilized Candida Lipase Abstract SImnobilized 1 ipase from a microorganism of the genus Candida which is 1n-iy bonded to epsilon-amino groups of the lysine by a.L, ',on with opened epoxide groups of an epoxide-activated macroscopic carrier, a process for its preparation, its use in a S o0 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 IJ 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. 5*55
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 |
| AT1715/91 | 1991-08-30 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2084492A AU2084492A (en) | 1993-03-04 |
| AU649347B2 true AU649347B2 (en) | 1994-05-19 |
Family
ID=3519425
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU20844/92A Ceased AU649347B2 (en) | 1991-08-30 | 1992-08-05 | Process for increasing the enantioselectivity 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 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Family Cites Families (3)
| 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 |
-
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
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH05207881A (en) | 1993-08-20 |
| EP0529424A3 (en) | 1993-11-10 |
| ATA171591A (en) | 1994-03-15 |
| ZA926022B (en) | 1993-03-10 |
| CZ252792A3 (en) | 1993-03-17 |
| EP0529424A2 (en) | 1993-03-03 |
| AT398310B (en) | 1994-11-25 |
| CA2075699A1 (en) | 1993-03-01 |
| NZ243708A (en) | 1993-10-26 |
| SK252792A3 (en) | 1995-11-08 |
| AU2084492A (en) | 1993-03-04 |
| KR930004462A (en) | 1993-03-22 |
| HUT63658A (en) | 1993-09-28 |
| YU76692A (en) | 1995-03-27 |
| SI9200192A (en) | 1993-03-31 |
| HU9202785D0 (en) | 1992-12-28 |
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