DE2121809A1 - Enzymatic catalyst, process for its preparation and its use for polymerizing nucleoside diphosphates - Google Patents

Description

DR.-ING. WALTER ABITZ DR. DIETER F. MORF DR. HANSA. BROWN

Patent attorneys

Munich, July 12, 1971 Postal address / Poito! Adclre «8 Munich 86, P.O. Box 860109

Pienienauerstraße 28 έ ^Telefcn 48 32 25 and 48 6415 ^z telegrams: Chemindus Munich

P 21 21 809.7 MERCK & CO. » INC. Rahway, New Jersey 07065, V, St.A.

Enzymatic catalyst, process for its preparation and its use to polymerize of nucleoside diphosphates

The invention relates to a new enzymatic catalyst sator in covalently bound form, a method of making this bound enzyme by treating

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an enzyme catalyst with activated cellulose, and a new process for the production of polynucleotides the liucleosiddiphosphate is polymerized with the help of this covalently bound enzymatic catalyst. The enzymatic one For this purpose, the catalyst polynucleotide phosphorylase (PNPase) is treated with cellulose activated by cyanogen halide, whereby the PNPase is bound to the cellulose. The covalently bound enzymatic PNPase catalyst obtained in this way is then treated with a solution of the nucleoside diphosphate in Brought into contact, forming the corresponding polynucleotide. For example, if one uses (a) inosine diphosphate, forms poly- (ribo) -inosinic acid, and if one uses (b) cytidine diphosphate, poly- (ribo) -cytidylic acid is formed.

The enzymatic catalyst can be used in a similar manner RNA polymerase in the acting as an enzymatic catalyst, convert covalently bound RNA polymerase, and when this is brought into contact with a solution, it becomes a mixture contains inosine diphosphate and cytidine diphosphate a polyribonucleotide copolymer with alternating inosinic acid units and cytidylic acid units as nucleotide units.

These polyribonucleotide copolymers with alternating units as well as the double-stranded complexes that result from mixing two homopolyribonucleotidones, such as the one mentioned above Poly- (ribo) -inosinic acid and the above-mentioned poly- (ribo) -cytidylic acid, are valuable in inducing interferon and host resistance when applied to living cells administered.

So far, polynucleotides were prepared by mixing in aqueous solution a Hucleosiddiphosphat odor, a mixture of nucleoside diphosphates with a rmoleosld-polymerisLerenden enzyme such as Polj-; MiolfiO TUL-PhosphoL'yiuse, RHS Polymsrase and dorgleiehon, This step zuutmmenbviiühbe * r'Le Polymeriua-

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tion continues to the desired level, and the enzyme catalyst was usually destroyed, whereupon the polynucleotide was separated from the inactivated enzyme material. Therefore went with In this known polymerization process, the enzyme catalyst itself, although it was not consumed in the reaction, lost in purification, and fresh catalyst was used to polymerize each new batch of nucleoside diphosphate necessary. Furthermore, the purification of the polynucleotide was made very difficult because it was difficult to obtain the polynucleotide from the inactivated enzyme protein material.

According to the invention, an aqueous PNPase solution is usually obtained by fermenting an aqueous nutrient medium Micrococcus lysodeikticua (now referred to as M. luteus), separating the cell pulp containing the PNPase enzyme, Homogenize the pulp in a homogenizer to break the cells and separate of the cells from the supernatant aqueous PNPase solution. Another method is to lyse the cells, preferably by treating them with lysozyme in aqueous medium, the cell concentration being about 100 g / l and the Lysozyme concentration is about 3 »5 x 10 units per liter. The aqueous PNPase solution (either the supernatant after centrifuging the homogenized cells or the lysate) is stored in two croissants, optionally in a frozen state.

