SI9200192A - Process for higher enantioselectivity of candid lypase by chiral alcohols estering and imobilised candid lypase - Google Patents

Process for higher enantioselectivity of candid lypase by chiral alcohols estering and imobilised candid lypase Download PDF

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SI9200192A
SI9200192A SI19929200192A SI9200192A SI9200192A SI 9200192 A SI9200192 A SI 9200192A SI 19929200192 A SI19929200192 A SI 19929200192A SI 9200192 A SI9200192 A SI 9200192A SI 9200192 A SI9200192 A SI 9200192A
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lipase
carrier
epoxy
alcohol
macroporous
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Kurt Faber
Brigitte Berger
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Chemie Linz Gesellschaft M.B.H.
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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
    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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

Abstract

Lipase from a microorganism of the genus Candida, which is covalently bonded to epsilon-amino groups of lysine by opened epoxide groups of an epoxide-activated macroscopic support by N-alkylation and thus immobilised, 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 centre of asymmetry 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 centre of asymmetry in the molecule, with the aid of the immobilised lipase in the presence of an enol ester.

Description

Postopek za zvišanje enantioselektivnosti Candida lipaze pri zaestrenju kiralnih alkoholov in imobilizirana Candida lipazaProcedure for Enantioselectivity Increase of Candida Lipase in Chiral Alcohol Disturbance and Immobilized Candida Lipase

Predloženi izum se nanaša na postopek za zvišanje enantioselektivnosti, aktivnosti in stabilnosti lipaze iz mikroorganizma rodu Candida v encimatskem postopku preestrenja v katerem kiralni alkohol, ki vsebuje vsaj en asimetričen center v molekuli zaestrimo enantioselektivnost z enolnim estrom, imobilizirana Candida lipaza in postopek za njeno pripravo.The present invention relates to a process for enhancing the enantioselectivity, activity and stability of a lipid from a microorganism of the genus Candida in an enzymatic esterification process in which a chiral alcohol containing at least one asymmetric center in the molecule esterifies enantioselectivity with an enol ester, immobilized Candida lipase and a process for its preparation.

Kiralni enantiomemo čisti alkoholi so pomembni za različne namene uporabe, npr. za sintezo farmacevtskih učinkovin ali agrarnih kemikalij. Njihova priprava lahko npr. poteka z enantioselektivnim preestrenjem, pri čemer enantiomemo zmes kiralnega alkohola presnovijo v prisotnosti estra karboksilne kisline s hidrolazo. Ker hidrolaze na splošno katalizirajo tako reakcijo v eno kot tudi v drugo smer nastane želeni končni produkt pri takem preestrenju pogosto le zelo počasi in z nezadostno optično čistoto. V M.Degueil-Castaing et al, Tetrahedron Letters, Vol 28, 9 (1987), 953-954 zato predlagajo uporabo enolnega estra karboksilne kisline, ker potem ne more več potekati povratna reakcija. Kot sicer izhaja iz Zakrzewska et al, Acta Med. Pol., 29, (1988), 1-2, stran 44, lahko aldehidi, ki nastanejo pri preestrenju iz enolnega estra, z reakcijo s terminalnimi funkcionalnimi skupinami amino kislin encima deak» * « · » > · · · » · · » » • * · >Chiral enantiome pure alcohols are important for various uses, e.g. for the synthesis of pharmaceutical ingredients or agrarian chemicals. Their preparation may be e.g. is carried out by enantioselective esterification, whereby the enantiomeric mixture of chiral alcohol is metabolized in the presence of a carboxylic acid ester by hydrolase. As hydrolases generally catalyze both the reaction in one direction and the other, the desired end product is formed in such a transester, often only very slowly and with insufficient optical purity. In M.Degueil-Castaing et al, Tetrahedron Letters, Vol 28, 9 (1987), 953-954, they therefore propose the use of the carboxylic acid enol ester, since then no reverse reaction can take place. As otherwise derived from Zakrzewska et al, Acta Med. Pol., 29, (1988), 1-2, page 44, aldehydes resulting from the esterification of the enol ester may be reacted with the terminal functional groups of amino acids of the enzyme deak "*" · »> · · ·» · · » »• * ·>

