CS263588B1 - Process for preparing d-galactopyranuroic acid - Google Patents
Process for preparing d-galactopyranuroic acid Download PDFInfo
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- CS263588B1 CS263588B1 CS879159A CS915987A CS263588B1 CS 263588 B1 CS263588 B1 CS 263588B1 CS 879159 A CS879159 A CS 879159A CS 915987 A CS915987 A CS 915987A CS 263588 B1 CS263588 B1 CS 263588B1
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Description
Vynález sa týká spósobu přípravy kyseliny D-galaktopyranurónovej účinkom imobilizovanej exo-D-galakturonázy..The present invention relates to a process for the preparation of D-galactopyranuronic acid by immobilized exo-D-galacturonase.
Kyselina D-galaktopyranurónová sa doteraz vyrába syntézou len v kapitalistických štátoch, na socialistickom trhu je nedostupná. Bola připravená vo viacerých laboratóriach a to cestou chemickej hydrolýzy: Hatanaka C., Ozawa J.: J. Gen. Chem. Soc. Japan 40, 98 (1960), Rexová — Benková li.: Chem. Zvěsti 21, 59 (1970), alelbo cestou enzýmovej hydrolýzy aplikováním rozpustných pektolytických enzýmov: Van Houndenhoven. F. Vitte P. D., Vister J.: Carbohydr. Res. 14, 113 (1974), Heinrichová K.: AO 220 296 (1986), resp. aplikováním imobilizovanej endo-D-galakturonanázy: Rexová — Benková li., Omelková J., Kubánek V.: AO 218 198 (1983). Postup přípravy kyseliny D-galaktopyranurónovej účinkom imobilizovanej exo-D-galakturonanázy nebol doteraz popísaný. Všetky doteraz popísané postupy sú viacstupňové a značné sa odlišujú heterogenitou získaného produktu. Spoločným rysom týchto postupov je, že vedú k malým výťažkom kyseliny D-galaktopyranurónovej popři výtažku ostatných oligogalakturónových kyselin. Získanie kyseliny D-galaktopyranurónovej zo zmesi oligogalakturónových kyselin, má mnohé nevýhody prácnosť, časová náročnost a viacstupňové izolácie.Until now, D-galactopyranuronic acid has been produced by synthesis only in capitalist countries, and is unavailable on the socialist market. It has been prepared in several laboratories by chemical hydrolysis: Hatanaka C., Ozawa J .: J. Gen. Chem. Soc. Japan 40, 98 (1960), Rex-Benkova: Chem. Rumors 21, 59 (1970), or via enzyme hydrolysis by applying soluble pectolytic enzymes: Van Houndenhoven. Vitte P. D., Vister J .: Carbohydr. Res. 14, 113 (1974), Heinrich K .: AO 220 296 (1986), respectively. by application of immobilized endo-D-galacturonanase: Rex-Benkova, Omelkova J., Kubanek V .: AO 218 198 (1983). The procedure for the preparation of D-galactopyranuronic acid by immobilized exo-D-galacturonanase has not been described so far. All the processes described so far are multistage and differ significantly in the heterogeneity of the product obtained. A common feature of these processes is that they lead to low yields of D-galactopyranuronic acid in addition to the yield of other oligogalacturonic acids. Obtaining D-galactopyranuronic acid from a mixture of oligogalacturonic acids has many drawbacks of labor, time-consuming and multi-step isolation.
Tieto nedostatky odstraňuje spósob přípravy kyseliny D-galaktopyranurónovej úČinkom imobilizovanej exo-D-galakturonanázy, ktorého podstata spočívá v tom, že sa na pektan sodný pósobí imobilizovaným pektolytickým enzýmom — exo-D-galakturonanázou, pri teplote 30 až 40 °C a pH 4,4 až 5,2, pričom jediným produktom enzýmom tkatalyzovanej reakcie je kyselina D-galaktopyranurónová, ktorá sa oddělí chromatografiou na kolóne Sephadexu G—10 a efluent sa· odpaří, alebo vysuší mrazovou sublimáciou.These drawbacks are overcome by the method of preparation of D-galactopyranuronic acid by the action of immobilized exo-D-galacturonanase, which consists in treating sodium pectane with an immobilized pectolytic enzyme, exo-D-galacturonanase, at a temperature of 30 to 40 ° C and pH 4, 4 to 5.2, the only product of the enzymatic reaction being D-galactopyranuronic acid which is separated by Sephadex G-10 column chromatography and the effluent is evaporated or freeze-dried.
Ďalšou výhodou tohoto postupu je kontinuálně a viacnásobné použitie preparátu imobilizovaného enzýmu v reakcii, čo oproti aplikácii rozpustného enzýmu vedie k značným úsporám na nákladech.Another advantage of this process is the continuous and multiple use of the immobilized enzyme preparation in the reaction, which leads to considerable cost savings over the application of the soluble enzyme.
Kyselina D-galaktopyranurónová je látka dóležitá z hlediska teoretického štúdia pektínových látok a pektolytických enzýmov 'a pre charakterizáciu v priemysle používaných komerčných pektináz.D-galactopyranuronic acid is an important substance for the theoretical study of pectin substances and pectolytic enzymes and for the characterization of commercial pectinases used in industry.
Spósob přípravy exo-D-galakturonanázy imobilizovanej kovalentnou vazbou je predmetom AO 257 830, autorov Heinrichová K. a Zliechovcová D. a spósob přípravy exo-D-galakturonanázy imobilizovanej hydrofóbnou adsorpciou je uvedený v AO 257 831 autor Heinrichová K.A method of preparation of exo-D-galacturonanase immobilized by covalent bond is the subject of AO 257 830 by Heinrichová K. and Zliechovcová D. and a method of preparation of exo-D-galacturonanase immobilized by hydrophobic adsorption is disclosed in AO 257 831 by Heinrichová K.