This aqueous PNPase solution, which, if it has been stored in the frozen state for a long time, is expediently centrifuged in order to separate deposited proteins, is added you add so much ammonium sulfate that the ammonium sulfate concentration of the solution is about 24 #, with the protein material (as well as the cell debris if the cells lysed have been) fails. The supernatant PNPase solution, which may be dialyzed to remove the ammonium sulfate

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is then diluted with a water-miscible organic solvent such as acetone or preferably a lower alkanol such as methanol, ethanol, isopropanol and the like, whereupon the majority of the PHPase separates out of the aqueous solution of the organic solvent. In order to achieve the maximum separation of the PNPase, the most favorable concentration of the organic solvent in the resulting aqueous mixture is about 30 to 80% for methanol , preferably about 50 ° β>, for isopropanol about 30 to 40 % and for ethanol about 35 to 45 #, preferably about 40 fo. It is usually advantageous to adjust the pH fourth of the PNPase aqueous solution to about 8.0 and to use BBA alcohol at a concentration of 40 fo . The slurry formed when the organic solvent is mixed with the aqueous PNPase solution is centrifuged in order to separate the precipitated PNPase. This PiTPase is then usually dissolved in a buffer solution with a pH value of 8.2 and the solution is dialyzed against fresh buffer in order to remove the remaining ammonium sulfate from it. The buffered PNPase solution obtained in this way is clarified by centrifugation and expediently stored in the frozen state. This PNPase solution is relatively free of inhibiting factors and generally has a specific PNPase activity of about 0.04 to 0.10 units per mg of protein.

Furthermore, the enzyme catalyst according to the invention is in a converted to covalently bound, water-insoluble form. When this bound catalyst with an aqueous solution of Nucleoside diphosphate is brought into contact, the nucleoside diphosphate polymerizes to the polynucleotide, which in the aqueous Phase remains and is easily removed, e.g. by centrifugation or decanting, from the covalently bound enzyme catalyst can be separated. The polynucleotide is thus obtained in aqueous solution practically free from the enzyme catalyst and of protein impurities that occur in the previously known

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Procedures have resulted in the inactivation of the enzyme. The separation The polynucleotide of this relatively pure aqueous solution is therefore significantly simplified, and the desired polynucleotide can easily be obtained in a highly pure form. Another feature of the invention lies in the fact that the covalently bound enzyme catalyst, which after separation from the one containing the polynucleotide aqueous phase remains, has practically not been changed during the polymerization reaction and therefore several times (at least 30 to 40 times) can be used to polymerize fresh batches of nucleoside diphosphate.

The covalently bound enzymatic catalyst is useful produced by bringing the enzymatic catalyst into contact with activated cellulose in an aqueous medium, whereby the enzyme binds covalently to the cellulose. The "activation" of the cellulose is usually carried out by the cellulose is first mercerized by treatment with aqueous alkali and the mercerized cellulose in aqueous medium, preferably at a pH of about 10 to 11, treated with cyanogen halide. The activated cellulose is adjusted to a pH value of 8 with water, a dilute alkaline solution and finally with a Washed buffer solution. The activated cellulose slurry in buffer solution thus obtained is then mixed with a solution a polymerizing enzyme such as polynucleotide phosphorylase (PNPase), and the slurry kept moving for several hours, whereby the polymerizing enzyme, such as PNPase, binds to the cellulose.

The polymerization of nucleoside diphosphate using this covalently bound enzymatic catalyst is carried out carried out by adding a slurry of the catalyst in the nucleoside diphosphate solution, preferably at about 37 C, keep moving until the desired degree of polymerization is reached. For example, if you have covalently bound

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PNPase as an enzymatic catalyst and a solution of Inosine diphosphate or cytidine diphosphate is used, the Polymerization reaction usually takes about 2 to 10 hours carried out, with a product of higher viscosity (relative viscosity about 8 compared to the Ijucleosidphosphat starting solution) and in the longer polymerization times a product of lower Viscosity (relative viscosity about 5) receives «, It will believed that this decrease in viscosity. due to a breakdown reaction in the case of longer polymerization times which usually takes place simultaneously with the polymerization.