tivirajo encim. V EP-A-0 321 918 je opisan postopek za encimatsko racematsko cepljenje enantiomemih zmesi racemnega alkohola z ali v vinilnem estru z lipazo, pri katerem sproščeni aldehid naj ne bi deaktiviral lipaze. Izkazalo se je, da pri ponovni uporabi Candida lipaze v takem postopku zelo hitro močno pade tako njena aktivnost kot tudi njena selektivnost. V C.J. Gray et al, Enzyme Microb. Technol., Vol 12 (1990), str. 800 do 807, je omenjeno, da se stabilnost Candida cylindracea lipaze lahko zviša z različnimi mobilizacijskimi tehnikami za ponovno uporabo. Preestrenja z uporabo enolnega estra pa tam niso omenjena.tivate the enzyme. EP-A-0 321 918 describes a method for the enzymatic racemic grafting of enantiomeric mixtures of racemic alcohol with or in a vinyl ester with lipase in which the released aldehyde is not expected to deactivate the lipase. It appears that Candida lipase re-use in such a process rapidly decreases both its activity and its selectivity. In C.J. Gray et al, Enzyme Microb. Technol., Vol 12 (1990), p. 800 to 807, it is mentioned that the stability of Candida cylindracea lipase can be enhanced by various re-mobilization techniques. However, the exaggerations using single-ester are not mentioned there.

Sedaj smo nepričakovano ugotovili, da se tako stabilnost proti aldehidom kot tudi aktivnost in neobičajno tudi selektivnost Candida lipaze zviša, če lipazo pred njeno uporabo v postopku preestrenja, ob uporabi enolnih estrov, s presnovo epsilon amino skupin lizina lipaze mobiliziramo z epoksidnimi skupinami makroporoznega nosilca, aktiviranega z epoksidom, pri čemer pride do N-alkiliranja. S tem imobiliziranjem se zviša tako obstojnost lipaze proti aldehidom kot tudi njena aktivnost in substratna selektivnost v pimerjavi z neimobilizirano lipazo, pri čemer substratna selektivnost pri ponovni uporabi ne pade ampak ostane popolnoma konstantna.It has now been unexpectedly found that both aldehyde stability and activity, and abnormally the selectivity of Candida lipase, is enhanced if the lipase is mobilized by the macroporous carrier epoxide groups by metabolizing the epsilon amino groups of the lysine lipase prior to its use in the esterification process using enol esters. activated by epoxide, resulting in N-alkylation. This immobilization increases both lipase resistance to aldehydes as well as its activity and substrate selectivity when compared with non-immobilized lipase, whereby substrate selectivity does not fall but remains completely constant when reused.

Predmet predloženega izuma je zato lipaza iz mikroorganizma rodu Candida, označena s tem, da so epsilon amino skupine lizina v lipazi z N-alkiliranjem kovalentno vezane preko odprtih epoksidnih skupin makroporoznega nosilca, aktiviranega z epoksidom, s čimer je lipaza mobilizirana.It is therefore an object of the present invention to provide a lipase from a microorganism of the genus Candida, characterized in that the epsilon amino groups of lysine in the lipase are N-alkylated covalently bonded via open epoxy groups of a macroporous epoxide-activated carrier, thereby mobilizing the lipase.

Pod lipaze iz mikroorganizma rodu Candida pri tem razumemo tako neočiščene suspenzije mikroorganizma rodu Candida kot tudi očiščene encimske frakcije. Vrste rodu Candida (C.) so, npr. C.antarctica, C.rugosa, C.cylindracea. Prednostno uporabimo lipazo iz C.cylindracea. Lipaze rodu Candida dobimo na tržišču v različnih stopnjah čistote.The term lipid from a microorganism of the genus Candida is understood to mean both the unpurified suspensions of the microorganism of the genus Candida and the purified enzyme fractions. The species of the genus Candida (C.) are, e.g. C.antarctica, C.rugosa, C.cylindracea. Preferably lipase from C.cylindrace is used. Candida lipases are commercially available in varying degrees of purity.

