CS232642B1 - Immobilization method of biopolymers and cells - Google Patents
Immobilization method of biopolymers and cells Download PDFInfo
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- CS232642B1 CS232642B1 CS835541A CS554183A CS232642B1 CS 232642 B1 CS232642 B1 CS 232642B1 CS 835541 A CS835541 A CS 835541A CS 554183 A CS554183 A CS 554183A CS 232642 B1 CS232642 B1 CS 232642B1
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- cells
- weight
- biopolymers
- polyethyleneimine
- hexane
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- 229920001222 biopolymer Polymers 0.000 title claims abstract description 12
- 238000000034 method Methods 0.000 title claims abstract description 10
- 229920002873 Polyethylenimine Polymers 0.000 claims abstract description 10
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 230000001588 bifunctional effect Effects 0.000 claims abstract description 3
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 3
- 239000007864 aqueous solution Substances 0.000 claims description 6
- ZWVMLYRJXORSEP-LURJTMIESA-N (2s)-hexane-1,2,6-triol Chemical compound OCCCC[C@H](O)CO ZWVMLYRJXORSEP-LURJTMIESA-N 0.000 claims description 3
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims description 3
- RRAMGCGOFNQTLD-UHFFFAOYSA-N hexamethylene diisocyanate Chemical compound O=C=NCCCCCCN=C=O RRAMGCGOFNQTLD-UHFFFAOYSA-N 0.000 claims description 3
- YVOFTMXWTWHRBH-UHFFFAOYSA-N pentanedioyl dichloride Chemical compound ClC(=O)CCCC(Cl)=O YVOFTMXWTWHRBH-UHFFFAOYSA-N 0.000 claims description 3
- 238000000265 homogenisation Methods 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 5
- 230000000813 microbial effect Effects 0.000 abstract description 4
- 102000004169 proteins and genes Human genes 0.000 abstract description 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 4
- 230000003100 immobilizing effect Effects 0.000 abstract description 3
- 239000000725 suspension Substances 0.000 abstract description 3
- -1 2-chloromethyloxirane Hexane Chemical compound 0.000 abstract description 2
- 239000013543 active substance Substances 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 abstract 3
- XVPOFVIGJRSQRJ-UHFFFAOYSA-N pentanedioic acid;hydrochloride Chemical compound Cl.OC(=O)CCCC(O)=O XVPOFVIGJRSQRJ-UHFFFAOYSA-N 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 7
- 108010015776 Glucose oxidase Proteins 0.000 description 6
- 235000019420 glucose oxidase Nutrition 0.000 description 6
- 239000004366 Glucose oxidase Substances 0.000 description 5
- 229940116332 glucose oxidase Drugs 0.000 description 5
- 230000007423 decrease Effects 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000002156 mixing Methods 0.000 description 3
- 102100033639 Acetylcholinesterase Human genes 0.000 description 2
- 108010022752 Acetylcholinesterase Proteins 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 108700040099 Xylose isomerases Proteins 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241001136506 Debaryomyces robertsiae Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229940022698 acetylcholinesterase Drugs 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 229920005601 base polymer Polymers 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
Vynález sa týká oboru biochémie, polymérnej chemie, mikrobiologie a biotechnologie. Vynález rieši sposob imobilizácie biopolymérov, predovšetkým proteinov peptidov, enzýmov a celých mikrobiálnych buniek. Uvedeného účelu sa dosiahne tým, že příslušné biopolymérne látky, resp. celé mikrobiálně buňky sa rozpustia alebo suspendujú v roztoku polyetylénimínu tak, aby koncentrácia polyetylénimínu bola v roztoku 15 až 25 % hmotnostných a koncentrácie biologicky aktívneho proteinu alebo mikrobiálnych buniek 0,25 až 5 % hmotnostných, načo sa homogenizovaný roztok alebo suspenzia zosieťuje bifunkčným sietovacím reagentom, najlepšie s 2-chlormetyloxiránom, 1,6-diizotiokyanátom hexanu, 1,6-diizokyanátom hexanu, gluteraldehydom alebo chloridom kyseliny glutarovej po dobu 10 až 60 minút pri teplote 10 až 30 °C. Vynález má využitie všade, kde je účelné imobilizovať biologicky aktivně látky, resp. celé buňky či už pre analytické, alebo priemyslové využitie.The invention relates to the field of biochemistry, polymeric chemistry, microbiology and biotechnology. The invention addresses the method of immobilizing biopolymers, especially peptide proteins, enzymes and whole microbial cells. That purpose is achieved by having appropriate biopolymer substances, respectively. whole microbial the cells are dissolved or suspended in a polyethyleneimine solution such that the concentration polyethyleneimine was in solution 15 up to 25% by weight and concentrations biologically active protein or microbial from 0.25 to 5% by weight of cells homogenize the solution or suspension crosslinks with a bifunctional crosslinking reagent best with 2-chloromethyloxirane Hexane 1,6-diisothiocyanate, 1,6-diisocyanate hexane, gluteraldehyde or chloride glutaric acid for 10-60 minutes at 10 to 30 ° C. The invention has utility where it is expedient immobilize biologically active substances, respectively. whole cells, either analytical or industrial use.