The aqueous polynucleotide solution is separated from the insoluble, covalently bound enzyme catalyst separated and the latter several times - for the polymerization of v / pus approaches of cucleoside diphosphate reused. 85 percent phenol is immediately added to the polynucleotide solution to remove any traces of enzyme catalyst which, along with the solution, may have separated from the insoluble, bound enzyme inactivate. The solution obtained in this way can optionally be extracted with phenol in order to remove traces of protein, even though this stage of the process is also used to purify polynucleotides, which have been polymerized with the aid of bound enzyme is usually not required. That in the Phenol remaining in the aqueous phase, which enters the aqueous phase during inactivation and / or during phenol extraction is extracted from it with ether. The aqueous polynucleotide solution obtained in this way is then first against aqueous, saline sodium citrate buffer solution and then against Dialyzed water, which deprived her of inorganic phosphates, unpolymerized substrate and any trace of protein will. The remaining aqueous solution is converted into the desired polynucleotide, e.g. Polyriboinosinsäure, Polyriboeytidyläure and the like, converted in practically pure form.

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example 1

A fermentation medium is made up of the following ingredients manufactured:

Difco yeast extract 4670.0 g

Mg SO ₄ .7H ₂ O 186.8 g

NaOl - 9.3 g

FeSO ₄ .71I ₂ O 9.3 g MnS0 ₄ c4H ₂ 0 ■ 7.4 g

NaHOO ₅ 3969.6 g

Dextrose 9,340.0 g Made up to 467 1 with water.

This medium is sterilized and with 1.5 1 of a culture of Micrococcus lysodeikticus (now also referred to as M. luteus; ATTG No. 4698) "inoculated in the course of Grown for 36 hours at 28 ° C in 1.5 l of the same medium has been. The inoculated fermentation liquid is then incubated with agitation and aeration for 20 hours at 28 ° C, whereupon the optical density of the fermented liquid approximately Is 2.0. The fermented liquid will pop up quickly Chilled 0 to 5 ° C and then at temperatures below about 5 ° C with a 11.4 cm Sharples centrifuge at a throughput of Centrifuged 3.8 l / min. The supernatant liquid is discarded and the cold cell pulp, which weighs 1.5 to 2.5 kg, suspended in ice-cold 0.5 percent saline solution using 1 1 saline solution per kg cell pulp. At 0 up to 5 ° C., 1 ml of 2-mercaptoethanol as an oxidation retarder is added to this slurry while stirring, and the mixture is filtered then pass the slurry through cheesecloth to remove unbroken lumps which are broken up again and added to the slurry will.

The resulting slurry (which is precooled to 0 to 5 ° C before each pass) is then heated three times at about 560 kg /

ρ
cm sent through a homogenizer in order to

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len to break. This homogenization is conveniently carried out with the laboratory homogenizer, model 15M5O-8TBA, manufactured by Manton-Gaulin Mfg.Co ₀ , Everett, Massachusetts, V.St ₀ A. The homogenized slurry is then 40 minutes at 11,000 g in centrifuged in a deep-frozen Sorvall centrifuge, the solid residue, namely the cell residues, is discarded and the clear supernatant liquid (about 2000 ml) is frozen. Analysis of this supernatant fluid reveals a protein content of about 3.6 mg / ml and a PNPase activity of 0.05 units / ml; 0.014 PNPase units per mg protein.

Some of this frozen liquid is thawed and, if necessary, centrifuged to remove a small amount Remove proteins. To 130 ml of this supernatant liquid, which contains a total of 24 PNPase units, 31 g of solid are slowly added at 0 to 5 ° C. with stirring Ammonium sulfate too. The ammonium sulfate concentration in the resulting solution is 24 #. The mixture is left for an additional 20 minutes stirred and then centrifuged at 23,000 g for 30 minutes. The precipitated solids, mainly proteins, will discarded. With this precipitate, 4.9 units of PNPase activity are lost. This centrifugation (if necessary together with the previous addition of ammonium sulfate) not only proteins are separated, but it becomes also achieved a significant increase in PNPase activity, which is likely due to the withdrawal of inhibitory factors is due.