Za imobiliziranje lipaze uporabimo makroporozni nosilec, aktiviran z epoksidom. Njegova priprava poteka npr. s suspenzijsko polimerizacijo vinilacetata in monomera, ki se ga da sopolimerizirati z vinilacetatom, npr. N,N’-diviniletilensečnino v vodi, delnim umiljenjem acetatnih v hidroksi skupine in zatem presnovo z epiklorhidrinom, ki reagira s prostimi hidroksi skupinami, pri čemer dobimo epoksidni obroč. Pri tem se v polimerizatu izoblikujejo prostorske skupine z reaktivnimi epoksidnimi skupinami, ki so primerne za vezavo nosilca z najrazličnejšimi substrati. Taki nosilci in njihove priprave so opisani, npr. v K. Burg et al, Angew. Makromol. Chem.An epoxide-activated macroporous support is used to immobilize the lipase. Its preparation takes place e.g. by suspension polymerization of vinyl acetate and a copolymerizable monomer with vinyl acetate, e.g. N, N'-divinylethyleneurea in water, partial saponification of acetate in hydroxy groups and then metabolised with epichlorohydrin, which reacts with free hydroxy groups to give the epoxy ring. In this case, polymeric groups are formed by space groups with reactive epoxy groups, which are suitable for bonding the carrier with a variety of substrates. Such carriers and their preparations are described, e.g. in K. Burg et al, Angew. Macromol. Chem.

157, (1988), str. 105 do 121. Dosegljivi so na tržišču. Posebno prednostno uporabimo kot nosilec Eupergit C (Rohm Pharma, Nemčija) ah VA-Epoxy-Biosynth, Riedel de Haen (Nemčija).157, (1988), p. 105 to 121. They are commercially available. Particularly preferably used as a carrier Eupergit C (Rohm Pharma, Germany) ah VA-Epoxy-Biosynth, Riedel de Haen (Germany).

V A.N. Glazer, The proteins, izd. H. Neurath in R.L. Hill, Academic press London, 1976, zv. II, str. 1-109, je navedeno, da pri tem načinu imobiliziranja encimov v prvi vrsti reagirajo epsilon amino skupine lizina z epoksidnimi skupinami nosilca, pri čemer se amino skupine lizina alkilirajo.In A.N. Glazer, The proteins, ed. H. Neurath and R.L. Hill, Academic Press London, 1976, p. II, p. 1-109, it is stated that in this mode of immobilization of enzymes, the epsilon of the lysine amino groups is first reacted with the epoxide groups of the carrier, with the lysine amino groups being alkylated.

Lipaza v smislu izuma je tudi alkilirana na epsilon amino skupinah lizina lipaze preko odprtih epoksidnih skupin nosilca, t.j. kovalentno vezana z nosilcem.The lipase of the invention is also alkylated on epsilon amino groups of lysine lipase via open epoxy groups of the carrier, i.e. covalently bonded to the carrier.

Predmet predloženega izuma je tudi postopek za pripravo imobilizirane lipaze v smislu izuma. Zato makroporozni nosilec, aktiviran z epoksidom, spravimo v stik z vodno encimsko raztopino Candida lipaze pri temperaturah od 15°C do temperature deaktiviranja lipaze, prednostno od 18 do 30°C, npr. s stresanjem v stresalni bučki. Razmerje nosilca in lipaze mora biti vsaj takšno, da pride v reakcjski zmesi na prosto amino skupino lizina v lipazi ena epoksidna skupina nosilca. Prednostno uporabimo na 1 g nosilca približno 0,05 do 0,1 g lipaze. Lipaza je pri tem lahko raztopljena v čisti vodi, raztopini pufra ali raztopini soli. Pri tem reagirajo epsilon amino skupine lizina v lipazi z epoksidnimi skupinami nosilca, pri čemer nastane N-alkiliranje, torej kovaletna vez med lipazo in nosilcem. Po končani reakciji imobilizirano lipazo, v danem primeru po dodatku soli, npr. NaCl v reakcijsko zmes, odfiltriramo in izperemo s pufrsko raztopino. Imobilizirano lipazo lahko shranimo v pufru ali vlažno ali tudi posušeno do uporabe.The subject of the present invention is also a process for the preparation of an immobilized lipase of the invention. Therefore, the macroporous epoxide-activated carrier is contacted with an aqueous enzyme Candida lipase solution at temperatures from 15 ° C to a lipase deactivation temperature, preferably from 18 to 30 ° C, e.g. by shaking in a shaking flask. The ratio of carrier to lipase should be at least such that one epoxide group of carrier exists in the reaction mixture to the free amino group of lysine in lipase. Preferably about 0.05 to 0.1 g of lipase is used on 1 g of carrier. The lipase can then be dissolved in pure water, buffer solution or brine. In doing so, the epsilon of the amino group of lysine in lipase reacts with the epoxy groups of the carrier, resulting in N-alkylation, which is a covalent bond between the lipase and the carrier. Upon completion of the reaction, immobilized lipase, optionally after addition of salt, e.g. NaCl into the reaction mixture was filtered off and washed with buffer solution. The immobilized lipase can be stored in buffer or moist or dried until used.