Příklad 1Example 1
100 ml 1 %-ného roztoku pektanu sodného v 0,1 mól. Γ1 octanového tlmivého roztoku pH 4,4 až 4,8 sa inkubuje so suspenziou hydrofóbnou adsorbciou imobilizovaného enzýmu, za miešania, v temperovanej nádobě s dvojitým plášťom. Hydrolýza prebieha pri 40 °G počas 16 až 24 hodin. Enzýmom katalyzovaná reakcia (po kontrole chromatografiou) sa ukončí odstránením imobilizovaného enzýmu odsátím fritou. Supernatant sa zahustí vákuovým odpařením prí 40 °C. 10 ml zahuštěného hydrolyzátu sa nanesie na štipec molekulového šita (Sephadexj G—10) a efluent s kyselinou D-galaktopyranurónovou sa odpaří alebo vysuší mrazovou sublimáciou.100 ml of a 1% solution of sodium pectane in 0.1 mol. Γ 1 acetate buffer pH 4.4 to 4.8 is incubated with the suspension by hydrophobic adsorption of the immobilized enzyme, with stirring, in a double-jacketed, tempered vessel. The hydrolysis is carried out at 40 ° C for 16 to 24 hours. The enzyme-catalyzed reaction (after chromatography control) is terminated by removing the immobilized enzyme by suction filtration. The supernatant is concentrated by vacuum evaporation at 40 ° C. 10 ml of the concentrated hydrolyzate is applied to a column of molecular sieve (Sephadex® G-10) and the D-galactopyranuronic acid effluent is evaporated or freeze-dried.
Výtažok kyseliny D-galaktopyranurónovej je 72 %,The yield of D-galactopyranuronic acid is 72%,
Příklad 2Example 2
Suspenzia exo-D-galakturonanázy imobilizovanej na lubovolný nosič v tlmivom roztoku o pH 4,4 až 5,2 sa naplní do kolóny cez ktorú sa filtruje 0,5 %-ný roztok pektanu sodného dovtedy, kým sa nezíská 90 %-ná konverzia substrátu. Enzýmom katalyzovaná reakcia prebieha pri 30 aC. Efluent z kolóny sa zahustí vákuovým odpařováním pri 40 °C. 10 ml hydrolyzátu sa nanesie na štipec molekulového šita — Sephadex G—10, frakcia kyselinou D-galaktopyranurónovou sa vysuší mrazovou sublimáciou.A suspension of exo-D-galacturonanase immobilized to any carrier in a pH 4.4 to 5.2 buffer is packed into a column through which 0.5% sodium pectane solution is filtered until 90% substrate conversion is obtained. . The enzyme catalyzed reaction proceeds at 30 and C. The effluent from the column is concentrated by vacuum evaporation at 40 ° C. 10 ml of hydrolyzate is applied to a column of molecular sieve Sephadex G-10, the fraction of D-galactopyranuronic acid is freeze-dried.
Výtažok kyseliny D-galaktopyranurónovej je až 82 %.The yield of D-galactopyranuronic acid is up to 82%.
Příklad 3 ml 0,75 %-ného roztoku pektanu sodného v 0,1 mól. I“1 octanovom tlmivom roztoku pH 5,0 až 5,2 sa inkubuje so suspenziou exo-D-galaktronanázy kovalentne viazanej na nosič polyakrylamidového typu. Enzýmom katalyzovaná reakcia prebieha za miešania a temperovania 24 hodin pri 35 °C. Enzýmová reakcia sa ukončí odsátím imobilizovaného enzýmu. Efluent obsahujúci kyselinu D-galaktopyranurónovú sa spracuje ďalej postupom uvedeným v příklade 1 a 2.Example 3 ml of a 0.75% sodium pectane solution in 0.1 mol. I "1 acetate buffer pH 5.0 to 5.2 is incubated with a suspension of exo-D-galaktronanázy covalently linked to the polyacrylamide-type support. The enzyme-catalyzed reaction is carried out under stirring and heating for 24 hours at 35 ° C. The enzyme reaction is terminated by aspirating the immobilized enzyme. The D-galactopyranuronic acid-containing effluent is further processed as in Examples 1 and 2.
Výtažok kyseliny D-galakturónopyranurónovej je 65 %.The yield of D-galacturonopyranuronic acid is 65%.
Schopnost imoibilizovaného enzýmu katalyzovať zmienenú degradáciu pektanu sodného je závislá na obsahu exo-D-galakturonanázy na nosiči a relatívnej aktivitě preparátu ako 1 od reakčných podmienok uvedených v příklade 1, 2 a 3. Preparáty použité v příklade 1 a 3, pri štiepení glykozidických vázieib pektanu sodného vykazovali 40 až 45 % z aktivity rozpustnej formy enzymu.The ability of the imoibilized enzyme to catalyze said degradation of sodium pectan is dependent on the exo-D-galacturonanase content on the carrier and the relative activity of the preparation as 1 from the reaction conditions given in Examples 1, 2 and 3. The preparations used in Examples 1 and 3 sodium showed 40 to 45% of the activity of the soluble form of the enzyme.
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| Application Number | Priority Date | Filing Date | Title |
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| CS879159A CS263588B1 (en) | 1987-12-14 | 1987-12-14 | Process for preparing d-galactopyranuroic acid |
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| CS879159A CS263588B1 (en) | 1987-12-14 | 1987-12-14 | Process for preparing d-galactopyranuroic acid |
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| CS263588B1 true CS263588B1 (en) | 1989-04-14 |
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