CS275662B6

CS275662B6 - Carrier for enzyme stabilization and method for its preparation

Info

Publication number: CS275662B6
Application number: CS664289A
Authority: CS
Inventors: Ludovit Ing Csc Kuniak; Angela Ing Matusova
Original assignee: Univ Slovenska Tech
Priority date: 1989-11-24
Filing date: 1989-11-24
Publication date: 1992-03-18

Abstract

Riešpnie sa týká nosiča na stabilizáciu enzýmov tia báze proteínov. Jeho podstatou je, že je tvořený práškovou formou proteinu, s výhodou vaječného bielka, alebo hovadzieho albuminu, alebo kazefnu, zosieteného epi- chlórhydrínom v množstve 15 až 25 X hmot. na hmotnost sušiny proteinu, pričom velkost .častíc nosiča je v rozsahu 40 až 300 mikro- metrov a hodnota napúčania vo vodě je v rozsahu 6 až 15 ml/g. Podstata spósobu přípravy spočívá v tom, že roztok proteinu, s výhodou vaječného albuminu sa zosietuje epi- chlórhydrínom v alkalickom prostředí při teplote 15 až 30 °C po dobu 12 až 20 hodin. Potom sa zosietený protein za mokra defi- bruje na částice o velkosti 40 až 300 mikro- metrov a po premytí sa z nich voda vytěsní etanolom, ktorý sa z gélu volné odpaří. Na vysušený nosič sa nanesie enzýmový roztok hydrolázy v pomere 0,75 ml enzýmového roztoku na 1 g suchého nosiča a nechá sa volné vysušit. Takto zakotvený enzým má vysokú stabilitu pri dlhodobom skladovaní. Rieše- nie má použitie všade, kde je potřebné dlho- dobo stabilizovat hlavně hydrolázové enzýmy . při dlhodobom skladovaní bez straty enzýmo- vej aktivity.The present invention relates to a carrier for the stabilization of protein base enzymes. It is based on the fact that it consists of a powdered form of a protein, preferably egg white or bovine albumin, or a epichlorohydrin cross-linked casin in an amount of 15 to 25% by weight. to the dry weight of the protein, wherein the carrier particle size is in the range of 40 to 300 microns and the swelling value in water is in the range of 6 to 15 ml / g. The method of preparation is based on the fact that the protein solution, preferably egg albumin, is crosslinked by epichlorohydrin in an alkaline medium at 15 to 30 ° C for 12 to 20 hours. Thereafter, the crosslinked protein is wet-wetted to a particle size of 40 to 300 microns, and after washing, the water is displaced with ethanol, which is freely evaporated from the gel. The hydrolase enzyme solution is applied to the dried carrier in a ratio of 0.75 ml of enzyme solution to 1 g of dry carrier and allowed to dry freely. Such an anchored enzyme has a high long-term stability. The solution is used wherever it is necessary to stabilize mainly hydrolase enzymes for a long time. long-term storage without loss of enzyme activity.

Description

Vynález sa týká nosiča na stabilizáciu enzýmov a spůsobu jeho přípravy.The invention relates to a carrier for stabilizing enzymes and to a process for its preparation.

Stabilizácia enzýmov je důležitá tak pře účely základného výskumu, ako i pre potřeby spoločenskej praxe. Mnohé enzýmy najma pri vysokých stupňoch čistoty sú velmi citlivé na stratu aktivity při skladovaní, a to nielen pri laboratórnych teplotách, ale i při znížených teplotách, čím sa znehodnocuje práca spojená s ich izoláciou a purifikáciou. Sú známe různé spůsoby stabilizácie enzýmov tak v roztoku, alebo v pevnom stave ich chemickou modifikáciou /R.Axén, P.Marin, Jonson J.: Biopolymers 9,401 /1970/, resp. immobilizáciou/ K. Mosbach: Meth. Enzymol. 44,453 /1976/. Společným nedostatkem známých postupov stabilizácie enzýmov je spravidla ich technologická náročnost’, ako i strata značnej časti aktivity enzýmu v modifikačnom stupni.Stabilization of enzymes is important both for the purposes of basic research and for the needs of social practice. Many enzymes, especially at high degrees of purity, are very sensitive to loss of activity during storage, not only at room temperatures but also at reduced temperatures, thus undermining the work associated with their isolation and purification. Various methods for stabilizing enzymes in solution or in the solid state by chemical modification are known (R. Oxen, P. Marin, Jonson J .: Biopolymers 9,401 (1970), resp. immobilization / K. Mosbach: Meth. Enzymol. 44,453 (1976). A common shortcoming of the known methods for stabilizing enzymes is usually their technological complexity, as well as the loss of a significant part of the enzyme activity in the modification step.