Na ta način pripravljeno lipazo uporabimo za enantioselektivno zaestrenje enanf tiomernih zmesi kiralnih alkoholov, s preestrenjem ob uporabi enolnih estrov;In this way, the prepared lipase is used for the enantioselective esterification of enan-thiomeric mixtures of chiral alcohols, by esterification using enol esters;

Predmet predloženega izuma je zato tudi postopek za enantioselektivno zaestrenje alkohola, ki vsebuje vsaj en asimetričen center v molekuli, označen s tem, da alkohol zaestrimo v prisotnosti enolnega estra in lipaze iz mikroorganizma rodu Candida, ki je kovalentno vezana na epsilon amino skupine lizina v lipazi z odprtimi epoksidnimi skupinami makroskopskega nosilca, aktiviranega z epoksidom, z N-alkiliranjem, nakar nastali ester kiralnega alkohola in v danem primeru nepresnovljeni alkohol izoliramo iz reakcijske zmesi.The present invention is therefore also a process for the enantioselective esterification of an alcohol containing at least one asymmetric center in a molecule, characterized in that the alcohol is esterified in the presence of an enol ester and a lipase from a microorganism of the genus Candida, which is covalently bound to the epsilon of the amino group of lysine in the lipase by open epoxy groups of the macroscopic carrier activated by epoxide, by N-alkylation, and then the resulting chiral alcohol ester and optionally the unprocessed alcohol is isolated from the reaction mixture.

) ·») · »

Uporabljeni alkohol ima vsaj en asimetričen center in je zato optično aktiven. Le-ta je lahko v obliki racemnih enantiomernih zmesi ali takšnih v katerih je en ali drug enantiomer obogaten.The alcohol used has at least one asymmetric center and is therefore optically active. It may be in the form of racemic enantiomeric mixtures or such in which one or the other enantiomer is enriched.

Z lipazo mobilizirano v smislu izuma lahko presnovimo tako primarne kot tudi sekundarne alkohole. Alkohol je lahko tudi dialkohol z dvema asimetričnima centroma v molekuli, pri čemer so lahko zmesi iz R,S-, S,R-, R,R- ali S,S-alkohola. Prednostno kot substrat uporabimo sekundarne alkohole z enim asimetričnim centrom v molekuli.Both the primary and secondary alcohols can be metabolized by the lipase mobilized according to the invention. Alcohol may also be a dialcohol with two asymmetric centers in the molecule, the mixtures of R, S-, S, R-, R, R- or S, S-alcohols. Preferably, secondary alcohols with one asymmetric center in the molecule are used as the substrate.

Kot enolni ester lahko uporabimo, npr. v EP-A-0 321 918 navedeni enolni ester. Prednostno uporabimo vinilni ester nižjih organskih kislin, posebno prednostno vinilacetat, vinilpropionat, vinilbutirat.As a single ester, it can be used, e.g. in EP-A-0 321 918 the enol ester indicated. Preferably, the vinyl ester of lower organic acids is used, especially preferably vinyl acetate, vinyl propionate, vinyl butyrate.

