DE4000247C2 - - Google Patents

Description

The invention relates to an enzyme reaction method with a the enzyme activity enhancing additive component, wherein the respective enzyme nucleotide-containing molecules, in particular Polynucleic acids as a substrate has.

Reaction solutions for the abovementioned enzyme reactions contain, in addition to the obligatory buffer components: pH-adjusted Good buffer, NaCl, MgCl ₂ , etc. (see: Maniatis, T., Fritsch, EF, Sambrook, J. (1982) "Molecular Cloning" , Pp. 104ff, 453ff), often also additives which, although not a cofactor or co-substrate, accelerate the enzyme reaction or stabilize the enzyme in the reaction solution (Davies, LG, Dibner, MD, Battey, JF (1986) "Basic Methods in Molecular Bio-logic "(Elsevier Science Publishing Co., Inc., New York) pp. 52-53; McCelland, M., Hanish, J., Nelson, M., Patel, Y. (1988) Nucl. Acids Res., 18 (1), 364ff; Dillely, AG, Woo, SLC (1985) Focus (Bethesda Research Laboratories, USA) 7 (2), 1ff). Known additives are, for example: bovine serum albumin, spermidine, polyethylene glycol, dithioerythritol, mercaptoethanol, inhibitors, such as placental RNAse inhibitor, protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), DNase inhibitors, etc.

Furthermore, individual components of the buffer solution, such as z. As sodium chloride, by physiologically related substances be replaced; instead of sodium chloride, for example, Kali umglutamate recommended. Such special supplements are only with conducive to certain individual enzyme reactions. For others Reaction types, on the other hand, usually have a harmful, d. H. inhibitory effect and can also complete a reaction constantly prevent. For example, promotes dibasic spermidine the ligation of oligonucleotides; but it inhibits the Re stricture of high-molecular-weight DNA because it causes DNA strands  be crosslinked to an insoluble precipitate. Some of the Additives, such. As potassium glutamate or PMSF, also influence in gel electrophoretic separation, the running behavior the reaction products, for. B. DNA fragments, so that they because the associated problems for general use unge are suitable.

Commercial enzyme manufacturers therefore avoid Son additives in the reaction solutions - or the regulations and recommendations - because there are always risks and disadvantages associated with this: ignorance of the users about the Be particularities of the individual enzymes; Confusion; Kon decontamination danger; additional effort in the production the reaction buffer; Insufficient degree of purity of the special components; Stock instabilities, etc. Besides, some are the special additives difficult to produce and very expensive.

In forensic and clinical investigations, however, must often large amounts of genomic DNA with different DNA re cleavage endonucleases. But since most Restriction enzymes are very expensive, it is for cost reasons desired, the enzyme activity by means of a special addition stabilize or increase if necessary, the additional components te should be relatively inexpensive. Because such enzyme Reactions often very difficult and only for large amounts of enzyme from Therefore, there was a need for a general better and cheaper enzyme reaction method.

This object is achieved by the enzyme reaction method according to claim 1 solved. Further embodiments of the invention Method emerge from the dependent claims.  

The additional component in the reaction process according to the invention consists of milk or its Ingredients (see claim 1). Under economic In view of this, cow's milk should be preferred.

The additional component in the reaction process according to the invention not only causes an increase in enzyme activity, especially in restriction endonucleases, but also that the reaction with the substrate proceeds more completely. So takes place, for example, in many cases, the restriction digestion of human genomic DNA incomplete - the reasons this is not completely understood - what then wrong Results in the study of restriction fragment length polymorphisms (RFLP's) in the genomic DNA. The invention used special component in the restriction buffer On the other hand, it stabilizes and increases the amount used Enzyme activity, so that the genomic DNA completely is split.

The additional component for the process according to the invention is inexpensive to win, easy to use, and they causes a significant reduction in enzyme costs scientific, clinical, forensic or other DNA / RNA studies, since the activity of the relatively expensive Enzymes can be better utilized.