Uvedené nedostatky rieši nosič na stabilizáciu enzýmov na báze proteínov podlá vynálezu, ktorého podstatou je, že je tvořený práškovou formou proteinu, s výhodou vaječného bielka alebo hovádzieho albuminu alebo kazeínu, zosieteného epichlórhydrínom v množstve 15 až 25 % hmot, na hmotnost’ sušiny proteinu, pričom velkost’ častíc nosiča je v rozsahu 40 až 300 mikrometrov a hodnota napúčania vo vodě je v rozsahu 6 až 15 ml/g. Podstatou spůsobu přípravy nosiča je, že vodný roztok proteinu o koncentrácii 10 až 13 % hmot., s výhodou čerstvý vaječný bielok, sa sieťuje v prostředí hydroxidu sodného o koncentrácii 0,8 až 1,2 % hmot, pri teplote 15 až 25 °C po dobu 12 až 20 h s epichlórhydrínom v množstve 1,5 až 2,5 % hmot, na hmotnost sletovaného proteinového roztoku, načo sa zosietený protein zmixuje v rýchloobrátkovom mixeri v etanole na velkost častíc 40 až 3.00 mikrometrov, suspenzia sa premyje na filtri destilovanou vodou do neutrálnej reakcie a nakoniec sa voda z gelu vytěsní alkoholem a alkohol sa odpaří volné na vzduch alebo vo vákuovej sušiarni pri teplote do 40 °C.These shortcomings are addressed by the protein-based enzyme stabilizing support according to the invention, which consists of a powdered form of a protein, preferably egg white or bovine albumin or casein, crosslinked with epichlorohydrin in an amount of 15 to 25% by weight, based on the dry weight of the protein. wherein the particle size of the carrier is in the range of 40 to 300 micrometers and the swelling value in water is in the range of 6 to 15 ml / g. The essence of the process for the preparation of the carrier is that an aqueous protein solution with a concentration of 10 to 13% by weight, preferably fresh egg white, is crosslinked in a sodium hydroxide medium with a concentration of 0.8 to 1.2% by weight, at a temperature of 15 to 25 ° C. for 12 to 20 hours with epichlorohydrin in an amount of 1.5 to 2.5% by weight, based on the weight of the ground protein solution, after which the crosslinked protein is mixed in a high-speed mixer in ethanol to a particle size of 40 to 3.00 micrometers, the suspension is washed on the filter with distilled water until neutral, and finally the water is displaced from the gel by the alcohol and the alcohol is evaporated free to air or in a vacuum oven at a temperature of up to 40 ° C.

Při využití nosiča podlá vynálezu nedochádza k poklesu aktivity enzýmu a spůsob přípravy nosiča je technologicky podstatné jednoduchší v porovnaní s doteraz známými postupmi.When using the carrier according to the invention, there is no decrease in the activity of the enzyme and the method of preparation of the carrier is technologically substantially simpler in comparison with the hitherto known methods.