The supernatant liquid obtained during this centrifugation, the volume of which is about 140 ml, is slowly added, with stirring, with 75 ml of BBA ethanol up to a final concentration of ethanol of 35 ° , whereby PNPase precipitates. The aqueous-ethanol mixture is stirred at 0 to 5 ° C. for 20 minutes and then centrifuged for 35 minutes at 23,000 g, whereby the precipitated PNPase is recovered. The protruding river

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fluid that still contains 3.5 units of PNPase activity, is discarded.

The precipitated PNPase is with a small volume (about 15 to 20 ml) cold (about 5 ° C) PNPase buffer solution of the following Mixing composition:

10 ml 1 molar tris (hydroxymethyl) aminomethane hydrochloride solution

1 ml 1 molar magnesium acetate solution 0.07 ml thioethanol

With water distilled from the glass flask to 1 1

filled up, and the pH of the buffer solution. i

is set to 8.2.

This solution is then dialyzed against fresh PNPase buffer solution at 5 ° C. for 15 hours. The dialysate is clarified by centrifugation (preferably in a deep-frozen Sorvall centrifuge) at 0 to 5 ° C. and the clear supernatant liquid is bottled, frozen and stored in a frozen state. Analysis of this supernatant liquid, the volume of which is 17.6 ml, shows a protein content of 2.8 mg / ml ₉ a PNPase activity of 0.273 units / ml; 0.094 PNPase units per mg protein. The total PNPase content of the supernatant liquid is 4.8 units, which corresponds to a 20 percent recovery of the PNPase activity from the supernatant liquid obtained during centrifugation of this homogenized cell suspension.

Example 2

_{A cell slurry of M 0} luteus obtained by resuspending a cold cell paste prepared according to Example 1 in ice-cold 0.5 percent saline solution, which contains 100 g of cells (dry weight) per liter of 0.5 percent saline solution, is adjusted to a pH of 8 and treated for 12 minutes at 37 ° 0 with lysozyme at a concentration of 3.5 x 10 units per liter of cell slurry.

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The lysed solution is rapidly cooled, mixed with half its volume of cold saturated ammonium sulfate solution and centrifuged. The supernatant liquid is dialyzed against PlTPase buffer solution for 15 hours. “The dialysate is mixed with 0.66 parts by volume of BBA ethanol, which on has been pre-cooled below -20 ° 0. The aqueous ethanol solution will stand at a temperature below 5 ° C. for 15 hours left and the precipitate centrifuged, resuspended in 100 ml PNPase buffer solution and fresh Dialyzed PNPase buffer solution. The dialysate is clarified by centrifugation to obtain a solution which 100 PNPase units per 100 g of that used as starting material Contains Mo luteus cells (dry weight).

Example 3

20 g of cellulose powder (preferably the Horroal variety from Whatman intended for analytical purposes) are slurried with four successive portions of approximately two parts by volume of demineralized water. Each slurry is allowed to settle and the supernatant liquid is decanted, whereby extremely finely divided material is removed. The suspension thus obtained is mixed with 500 ml of 5 N aqueous Katronlauge and the mixture stirred for 15 hours at 0 ° C un ~ W ter nitrogen gently. The aqueous alkaline solution is decanted from the cellulose powder, after which it is filtered on a coarse glass frit plate (without suction) and washed thoroughly with demineralized water. A water-washed slurry of the merited cellulose, in which the cellulose powder has swollen to form a suspension of about 350 ml of looker-settled material, is then bracketed to a volume of 600 al each contains 3 ml of slurry (half of the original fibers is in the - Terfahrsn as fine-grained material oeis ^ ^j i: i solution of gone cellules © Terlorengegangesi) "