Description
Přihláška vynálezu sa týká sposobu imobilizácie biologicky aktívnych biopolymérov, predovšetkým proteínov, peptidov, enzýmov a celých mikrobiálnych buniek.The invention relates to a method of immobilizing biologically active biopolymers, in particular proteins, peptides, enzymes and whole microbial cells.
Spósoby imobilizácie biologicky aktívnych biopolymérov ako aj celých buniek sú povačšine viacstupňové procesy, pozostávajúce z přípravy a aktivácie matrice k imobilizácii, adjustácie matrice pre vlastnú imobilizačnú reakciu a z odstránenia nezreagovaných východzích látok a reakčných splodín. Nedostatkom vačšiny nosičov je poměrně nízký obsah funkčných skupin využitelných k imobilizácii, resp. ich nerovnoměrná distribúcia. Uvedené nedostatky čiastočme eliminuje využitie polyetylénimínu ako enzýmového nosiče [E. Kuniak, J. Zemek: AO č. 159,063; J. Zámocký, J. Zemek, E. Kuniak, S. Kučár: AO 198 554; J. Zemek, E. Kuniak, P. Gemeiner, J. Zámocký, Š. Kučár: Enzyme Microb. Technol. 4, 233, (1982)], použité postupy sú však poměrně pracné, zosietený skelet enzýmového nosiča neumožňuje vytvorenie membrány alebo filmu s imobillzovaným enzýmom a v případe, že by mali byť imobilizované celé buňky, výťažnosť imobilizačnej reakcie by bola velmi nízká.Methods of immobilizing biologically active biopolymers as well as whole cells are largely multistep processes consisting of preparing and activating the matrix for immobilization, adjusting the matrix for the actual immobilization reaction, and removing unreacted starting materials and reaction products. The disadvantage of most carriers is the relatively low content of functional groups usable for immobilization, respectively. their unequal distribution. These drawbacks are partially eliminated by the use of polyethyleneimine as an enzyme carrier [E. Kuniak, J. Zemek: AO no. 159.063; J. Zamocky, J. Zemek, E. Kuniak, S. Kucar: AO 198 554; Zemek J., Kuniak E., Gemeiner P., Zámocký J., Š. Curly: Enzyme Microb. Technol. 4, 233, (1982)], but the procedures used are relatively laborious, the cross-linked skeleton of the enzyme support does not allow the formation of a membrane or film with an immobilized enzyme and, if whole cells are to be immobilized, the yield of the immobilization reaction would be very low.
Uvedené nedostatky rieši principiálně nový vynález, a to rozpuštěním alebo suspendováním imobilizovanej látky v roztoku polyetylénimínu a masledovným zosietením uvedeného polymeru. Uvedené nevýhody v podstatnej miere odstraňuje spósob imobilizácie biopolymérov a buniek podlá vynálezu, ktorého podstata spočívá v tom, že do 100 hmotnostných dielov vodného roztoku polyetylénimínu o koncentrácii 30 až 50 % hmotnostných sa přidá 100 hmotnostných dielov vodného roztoku alebo: suspenzie obsahujúce 0,5 až 10 % hmotnostných biopolymérov alebo celých buniek a po zhomogenizovaní reakčnej zmesi sa přidá 5 až 20 hmotnostných dielov bifunkčného sietovacieho reagentu ako 2-chlormetyloxiránu, 1,6-diizokyanátu hexanu, 1,6-diizotiokyanátu hexanu, glutaraldehydu alebo chloridu kyseliny glutarovej, pričorn reakcia prebieha 10 až 60 minút pri teplote 10 až 30 °C.In principle, the above-mentioned drawbacks are solved by the new invention by dissolving or suspending the immobilized substance in a polyethyleneimine solution and by cross-linking said polymer. The above-mentioned disadvantages are substantially eliminated by the method of immobilization of the biopolymers and cells according to the invention, which consists in adding to 100 parts by weight of an aqueous solution of polyethyleneimine at a concentration of 30 to 50% by weight 100 parts by weight of an aqueous solution or: 10% by weight of biopolymers or whole cells and 5 to 20 parts by weight of bifunctional crosslinking reagent such as 2-chloromethyl oxirane, hexane-1,6-diisocyanate, hexane-1,6-diisothiocyanate, glutaraldehyde or glutaric acid chloride are added after homogenization of the reaction mixture. 10 to 60 minutes at 10 to 30 ° C.