Výsledný produkt, biely prášok s velkosťou častíc 40 až 300 mikrometrov je vhodný ako pevný nosič na stabilizáciu najma hydrolázových enzýmov. Stabilizácia enzýmu na pripravenom nosiči sa robí jednoducho tak, že enzýmový roztok nalejeme na suchý nosič najlepšie v pomere 0,75 ml roztoku enzýmu na 1 g suchého nosiča, dobré rozmiešame necháme 5 až 10 minút nasat kvapalnú fázu do pórov nosiča a potom zvlhčený nosič necháme volné na vzduchu vysušit .... v tenkej vrstvě. Vysušený nosič s enzýmom sa může skladovat při laboratórnej teplote, resp. u citlivějších enzýmov pri teplote 4 až 8 °C dlhú dobu bez zjavnej straty enzýmovej aktivity. Enzým podlá potřeby uvolníme z nosiča miešaním v 20 až 50 násobku destilovanej vody pri izbovej teplote po dobu 15 minút. Zosietený gél sa odcentrifuguje alebo odfiltruje. Supernatant, resp. filtrát s rozpuštěným enzýmom o původnej aktivitě použijeme podlá potřeby.The resulting product, a white powder with a particle size of 40 to 300 micrometers, is suitable as a solid carrier for stabilizing especially hydrolase enzymes. Stabilization of the enzyme on the prepared support is done simply by pouring the enzyme solution onto the dry support, preferably in a ratio of 0.75 ml of enzyme solution per 1 g of dry support, mixing well, letting the liquid phase into the pores of the support for 5 to 10 minutes and then letting the moistened support. free to air dry .... in a thin layer. The dried enzyme carrier can be stored at room temperature or for more sensitive enzymes at 4 to 8 ° C for a long time without apparent loss of enzyme activity. The enzyme is released from the support as needed by stirring in 20 to 50 times distilled water at room temperature for 15 minutes. The crosslinked gel is centrifuged or filtered. Supernatant, resp. The filtrate with the dissolved enzyme of the original activity is used as needed.

Pochopitelné roztok enzýmu před štabilizáciou s ohladom na pH a ionovú silu upravíme na optimálně hodnoty konkrétného enzýmu. Pri navrhovanom spůsobe stabilizácie enzýmov na pevnej bielkovinovej matrici nedochádza ku kovalentnej vazbě enzýmu na pevný nosič, ale jedná sa tu o fyzikálnu adsorbciu enzýmu na pevný nosič, ktorý má protektorové vlastnosti oproti molekule enzýmu, ktorá sa při vysušování orientuje na povrchproteinu tak, že aktivně centra uchovávaného enzýmu sú chráněné proti fyzikálno-chemickej dezakťivácii.We can adjust the understandable enzyme solution to the optimal values of the specific enzyme before stabilization with regard to pH and ionic strength. The proposed method of stabilizing enzymes on a solid protein matrix does not covalently bind the enzyme to a solid support, but involves physical adsorption of the enzyme to a solid support, which has protector properties over the enzyme molecule, which orients on the protein surface of the stored enzyme are protected against physico-chemical deactivation.

Na přípravu navrhovaného nosiča možno použit aj iný typ proteinu, napr. hovadzí albumin, kazeín a.i., no ekonomicky je najvýhodnejší vaječný bielok.Another type of protein can be used to prepare the proposed carrier, e.g. bovine albumin, casein a.i., but egg white is the most economically advantageous.

Hlavnou výhodou navrhovaného spůsobu přípravy nosiča na stabilizáciu enzýmov je jeho všeobecná dostupnost’, jednoduchost přípravy a vysoká účinnost stabilizácie hydrolázových enzýmov.The main advantages of the proposed process for the preparation of a carrier for the stabilization of enzymes are its general availability, ease of preparation and high efficiency of stabilization of hydrolase enzymes.

Příklad 1 .Example 1.