Za izvedbo reakcije zmešamo na gram enantiomeme zmesi kiralnega alkohola 2 do 30 g, prednostno 6 do 10 g mobilizirane lipaze in vsaj 5 ekvivalentov enolnega estra, v danem primeru z razredčilom, inertnim pri reakcijskih pogojih in ob mešanju ali stresanju pri temperaturah od 15°C do temperature deaktivacije lipaze, prednostno pri sobni temperaturi, pustimo reagirati.To carry out the reaction, the gram of enantiomeric mixture of chiral alcohol 2 to 30 g, preferably 6 to 10 g of mobilized lipase and at least 5 equivalents of the ester ester is mixed to the reaction, optionally with a diluent inert under the reaction conditions and stirred or shaken at 15 ° C. to the lipase deactivation temperature, preferably at room temperature, is allowed to react.

Reakcijo lahko izvedemo v topilu, inertnem pri reakcijskih pogojih, ali za preestrenje uporabljen enolni ester uporabimo z visokim prebitkom tudi kot razredčilo. Prednostno reakcijo ne izvedemo v topilu, inertnem pri reakcijskih pogojih, ampak v enolnem estru uporabljenem za presnovo.The reaction can be carried out in a solvent inert under the reaction conditions, or the enol ester used for high esterification can also be used with high excess as a diluent. Preferably, the reaction is not carried out in a solvent inert under the reaction conditions, but in the enol ester used for the metabolism.

Reakcijo izvedemo smotrno pri temperaturah pri katerih imajo lipaze njihovo najvišjo aktivnost. Ta temperatura je navedena od ponudnika ali jo lahko določimo z rThe reaction is conveniently carried out at temperatures at which lipases have their highest activity. This temperature is specified by the provider or can be determined by r

enostavnim poskusom. Pri tem nastane glede na uporabljeno alkoholno zmes, enolni ester in specifičnost uporabljene lipaze pretežno R- ali pretežno S- oz. pretežno R,Sali pretežno S,R-ester uporabljenega alkohola, medtem ko se ustrezni S- ali R- oz. S,R-, R,S-, R,R- ali S,S-alkohol ne presnovi ali samo z majhnim deležem.by simple experiment. Thus, depending on the alcohol mixture used, the enol ester and the specificity of the lipase used, it is predominantly R- or predominantly S- or. predominantly R, Sali predominantly S, the R-ester of the alcohol used, whereas the corresponding S- or R- resp. S, R-, R, S-, R, R- or S, S-alcohol is not metabolised or only by a small fraction.

Potek reakcije spremljamo s primernimi in v encimski kemiji znanimi postopki, npr. s plinsko-kromatografskim postopkom.The course of the reaction is monitored by suitable and known enzymatic chemistry methods, e.g. by gas-chromatographic process.

Reakcijo prekinemo ko dosežemo želeno enantiomemo čistoto produkta in reakcijsko zmes obdelamo. Zato imobilizirano lipazo odfiltriramo in matično lužnico » > · · 11 It ) ’ » * « tl tThe reaction is stopped when the desired enantiomeric purity of the product is reached and the reaction mixture is treated. Therefore, the immobilized lipase is filtered off and the mother liquor "> · · 11 It) '" * "tl t

11·· 1 » T » ’ » ’ 1 1 1 1 ) » > » t , , > i » » 1 » 11)1 podvržemo primerni operaciji za ločenje pri kateri izoliramo želene produkte. Ločitev želenih produktov iz reakcijske zmesi lahko poteka kot običajno npr. z ekstrakcijo, destilacijo in kromatografijo.11 ·· 1 »T» '»' 1 1 1 1)»> »t,,> i» »1» 11) 1 undergo a suitable separation operation in which we isolate the desired products. Separation of the desired products from the reaction mixture can be carried out as usual e.g. by extraction, distillation and chromatography.

V prednostni izvedbeni obliki mešamo ali stresamo racemno zmes alkohola s Candida cylindracea lipazo, imobilizirano z VA-Epoxy-Biosynth, Riedel-de-Haen, Nemčija, v vinilacetatu pri sobni temperaturi. Ko dosežemo želen enantiomerni prebitek nastalega R- ali S- oz. R,S- ali S,R-estra, encim odfiltriramo in želene produkte odločimo destilacijsko ali kromatografsko.In a preferred embodiment, the racemic alcohol mixture is mixed or shaken with Candida cylindracea lipase immobilized with VA-Epoxy-Biosynth, Riedel-de-Haen, Germany, at vinyl acetate at room temperature. When the desired enantiomeric excess of the resultant R- or S- or. The R, S- or S, R-esters are filtered off and the desired products are distilled or chromatographed.