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3 ml of this mercerized cellulose are centrifuged and 2 ml of supernatant liquid are decanted off, 50 mg of "dry equivalent ¹¹ " being obtained in a volume of 1 ml, which is cooled to 0 ° C. 150 mg of cyanogen bromide are mixed with 2 ml of water and as quickly as The aqueous cyanogen bromide solution is combined in one shot with the cold aqueous slurry of the mercerized cellulose and the mixture is brought to pH with stirring with 5 normal aqueous sodium hydroxide solution (about 0.1 to 0.2 ml) set from 10 to 11. Within the first 5 to 10 minutes, the alkali is consumed more quickly and the reaction is complete in 15 minutes. 0.1 molar aqueous sodium bicarbonate solution, then washed again with ice-cold water and then twice with 0.01 molar PNPase buffer solution of the following composition en:

10 ml 1 molar tris (hydroxymeth.yl) aminoinethane hydrochloride solution

1 ml 1 molar magnesium acetate solution 0.07 ml thioethanol

Made up to 1 liter with water distilled from a glass flask, and the pH of the slurry is set to 8.2.

The washed "activated" cellulose is dissolved in 2 ml of PNPase buffer solution slurried.

This "activated" cellulose slurry (which contains 50 mg "dry equivalent" of fibers) is placed in a centrifuge tube combined with 1 ml of PNPase of about "1 unit" activity (one unit of PNPase activity is that amount of enzymatically active protein that is capable of 1 μΜοΙ nucleoside diphosphate per minute at a substrate concentration of 1 μΜοΙ / ml to an acid-insoluble polymer to bind at 37 ° C). The centrifuge glass is with

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Purged with nitrogen and the slurry slowly stirred at 5 ° for 15 hours. The resulting slurry is centrifuged and washed twice with 2 ml of fresh PNPase buffer solution each time. The covalently bound enzymatic PNPase catalyst obtained in this way is suspended in 2 ml of PNPase buffer solution and the suspension is stored at 0.degree. 1 ml of this suspension results in a sufficient polymerization rate _{β with 1 ml of 0.06 roolar nucleoside diphosphate solution}

Example 4

310 mg of crystalline inosine diphosphate are in 5 ml from the Glass flask of distilled water is dissolved, and the solution is mixed with 0.75 ml of a 2 molar buffer solution (pH 8.2) of tris (hydroxymethyl) aminomethane hydrochloride, 0.1 ml of a 0.1 molar solution of the sodium salt of ethylenediaminetetraacetic acid, 0.1 ml of a 1 molar magnesium acetate solution and 6 μΐ non-fractionated oligoadenylic acid as initiator (concentration about 5 μΜοΙ / ml) added. The volume of this solution is brought to 10 ml with water distilled from the glass flask and the pH is adjusted to 8.2. This is how you get one Inosine diphosphate staram solution of approximately the following composition:

0.06 moles of inosine diphosphate

0.15 moles of tris (hydroxymethyl) aminomethane hydrochloride as a buffer

0.01 moles of magnesium acetate

0.001 moles of ethylenediaminetetraacetic acid 3 x 10 "moles of oligoadenic acid.

1 ml of the, as described above, produced, 'covalently bound PXTPase suspension is centrifuged in a centrifuge tube for 1 minute in a clinical centrifuge and the Separated supernatant liquid. Then add 1 ml of the inosine diphosphate stock solution (0.06 molar of inosine diphosphate) to, stir the mixture, centrifuged, separates the inosine diphosphate

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phat washing solution and add 1 ml of fresh inosine diphosphate solution to. The resulting slurry of covalently bound PNPase in inosine diphosphate solution is stirred at 37 C until the relative viscosity of the supernatant liquid is in with respect to the inosine diphosphate starting solution is in the range from 2 to 8, and the supernatant liquid (the polyinosinic acid contains) is separated from the covalently bound PNPase. A polyinosinic acid solution is obtained that is practical is free from PNPase and other proteins.