K 100 g vaječného bielka so sušinou 12,6 % sa přidá 1,5 ml epichlórhydrínu a mieša sa pri laboratórnej teplote 30 min., kedy je epichlórhydrín homogénne rozpuštěný v celom objeme bielka. Potom sa přidá za zrýchleného miešania 1,5 ml 17,5 % hydroxid sodný a nechá sa CS 275 662 B6 2 při laboratórnej teplote sletovat 20 hodin. Po zosietení sa pevný gél mixuje v mixeri pri otáčkách 1500/min v 200 ml etanolu po dobu 2 minut. Zmixovaný gél sa potom odfiltruje cez sklený filter a na filtri sa premýva destilovanou vodou do neutrálnej reakcie filtrátu. Voda sa z gélu vytěsní etanolom a etanol sa odpaří na vzduchu z tenkej vrstvy gelu na filtračnom papieri, alebo sa odpaří vo vákuovej sušiarni při teplote do 40 °C. Vysušený gél sa presituje cez šitá 0,04 a 0,3 mm. Hrubšie částice, ktoré ostáné na site 0,3 mm, sa zo melú za sucha tak, až celý podiel častíc přejde cez šito 0,3 mm. Frakcia, ktorá přejde sitom 0,04 sa pře účely stabilizácie enzýmov nepoužije, ale len frakcia v rozsahu 0,04 až 0,03 mm. Výťažok použitelnej frakcie sa pohybuje v rozsahu 90 až 95 % hmot, na hmotnost sušiny použitého proteinu. Stabilizácia enzýmu: Na 10 g připraveného suchého proteinového nosiča sa naleje naraz 7,5 ml roztoku <?6-amylázy pankreatickej o aktivitě 6,0 yukat vo fosfátovom pufri 0,05 M o pH 7,0 a dobře sa zamieša, aby sa kvapalná fáza rovnoměrně rozdělila na pevnom nosiči. Po 15 min možno vzorku v tenkej vrstvě na celofáne nechať vysušit cez noc při izbovej teplote. Po vysušení sa vzorka homogenizuje v trecej miske a uskladní při izbovej teplote. Po polročnom skladovaní aktivita enzýmu poklesla len o 4,5 %. .1.5 ml of epichlorohydrin are added to 100 g of egg white with a dry matter of 12.6% and stirred at room temperature for 30 minutes, during which time epichlorohydrin is homogeneously dissolved in the entire volume of white. Then 1.5 ml of 17.5% sodium hydroxide are added with rapid stirring and the CS 275 662 B6 2 is allowed to solder at room temperature for 20 hours. After crosslinking, the solid gel is mixed in a mixer at 1500 rpm in 200 ml of ethanol for 2 minutes. The mixed gel is then filtered through a glass filter and washed on the filter with distilled water until the filtrate is neutral. The water is displaced from the gel with ethanol and the ethanol is evaporated in air from a thin layer of gel on a filter paper or evaporated in a vacuum oven at a temperature of up to 40 ° C. The dried gel is sieved through sieves 0.04 and 0.3 mm. The coarser particles remaining on the 0.3 mm sieve are ground dry until the entire proportion of particles passes through the 0.3 mm sieve. The fraction passing through the 0.04 sieve is not used for enzyme stabilization purposes, but only the fraction in the range of 0.04 to 0.03 mm. The yield of the usable fraction is in the range of 90 to 95% by weight, based on the dry weight of the protein used. Enzyme stabilization: 7.5 ml of a 6-yucat pancreatic β-amylase solution with an activity of 6.0 yucate in 0.05 M phosphate buffer at pH 7.0 are poured at once onto 10 g of the prepared dry protein carrier and mixed well so that the liquid phase evenly distributed on a solid support. After 15 minutes, the sample can be allowed to dry in a thin layer on cellophane overnight at room temperature. After drying, the sample is homogenized in a mortar and stored at room temperature. After six months of storage, the enzyme activity decreased by only 4.5%. .

Ak sa rovnaký roztok enzýmu naniesol na rovnaké množstvo suchej mikrokryštalickej celulózy a nechal sa rovnako vysušit, tak sa regenerovalo ihned po vysušení už len 2 % pfívodnej aktivity, tj. mikrokryštalická celulóza nemá žiadné protektorové vlastnosti pře uchovávanie enzýmovej aktivity pri skladovaní enzýmov.If the same enzyme solution was applied to the same amount of dry microcrystalline cellulose and allowed to dry in the same way, only 2% of the feed activity was recovered immediately after drying, i. microcrystalline cellulose has no protector properties to retain enzyme activity during enzyme storage.

Příklad 2Example 2

Postup ako v příklade 1 s tým rozdielom, že na sieťovanie sa použije 2,5 ml epichlórhydrínu a sieťovanie trvá 12 hodin pri laboratórnej teplote. Výsledný produkt má obdobné vlastnosti ako produkt v příklade 1.The procedure is as in Example 1, except that 2.5 ml of epichlorohydrin are used for crosslinking and the crosslinking lasts 12 hours at room temperature. The resulting product has similar properties to the product in Example 1.

Příklad 3Example 3

Postup ako v příklade 1 s tým rozdielom, že ako protein sa použije hovádzí albumin rozpustný v destilovanej vodě na koncentráciu 12,0 % hmot. Výsledný produkt má porovnatelné vlastnosti ako produkt z příkladu 1.The procedure is as in Example 1, except that bovine albumin soluble in distilled water is used as the protein to a concentration of 12.0% by weight. The resulting product has comparable properties to the product of Example 1.

Příklad 4 .Example 4.