Izkazalo se je, da pri reakciji v smislu izuma v primerjavi z neimobilizirano lipazo naraste tako obstojnost lipaze proti acetaldehidu kot tudi njena aktivnost in selektivnost. Posebno presenetljiv je več kot 5-kratni porast selektivnosti, ki ostane pri ponovni uporabi imobilizirane lipaze, popolnoma konstanten.It has been shown that in the reaction of the invention, in comparison with non-immobilized lipase, both lipase resistance to acetaldehyde and its activity and selectivity increase. Particularly striking is the more than 5-fold increase in selectivity, which remains completely constant when immobilized lipase is reused.

Imobilizirana lipaza in njena uporaba pri enantioselektivnem zaestrenju v smislu izuma zato predstavljata obogatitev tehnike.Immobilized lipase and its use in enantioselective esterification according to the invention therefore constitute an enriching technique.

» » · » ) > » t » » » » > T » • *»» · »)>» T »» »»> T »• *

Primer 1Example 1

10,0 g VA-Epoxy-Biosynth (Riedel-de-Haen, Nemčija) suspendiramo v 70 ml 0,1 n fosfatnega pufra, pH 7,00 in po dodatku 300 mg lipaze Candida cylindracea (ΑΥ-30, firma Amano Pharm. Co., Japonska), stresamo 3 dni pri 27°C in 80 obr./min. Po dodatku 70 ml raztopine natrijevega klorida odfiltriramo in po 2-krat speremo s po 30 ml 0,05 n fosfatnega pufra, pH 7 in posušimo.10.0 g of VA-Epoxy-Biosynth (Riedel-de-Haen, Germany) was suspended in 70 ml of 0.1 n phosphate buffer, pH 7.00 and after the addition of 300 mg of Candida cylindracea lipase (ΑΥ-30, by Amano Pharm. Co., Japan), shaken for 3 days at 27 ° C and 80 rpm. After the addition of 70 ml of sodium chloride solution, it is filtered off and washed twice with 30 ml of 0.05 n phosphate buffer, pH 7 each time, and dried.

Specifično aktivnost na ta način pripravljene imobilizirane lipaze določimo s titracijo ocetne kisline z 0,1 n natrijevim hidroksidom, nastale med hidrolizo triacetina v 0,1 n fosfatnem pufru, pH 7,00 in znaša 5,70 μιηοΐ na min'1 na g'1. Specifična aktivnost neimobilizirane lipaze je pri enakih pogojih 13 μ-mol min4 na g'1.The specific activity of the immobilized lipase thus prepared is determined by titrating the acetic acid with 0.1 n sodium hydroxide formed during the hydrolysis of triacetin in 0.1 n phosphate buffer, pH 7.00, and amounting to 5.70 μιηοΐ per min ' 1 per g' 1 . The specific activity of non-immobilized lipase is 13 μmol min 4 per g ' 1 under the same conditions.

Primer 2 g racemnega endo-norborn-5-en-2-ola (9 mmol) zmešamo z 200 mg Candida cylindracea lipaze (ΑΥ-30, firma Amano Pharm. Co., Japonska) in stresamo v 10 ml vinilacetata pri 20°C in 200 obr./min. Potek reakcije spremljamo s plinsko kromatografijo. Po 4 urah reakcijo prekinemo. Lipazo odfiltriramo in matično lužnico uparimo v vakuumu. Po običajni kolonski kromatografiji ostanka preko kremeničnega gela dobimo (+)-endo-norborn-5-en-2-il acetat in (-)-endo-norborn5-en-2-ol).Example 2 2 racemic endo-norborn-5-en-2-ol (9 mmol) was mixed with 200 mg of Candida cylindracea lipase (ΑΥ-30, Amano Pharm. Co., Japan) and shaken in 10 ml of vinyl acetate at 20 ° C. and 200 rpm. 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 gave (+) - endo-norborn-5-en-2-yl acetate and (-) - endo-norborn5-en-2-ol).