The remaining, covalently bound PNPase is used to polymerize, tion of further inosine diphosphate used by the method described above, and this polymerization method can be carried out repeatedly without a significant amount even after 30 to 40 individual polymerization processes Loss of activity of the covalently bound PNPas'e enzyme makes noticeable.

The polyinosinic acid solution is immediately with 85 percent Phenol is added, which contains sodium dodecyl sulfate to deactivate any traces of PNPase, then cooled to 0 ° 0 and extracted with phenol to remove the remaining proteins. The phenol becomes the aqueous phase extracted with ether, and the aqueous solution thus obtained f in the cold, first thoroughly against saline sodium citrate buffer solution (0.04 molar in sodium chloride, 0.005 molar in sodium citrate, adjusted to pH von 7 | 0) and then dialyzed against water, whereby its inorganic Phosphates, non-polymerized substrate, etc. removed will. The remaining aqueous solution is converted into polyinosinic acid by freeze-drying, which is a white, fibrous mass accumulates.

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Example 5

If, in the method of Example 4, cytidine diphosphate Used instead of the inosine diphosphate, polycytidylic acid is obtained.

Example 6

Some of the covalently bonded ones produced according to Example 3 PNPase is repeated to form batches of 60 μΜοΙ inosine diphosphate in buffer solution (inosine diphosphate stock solution, prepared according to Example 4) added after the previous Yield has been separated by centrifugation as a supernatant solution, and the enzymatic polymerization is carried out carried out according to Example 4. In this way, the Stirring, recovering and reconnecting the catalyst with fresh solution more than twenty times carried out, the suspension of the covalently bound PNPase and the substrate with each other for 2 to 10 hours be stirred. The relative viscosity of the supernatant liquid varies according to the polymerization processes in the area from about 3 to 8. The polymerization attempts last six days with interrupted operation at 37 ° 0? with everyone The experiment turns out to be 85% immediately after the polymerization Phenol and sodium dodecyl sulfate were added and the supernatant liquid was stored at 0 ° C.

Nine polymerization products of higher viscosity (polymerization time about 2 hours) are combined and extracted three times with the phenol phase, whereupon the aqueous Layer is separated. The aqueous layer is then freed from dissolved phenol by extracting with ether and thoroughly in the cold three times for 4 hours each against saline Citrate buffer solution and dialyzed four times against water distilled from the glass flask. By freeze drying 62 mg of polyinosinic acid are obtained (yield 25%)

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Eleven polymerization products of lower viscosity (polymer isation duration about 8 to 10 hours) are combined and treated in a similar manner, 87 mg Polyinosinic acid obtained (yield 20 #). '"

These polyinosinic acid fractions are complexed separately with equimolecular amounts of polycytidylic acid, resulting in a double strand of poly (I: C) complex-bound polymers (hypochromicity $ 37 or $ 38), which induces interferon when administered to living cells .

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Claims (7)

P 21 21 809.7 / mtV fi / il * VAATtTnJ July 12, 1971 ^ 1 21 Merck & Co., Inc. <. /, Ί. / 7 * 14 30? New patent claims 1. Enzymatic rather catalyst in covalently bound form. 2. Enzymaticeher catalyst according to claim 1, characterized in that φ indicates that polynucleotide phosphorylase is covalently attached activated cellulose is bound. 3. Enzymatic catalyst according to claim 1, characterized in that that ribonucleic acid polymerase is covalently bound to activated cellulose. 4. Process for the preparation of the covalently bound enzymatic Catalyst according to claim 1 to 3> characterized in that one in an aqueous medium brings together enzymatic catalyst with activated cellulose, whereby the enzyme covalently attaches itself to the cell ^ loosely ties. 5. The method according to claim 4, characterized in that one Polynueleotid-Phosphorylase with by halogen cyanogen brings together activated cellulose, 6. The method according to claim 4, characterized in that ribonucleic acid polymerase with by treating with Bringing together cyanogen activated cellulose. 7. Use of a covalently bound catalyst according to claim 1 to 3 for homo- or copolymerization of nucleoside diphosphates. 5-2V