Postup ako v příklade 1 s tým rozdielom, že ako enzým na štabilizáciu sa použije slinná alfa-amyláza. Stabilita enzýmu je obdobná po pol roku ako v příklade pankreatickej alfa amylázy.The procedure is as in Example 1, except that salivary alpha-amylase is used as the stabilizing enzyme. The stability of the enzyme is similar after half a year as in the example of pancreatic alpha amylase.

Vynález má použitie všade, kde je potřebné skladovat hydrolázové enzýmy bez straty aktivity, napr. při přípravě enzýmových štandardov slinnej a pankreatickej alfa amylázy pri diagnostikovaní funkčných porúch pankreasu v humánněj medicine.The invention has application wherever it is necessary to store hydrolase enzymes without loss of activity, e.g. in the preparation of salivary and pancreatic alpha amylase enzyme standards in the diagnosis of functional disorders of the pancreas in human medicine.

Claims

CS 275 662 B6 2 for 20 hours. After crosslinking, the solid gel is mixed in a 1500 / min mixer in 200 ml ethanol for 2 minutes. The blended gel is then filtered off through a glass filter and washed on the filter with distilled water to neutralize the filtrate. The water is displaced from the gel with ethanol and the ethanol is evaporated in air from a thin layer of gel on filter paper, or evaporated in a vacuum oven at a temperature below 40 ° C. The dried gel is sieved through 0.04 and 0.3 mm sutures. The coarser particles remaining on the 0.3 mm sieve are dry-dried until all of the particles pass through a 0.3 mm sieve. The 0.04 fraction is not used for enzyme stabilization purposes, but only a fraction of 0.04-0.03 mm. The yield of the usable fraction ranges from 90 to 95% by weight. the weight of the protein used. Enzyme Stabilization: Pour 10 ml of the pancreatic o-amylase solution of 6.0 yukat in pH 0.05 pH 7.0 phosphate buffer into a 10 g dry protein carrier prepared at once and mix well. the liquid phase is evenly distributed over the solid support. After 15 min, the sample can be dried in a thin layer over cellophane over night at room temperature. After drying, the sample is homogenized in a mortar and stored at room temperature. After half-year storage, enzyme activity decreased by only 4.5%. If the same enzyme solution was applied to the same amount of dry microcrystalline cellulose and allowed to dry as well, only 2% of the initial activity was regenerated immediately after drying, i.e. microcrystalline cellulose has no retector properties for storing enzyme activity when storing enzymes. Example 2 Procedure as in Example 1 except that 2.5 ml of epichlorohydrin is used for crosslinking and the crosslinking lasts 12 hours at room temperature. The resulting product has similar properties to that of Example 1. EXAMPLE 3 Procedure as in Example 1 except that bovine albumin dissolved in distilled water is used as a protein to a concentration of 12.0% by weight. The resulting product has comparable properties to that of Example 1. EXAMPLE 4 Procedure as in Example 1 except that saline alpha amylase is used as the enzyme for stabilization. The stability of the enzyme is similar after half a year as in the example of pancreatic alphaamylase. The invention has utility where it is desired to store the hydrolase enzymes without loss of activity, e.g., in the preparation of salivary and pancreatic alpha amylase enzyme standards, by diagnosing pancreatic functional disorders in human medicine. PATENT CLAIMS

What is claimed is: 1. A carrier for the stabilization of protein-based enzymes comprising a powder form of a protein, preferably egg white or bovine albumin or casein crosslinked with 15 to 25% by weight of epichlorohydrin. to the dry weight of the protein, the particle size of the carrier being in the range of 40 to 300 microns and the swelling value in water being in the range of 6 to 15 ml / g.

2. A method of preparing a carrier for stabilizing enzymes according to claim 1, wherein the aqueous protein solution at a concentration of 10 to 13% by weight, preferably fresh egg white, is sieved in a sodium hydroxide solution at a concentration of 0.8 to 1. 2 wt. at 15 to

For about 12 to 20 hours with epichlorohydrin in an amount of 1.5 to 2.5% by weight. to the weight of the protein solution to be ground, whereupon the crosslinked protein is mixed in ethanol to a particle size of 40 to 300 microns, the suspension is washed on the filter with distilled water to neutralize, the water is displaced from the gel by alcohol and the alcohol is evaporated at a temperature of up to 40 ° C.