Výhodou nového spósobu imobilizácie biopolymérov a celých buniek je jeho jednoduchost, nakoíko netřeba reakčnú zmes špeciálne upravovat, reakcia imobilizácie prebieha pri teplotách běžných v laboratóriách po krátku dobu, po skončení imobilizácie netřeba gél nosiča osobitne adjustovať. Nakolko pri imobilizácii dochádza jednak k imobilizácii kovalentnou vazbou, jednak k enkapsulácii biopolymérov do vnútomej štruktúry nosiča v dosledku čoho je hmotnostný podiel biopolymérov, resp. celých buniek v polyetylénovom nosiči velmi vysoký, až 30 % a tým aj vysoká účinnost imobilizácie. Nezanedbatelnou výhodou je aj nízká cena základného' polyméru.The advantage of the new method of immobilization of biopolymers and whole cells is its simplicity, since the reaction mixture does not need to be specially treated, the immobilization reaction takes place at temperatures common in laboratories for a short time, after the immobilization the carrier gel does not need to be separately adjusted. Since the immobilization occurs both on the one hand by the covalent bond and on the other hand by the encapsulation of the biopolymers into the internal structure of the carrier, as a result of which the mass fraction of the biopolymers, respectively. of whole cells in polyethylene carrier very high, up to 30% and hence high immobilization efficiency. A considerable advantage is also the low cost of the base polymer.
Uvedený spósob imobilizácie nie je však vyhovujúci pre tie biosystémy, ktoré sú v alkalickom prostředí polyetylénimínu dezaktivované (napr. pepsin).However, this method of immobilization is not suitable for those biosystems which are inactivated in the alkaline environment of polyethyleneimine (e.g. pepsin).
Příklad 1Example 1
2,25 g polyetylénimínu (0,02 mol) o koncentrácii 40 % hmotnostných a stredne] molekulovej hmotnosti 40 000 sa zmieša s 2,25 gramu suspenzie enzýmu glukózooxidázy (EC 1.1.3.4), obsahujúcej 0,0225 g enzýmového' glykolproteinu, 0,05 g albuminu hovádzieho séra vo fosfátovom pufri (10,0 mmiól/ /1; pH 7,8) a po dókládnom premiešaní na elektromagnetickej miešačke (10 min.) sa přidá 0,112 g 2-chlormetyloxiránu. Po 10 minútach a pri teplote 30 °C sa ztuhlý gél imobilizovaného enzýmu dezintegruje v mixéri, premyje na filtri so 100 ml destilovanej vody a nechá sa vysušit. Výtažok proteinu pri imobilizácii 0,021 g, t. j. 93 °/o. Pokles celkovej aktivity glukózooxidázy v priebehu imobiliácie je z 0,2 jukat na 0,17,ukat.2.25 g of polyethyleneimine (0.02 mol) with a concentration of 40% by weight and a mean molecular weight of 40,000 are mixed with 2.25 g of a glucose oxidase enzyme suspension (EC 1.1.3.4) containing 0.0225 g of enzyme glycolprotein. 0.05 g of bovine serum albumin in phosphate buffer (10.0 mmol / l; pH 7.8) and after thorough mixing on an electromagnetic stirrer (10 min.) Were added 0.112 g of 2-chloromethyl oxirane. After 10 minutes at 30 ° C, the solidified immobilized enzyme gel is disintegrated in a mixer, washed on a filter with 100 ml of distilled water and allowed to dry. Protein yield upon immobilization 0.021 g, t. j. 93 ° The decrease in total glucose oxidase activity during immobilization is from 0.2 jucat to 0.17, ukat.
