CS211694B1 - Method of preparing immobilized endopolygalacturonase - Google Patents
Method of preparing immobilized endopolygalacturonase Download PDFInfo
- Publication number
- CS211694B1 CS211694B1 CS172980A CS172980A CS211694B1 CS 211694 B1 CS211694 B1 CS 211694B1 CS 172980 A CS172980 A CS 172980A CS 172980 A CS172980 A CS 172980A CS 211694 B1 CS211694 B1 CS 211694B1
- Authority
- CS
- Czechoslovakia
- Prior art keywords
- endopolygalacturonase
- enzyme
- immobilized
- preparation
- preparing immobilized
- Prior art date
Links
- 108010059820 Polygalacturonase Proteins 0.000 title claims description 10
- 238000000034 method Methods 0.000 title claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 9
- 230000008569 process Effects 0.000 claims description 5
- 150000001991 dicarboxylic acids Chemical class 0.000 claims description 2
- 150000002009 diols Chemical class 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims description 2
- KKEYFWRCBNTPAC-UHFFFAOYSA-L terephthalate(2-) Chemical compound [O-]C(=O)C1=CC=C(C([O-])=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-L 0.000 claims 1
- -1 hydroxyalkyl methacrylate Chemical compound 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 229920000139 polyethylene terephthalate Polymers 0.000 description 4
- 239000005020 polyethylene terephthalate Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 229920002230 Pectic acid Polymers 0.000 description 2
- KKEYFWRCBNTPAC-UHFFFAOYSA-N Terephthalic acid Chemical compound OC(=O)C1=CC=C(C(O)=O)C=C1 KKEYFWRCBNTPAC-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 2
- 229920001277 pectin Polymers 0.000 description 2
- 239000001814 pectin Substances 0.000 description 2
- 235000010987 pectin Nutrition 0.000 description 2
- 239000010318 polygalacturonic acid Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 1
- 229920002125 Sokalan® Polymers 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 238000009924 canning Methods 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000010908 decantation Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 125000004185 ester group Chemical group 0.000 description 1
- 108010093305 exopolygalacturonase Proteins 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002351 pectolytic effect Effects 0.000 description 1
- 239000004584 polyacrylic acid Substances 0.000 description 1
- 229920000307 polymer substrate Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000003335 steric effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Description
Vynález sa týká sposobu přípravy imobilizovaného pektolytického enzýmu endopolygalakturonázy /poly-alfa-1,4-D-galacturonid glycanohydrolase, E. C. 3. 2. 1. 15/, ktorá specificky katalyzuje náhodné stiepenie vnútorných alfa-1,4 glykoxidických vazieb v molekule kyseliny pektovej, resp. pektínu a sposobuje tým výrazné zníženie viskozity ich roztokov.The invention relates to a process for the preparation of immobilized pectolytic enzyme endopolygalacturonase (poly-alpha-1,4-D-galacturonide glycanohydrolase, EC 3.2.1.15), which specifically catalyzes the random cleavage of internal alpha-1,4 glycoxidic bonds in a pectic acid molecule , respectively. % of pectin and thereby significantly reduces the viscosity of their solutions.
Doteraz známe imobilizácie tohto enzýmu sa zakladajú na kovalentnom viazaní na nerozpustné nosiče typu Sepharozy, hydroxyalkylmetakrylátu, alebo celulózy, po ich aktivácii brómkyanom, připadne na gél polyakrylovej kyseliny pomocou karbodiimidu.Previously known immobilizations of this enzyme are based on covalent binding to insoluble carriers of the type Sepharose, hydroxyalkyl methacrylate, or cellulose, upon activation with cyanogen bromide, or onto a polyacrylic acid gel with carbodiimide.
Obecnou nevýhodou týchto postupov je operačná zložitosť, podmienená nutnosíou aktivácie nosiča před imobilizáciou enzýmu. U nosičov aktivovaných brómkyanom naviac dochádza často k postupnému uvolňovaniu zakotveného enzýmu do roztoku.The general disadvantage of these procedures is the operational complexity, due to the necessity of activation of the carrier prior to enzyme immobilization. In addition, cyanogen bromide activated carriers often release the anchored enzyme gradually into solution.
dalším nedostatkom je změna sposobu účinku kovalentně viazaného enzýmu na polymérne substráty, podmienená stérickými efektami, ktorá sa prejavuje pomalším znižovaním viskozity ich roztokov než rozpustným enzýmom pri rovnakom stupni degradácie.another drawback is the change in the mode of action of the covalently bound enzyme on polymer substrates, due to steric effects, which results in a slower decrease in the viscosity of their solutions than a soluble enzyme at the same degree of degradation.
Uvedené nedostatky sa odstraňujú postupom podlá vynálezu, ktorého podstatou je, že sa enzým adsorbuje pri pH 3,8 až 4,6 a laboratórnej teplote na polymér obsahujúci v hlavnom reťazci esterová skupinu, na bázi diolov a dikarboxylových kyselin, s výhodou na polyetyléntereftalát.These drawbacks are overcome by the process according to the invention, which is based on the fact that the enzyme is adsorbed at pH 3.8 to 4.6 and room temperature to a polymer containing an ester group in the main chain, based on diols and dicarboxylic acids, preferably polyethylene terephthalate.