Rezultati raziskav, ki se nanašajo na aktivnost lipaze, presnovo in enantiomerno razmerje, so zbrani v tabeli 1.The results of studies regarding lipase activity, metabolism and enantiomeric ratio are summarized in Table 1.

Primer 3Example 3

Izvedemo kot primer 2 z razliko, da uporabimo imobilizirano lipazo po primeru 1. Rezultati raziskav, ki se nanašajo na lipazno aktivnost, presnovo in enantiomerno razmerje, so zbrani v tabeli 1.We perform as Example 2 with the difference that immobilized lipase is used according to Example 1. The results of the studies regarding lipase activity, metabolism and enantiomeric ratio are summarized in Table 1.

1 , ·1, ·

» » »»» »

Tabela 1Table 1

A A Lipaza Lipase Akt. Act. %ee (-)Alk. % ee (-) Alk. %ee (+) Est % ee (+) Est S S 1. 1. V V 39 39 51,4 51,4 66,5 66,5 8 8 2. 2. V V 1 1 11,0 11,0 54,0 54.0 4 4 3. 3. V V - - - - - - - - 1. 1. I I 100 100 62,2 62,2 91,3 91,3 42 42 2. 2. I I 50 50 66,0 66,0 91,7 91,7 42 42 3. 3. I I 23 23 59,1 59,1 91,8 91,8 42 42

V tabeli pomenijoIn the table, they mean

A: število uporabA: number of uses

V primerjavaIn comparison

I: imobilizirana lipazaI: immobilized lipase

Akt: relativna aktivnost, določena s primerjavo porasta poteka reakcije po prvih 20 % reakcije (tangentna metoda) %ee (-)Alk: enantiomemi prebitek nepresnovljenega (-)-endo-norbom-5-en-2-ola %ee (+)Est: enantiomemi prebitek nastalega (+)-endo-norbom-5-en-2-il-acetata.Act: relative activity determined by comparing the course of the reaction after the first 20% of the reaction (tangent method)% ee (-) Alk: enantiomyemic excess of unconverted (-) - endo-norbom-5-en-2-ol% ee (+) Est: enantiomeric excess of (+) - endo-norb-5-en-2-yl-acetate formed.

Vsakokratni enantiomemi prebitek določimo s plinsko-kromatografskim ločenjem ustreznega metilkloroformata, ki ga pripravimo z derivatiziranjem z (-)-mentilklorformatom po B. Berger et al, Tetrahedron: Asymmetry, 1 (1990), str. 541-546.Each enantiomeric excess is determined by gas chromatographic separation of the corresponding methyl chloroformate, which is prepared by derivatization with (-) - menthylchloroformate according to B. Berger et al, Tetrahedron: Asymmetry, 1 (1990), p. 541-546.

ff

S: enantioselektivnost reakcije, določena po postopku Chen et al.;S: enantioselectivity of the reaction determined by the method of Chen et al .;

J.Am.Chem. Soc, 104 (1982), str.7294-7299.J.Am.Chem. Soc, 104 (1982), pp.7294-7299.

ZaFor

Chemie Linz Gesellschaft m.b.H.Chemie Linz Gesellschaft m.b.H.

Claims (10)