Příklad 2 g vodného roztoku polyetylénimínu o koncentrácii 10 % hmotnostných sa zmieša s 0,095 g suspenzie permeabilizovaných buniek Wingea robertsii s xylózoizomerázovou aktivitou (4/xkat/g biomasy) v 0,9 ml destilovanej vody. Po dókládnom premiešaní na elektromagnetickej miešačke sa přidá 1,8 g 1,6-diizotiokyanátu hexanu a nechá sa reagovat po dobu 15 minút pri teplote 10 °C. Po vysušení sa získá 3,1 g suchého gélu o: aktivitě xylózaizomerázy 90 nkat/g získaného gélu.Example 2 g of an aqueous solution of 10% by weight polyethyleneimine are mixed with 0.095 g of a suspension of permeabilized Wingea robertsii cells with xylose isomerase activity (4 / xkat / g biomass) in 0.9 ml of distilled water. After thorough mixing on an electromagnetic stirrer, 1.8 g of hexane 1,6-diisothiocyanate is added and reacted for 15 minutes at 10 ° C. After drying, 3.1 g of a dry gel having a xylose isomerase activity of 90 nkat / g of the gel obtained is obtained.
P r i k 1 a d 3Example 1 and d 3
Ták ako je uvedené v příklade 1 s tým rozdielom, že sa vychádza z 5 g vodného roztoku polyetylénimínu a vlastná imobilizácia sa prevedie s 0,2 g 1,6-diizokyanátu hexanu, pri teplote 22 CC po dobu 12 minút. Pokles aktivity glukózooxidázy v priebehu imobilizácie je z 0,2 ^kat na 0,18 ,ukat.The same as in Example 1, except that starting from 5 g of an aqueous polyethyleneimine solution and the actual immobilization is carried out with 0.2 g of hexane 1,6-diisocyanate, at 22 DEG C. for 12 minutes. The decrease in glucose oxidase activity during immobilization is from 0.2 µm to 0.18 µm.
P r í k 1 a d 4Example 1 4
Tak ako je uvedené v příklade 1 s tým rozdielom, že sa vychádza v 10 g vodného roztoku ipolyetylénimínu, přidá sa 2,5 g glukózooxidázy a po dókládnom premiešaní na elektromagnetickej miešačke sa přidá 0,2 g glutaraldehydu. Po 15 minútach a pri teplote 30 °C je reakcia skončená, pričorn pokles celkovej aktivity glukózooxidázy je z 0,2 ^kat na 0,15 ^kat.As in Example 1, except that starting in 10 g of an aqueous solution of ipolyethyleneimine, 2.5 g of glucose oxidase is added and, after thorough mixing on an electromagnetic mixer, 0.2 g of glutaraldehyde is added. After 15 minutes at 30 ° C, the reaction is complete, while the total glucose oxidase activity decreases from 0.2 µm to 0.15 µm.
Příklad 5Example 5
Tak ako je uvedené v příklade 4 s tým rozdielom, že namiesto glutaraldehydu sa použije 0,2 g chloridu kyseliny glutarovej a namiesto glukózooxidázy sa použije ace232642 tylcholínesteráza (0,1 g EC 3.1.1.7) o špecifickej aktivitě nkat/mg. Pokles celkovej aktivity acetylcholínesterázy v priebehu imobilizácie bol za 7 ^kat na 5,94 ^kat, t. j. o 15 %.As described in Example 4, except that 0.2 g glutaric chloride is used in place of glutaraldehyde and ace232642 tylcholine esterase (0.1 g EC 3.1.1.7) with a specific activity of nkat / mg is used instead of glucose oxidase. The decrease in total acetylcholinesterase activity during immobilization was 7 kat cat to 5.94 kat cat, i. j. by 15%.
Vynález má využitie všade, kde je potřebné imobilizovať biologicky aktivně látky, resp. celé buňky či už pre analytické, alebo priemyslové využitie.The invention has applications wherever it is necessary to immobilize biologically active substances, respectively. whole cells, either for analytical or industrial use.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS835541A CS232642B1 (en) | 1983-07-25 | 1983-07-25 | Immobilization method of biopolymers and cells |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CS835541A CS232642B1 (en) | 1983-07-25 | 1983-07-25 | Immobilization method of biopolymers and cells |
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| Publication Number | Publication Date |
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| CS554183A1 CS554183A1 (en) | 1984-06-18 |
| CS232642B1 true CS232642B1 (en) | 1985-02-14 |
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