Výhoda spósobu přípravy imobilizovanej endopolygalakturonázy spočívá v operačnej jednoduchosti postupu a stálosti zakotvenía, čo pri dodržiavaní optimálnych podmienok enzýmovej reakcie umožňuje používať zmobilizovaný enzym opakované, alebo v dlhodobých kontinuálnych procesoch.The advantage of the process for the preparation of immobilized endopolygalacturonase lies in the operational simplicity of the procedure and the stability of the anchoring, which, in keeping with the optimum conditions of the enzyme reaction, makes it possible to use the mobilized enzyme repeatedly or in long-term continuous processes.
Ďalšou přednostou preparátu připraveného podía postupu je, že spósob jeho účinku na polymérne substráty nie je tak výrazné pozměněný oproti nezakotvenému enzýmu ako v případe preparátov, v ktorých bol enzym iraobilizovaný kovalentnýra viazaníro.A further advantage of the preparation prepared according to the process is that its mode of action on the polymeric substrates is not as significantly altered from the non-anchored enzyme as in the case of preparations in which the enzyme has been immobilized by covalent binding.
Spósob přípravy podía vynálezu je vysvětlený na nasledujúcich príkladoch.The preparation method of the invention is explained in the following examples.
PřikladlEXAMPLE
Endopolygalakturonáza přítomná v technických preparátoch pektináz, alebo z nich připravená lubovolným postupom, napr. afznztnou ehromatografiou na szeťovanej kyselině pektovej, bola zmobilizovaná na polyety1éntereftalát připravený z etylénglykolu a kyseliny tereftalovej peřd Ia následuj úceho postupu: k 1 g polyetylénteref talátu premytom 0,1 M octanovým tlmivým roztokom o pH medzi 3,8 a 4,6 a suspendovanému v 10 ml tohto tlmivého roztoku sa přidá 10 mg endopolygalakturonázy rozpustenej v tlmivom roztoku. Po 4 hodinách miešania pri laboratórnej teplote sa gél oddělí vhodným spósobom /fíltrácia, dekantácia, centrifugácia/.Endopolygalacturonase present in or prepared by technical preparations of pectinases, e.g. and by cross-linked chromatography on cross-linked pectic acid, it was mobilized to polyethylene terephthalate prepared from ethylene glycol and terephthalic acid according to the following procedure: to 1 g of polyethylene terephthalate washed with 0.1 M acetate buffer at pH between 3.8 and 4.6 and suspended ml of this buffer is added 10 mg of endopolygalacturonase dissolved in the buffer. After stirring at room temperature for 4 hours, the gel is separated by a suitable method (filtration, decantation, centrifugation).
Příklad 2Example 2
Polyetyléntereftalát suspendovaný vo vodě alebo tlmivom roztoku o pH medzi 3,8 a 4,6 sa naplní do kolony. Cez kolonu sa filtruje roztok obsahujúci endopolygalakturonázu dovtedy, kým efluent nevykazuje enzýmovú aktivitu. Kolona sa potom premyje octovým tlmivým roztokom pH 4,0 až 4,4.The polyethylene terephthalate suspended in water or buffer having a pH between 3.8 and 4.6 is packed into the column. A solution containing endopolygalacturonase is filtered through the column until the effluent shows enzyme activity. The column is then washed with acetic buffer pH 4.0 to 4.4.
Schopnosť zmobilizovaného enzýmu katalyzovať zmienenú degradáczu pektínových látok je závislá na obsahu adsorbovateIných kontarainujúcich látok vo východzom enzýmovom preparáte a tvoří přibližné 35 až 60 Z schopnosti nezakotveného enzýmu.The ability of the mobilized enzyme to catalyze said degradation of pectin substances is dependent on the content of adsorbable contaminants in the starting enzyme preparation and constitutes approximately 35 to 60% of the ability of the non-anchored enzyme.
Význam vynálezu spočívá v tom, že zmobilizovaná endopolygalakturonáza má využitie v konzervárenskom priemysle pri príprave štiav a muštov.The importance of the invention is that the mobilized endopolygalacturonase has utility in the canning industry in the preparation of juices and musts.
Claims (2)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS172980A CS211694B1 (en) | 1980-03-13 | 1980-03-13 | Method of preparing immobilized endopolygalacturonase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS172980A CS211694B1 (en) | 1980-03-13 | 1980-03-13 | Method of preparing immobilized endopolygalacturonase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CS211694B1 true CS211694B1 (en) | 1982-02-26 |
Family
ID=5352387
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CS172980A CS211694B1 (en) | 1980-03-13 | 1980-03-13 | Method of preparing immobilized endopolygalacturonase |
Country Status (1)
| Country | Link |
|---|---|
| CS (1) | CS211694B1 (en) |
-
1980
- 1980-03-13 CS CS172980A patent/CS211694B1/en unknown
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