1. Lipaza iz mikroorganizma rodu Candida, označena s tem, da so epsilon amino skupine lizina v lipazi z N-alkiliranjem kovalentno vezane preko odprtih epoksidnih skupine makroporoznega nosilca, aktiviranega z epoksidom, pri čemer se lipaza imobilizira.A lipase from a microorganism of the genus Candida, characterized in that the epsilon of the amino group of lysine in the lipase by N-alkylation is covalently linked via open epoxy groups of a macroporous epoxide-activated carrier, wherein the lipase is immobilized. 2. Lipaza po zahtevku 1, označena s tem, da pripravimo makroskopski nosilec s suspenzijsko polimerizacijo vinilacetata in monomera, ki se da sopolimerizirati z vinilacetatom, v vodi, delnim umiljenjem acetatnih v hidroksi skupine in zatem presnovo z epiklorhidrinom, ki reagira s prostimi hidroksi skupinami, pri čemer dobimo epoksidni obroč.Lipase according to claim 1, characterized in that a macroscopic carrier is prepared with suspension polymerization of vinyl acetate and monomer that can be copolymerized with vinyl acetate in water, partial saponification of acetate in hydroxy groups and then metabolised with epichlorohydrin reacting with free hydroxy groups , yielding an epoxy ring. 3. Postopek za imobiliziranje lipaze iz mikroorganizma rodu Candida, označen s tem, da makroporozni nosilec, epoksidiran z epoksidom presnovimo z raztopino Candida lipaze v vodi, pufrski raztopini ali raztopini soli pri temperaturah od 15°C do temperature deaktivacije lipaze, pri čemer z N-alkiliranjem nastanejo kovalentne vezi med epsilon amino skupinami lizina v lipazi z odprtimi epoksidnimi skupinami makroporoznega nosilca aktiviranega z epoksidom.Process for immobilizing lipase from a microorganism of the genus Candida, characterized in that the macroporous epoxy-oxidized carrier is reacted with Candida lipase solution in water, buffer solution or salt solution at temperatures from 15 ° C to lipase deactivation temperature, with N -alkylation results in covalent bonds between the epsilon amino groups of lysine in lipase with open epoxy groups of the macroporous epoxide-activated carrier. 4. Postopek po zahtevku 3, označen s tem, da na g makroporoznega nosilca uporabimo 0,05 do 0,1 g lipaze.Process according to claim 3, characterized in that 0.05 to 0.1 g of lipase is used per g of macroporous carrier. 5. Uporaba lipaze po zahtevku 1, označena s tem, da enatioselektivno zaestrimo kiralni alkohol z enolnim estrom.Use of a lipase according to claim 1, characterized in that enatioselectively esterifies the chiral alcohol with the enol ester. rr 6. Postopek za enantioselektivno zaestrenje kiralnega alkohola, označen s tem, da alkohol zaestrimo v prisotnosti enolnega estra in lipaze iz mikroorganizma rodu Candida, ki je z N-alkiliranjem kovalentno vezan preko epsilon amino skupine lizina z epoksidnimi skupinami makroporoznega nosilca, aktiviranega z epoksidom, nakar nastali ester kiralnega alkohola in v danem primeru nepresnovljen alkohol izoliramo iz reakcijske zmesi.6. A process for the enantioselective esterification of chiral alcohol, characterized in that the alcohol is esterified in the presence of an enol ester and a lipase from a microorganism of the genus Candida, which is covalently linked via N-alkylation via the epsilon amino group of lysine with epoxy groups of a macroporous carrier activated by an epoxy carrier activated by epoxy carrier then the resulting chiral alcohol ester and optionally the unprocessed alcohol is isolated from the reaction mixture. 7. Postopek po zahtevku 6, označen s tem, da uporabimo na g kiralnega alkohola vsaj 5 ekvivalentov enolnega estra in 2 do 30 g imobilizirane lipaze.Process according to claim 6, characterized in that at least 5 equivalents of the enol ester and 2 to 30 g of immobilized lipase are used per g of chiral alcohol. »»»t»» »T 8. Postopek po enem od zahtevkov 6 ali 7, označen s tem, da kot razredčilo za zaestrenje uporabimo tak enolni ester, kije uporabljen tudi kot reakcijski partner.Process according to one of Claims 6 or 7, characterized in that such a single ester is used as the diluent for esterification, which is also used as a reaction partner. 9. Postopek po enem od zahtevkov 6 do 8, označen s tem, da kot enolni ester uporabimo vinilacetat, vinilpropionat ali vinilbutirat.Process according to one of Claims 6 to 8, characterized in that vinyl acetate, vinyl propionate or vinyl butyrate are used as the enol ester. 10. Postopek po enem od zahtevkov 6 do 9, označen s tem, da kot lipazo uporabimo lipazo iz mikroorganizma iz vrste Candida cylindracea.Method according to one of Claims 6 to 9, characterized in that lipase from a microorganism of the species Candida cylindracea is used as a lipase. ZaFor Chemie Linz Gesellschaft m.b.H.:Chemie Linz Gesellschaft m.b.H .:
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