Enzymes, or reactions which are favored by the additional component in the reaction buffer, are DNA restriction endonucleases, in particular type II restriction endonucleases, but also type I restriction endonucleases which recognize complex DNA patterns, or also type III restriction endonucleases, which cut outside the recognition sequence. So, for example, the following restriction endonucleases:
Aac I, Aae I, Aat I, Aat II, Aca I, Acc I, Acc II, Acc III, Acy I, Afl I, Afl II, Afl III, Aha I, Aha II, Aha III, Aim I, Alu I , Ama I, Aor I, Aos I, Aos II, Apa I; Apy I, Aqu I, Asp AI, Ast AI, Asu I, Asu II, Asu III, Atu II, Atu AI, Atu BI, Atu BVI, Atu CI, Atu HAMI, Ava I, Ava II, Ava III, Avr I , Avr II, Bac I, Bal I, Bam FI, Bam HI, Bam KI, Bam NI, Bam Nx, Ban I, Ban II, Bam III, Bb I, Bb II, Bb AI, Bb AII, Bbi I, Bbi II, Bbi III, Bbi IV, Bbr I, Bbv SI, Bbv I, Bce 170, Bce 1229, Bce14579, Bce FI, Bce R, Bcl I, Bcn I, Bgl I, Bgl II, Bin I, Bin SI, Bin SII, Blo I, Blu I, Blu II, Bme I, Bme 205, Bme 899, Bpe I, Bpr I, Bpu I, Bsa PI, Bse I, Bse II, Bse PI, Bsm I, Bso PI, Bsp 1286, Ex: RI, Bsr HI, Bsr PI, Bsr PII, Bss CI, Bss GI, Bss GII, Bss HI, Bss HII, Bss PI, Bst I, Bst AI, Bsr CI, Bst EI, Bst EII, Bst EIII, Bst GI Bst GII Bst HI Bst NI Bst PI Bst TI Bst XI Bst XII Bsu 1076 Bsu 1114 Bsu 1145 Bsu 1192I Bsu 1192II Bsu 1193 Bsu 1247 Bsu A259 Bsu 6633 Bsu EII, Bsu FI, Bsu M, Bsu RI, Bth I, Bth II, Bvu I, Cal I, Cau I, Cau II, Cau III, Ccr I, Ccr II, Cfo I, Cfr I, Cfr 4I, Cfr 5I, Cfr 6I, Cfr 7I, Cfr 8I, Cfr 9I, Cfr 10I, Cfr 11I, Cfr 13I, Cfr 14I, Cfu I, Chi I, Chu I, Chu II, Cla I, Clm I, Clm II, Clt I, Clu I, Cpa I, Cpe I, Cpf I, Csc I, Cve I, Cvi I , Cvn I, Dde I, Dde II, Dds I, Dpn I, Dpn II, Dra I, Dra II, Dra III, Eae I, Eae PI, Eca I, Eca II, Ecc I, Ecl I, Ecl II, Eco A, Eco B, Eco DXI, Eco ICRI, Eco K, Eco PI, Eco P15, Eco RI, EcoRI ⁽ⁱ⁾ , Eco RII, Eco RV, Eco 24I, Eco 25I, Eco 26I, Eco 27I, Eco 32I, Eco 35I, Eco 36I, Eco 38I, Eco 39I, Eco 40I, Eco 41I, Eco 47I, Eco 47II, Eco 47III, Eco 48I, Eco 49I, Eco 50I, Fdi I, Fdi II, Fnu 48I, Fnu 4HI, Fnu AI, Fnu AII, Fnu CI, Fnu DI, Fnu DII, Fnu DIII, Fnu EI, Fok I, Gdi I, Gdi II, Gdo I, Ggl I, Gin I, Gox I, Gsb I, Gsu I, Hae I, Hae II , Hae III, Hag I, Hap I, Hap II, Hga I, Hgi AI, Hgi BI, Hgi CI, Hgi CII, Hgi CIII, Hgi DI, Hgi DII, Hgi EI, Hgi EII, Hgi FI, Hgi GI, Hgi HI, Hgi HII, Hgi HIII, Hgi JI, Hgi JII, Hgi KI, Hha I, Hha II, Hhg I, Hin 173, Hin 1056I, Hin 1056II, Hin bIII, Hin cII, Hin dI, Hin dII, Hin dIII, Hin dIV, Hin eI, Hin fI, Hin fII, Hin fIII, Hin G UI, Hin GUII, Hin HI, Hin JCI, Hin JCII, Hin P₁I, Hin S₁I, Hin S₂, Hpa I, Hpa II, Hph I, Hsu I, Kpn I, Mae I, Mae II, Mae III, Mau I, Mbo I, Mbo II, Mbv I, Mgl I, Mgl II, Mki I, Mla I, Mlu I, Mni I, Mni II, Mnl I, Mnn I, Mnn II, Mnn III, Mnn IV, Mno I, Mno II , Mno III, Mos I, Mph I, Mra I, Msi I, Msi II, Msp I, Mst I, Mst II, Mth I, Mva I, Mvi I, Mvi II, Nae I, Nam I, Nar I, Nba I, Nbl I, Nbr I, Nca I, Nci I, Nco I, Ncu I, Nda I, Nde I, Nde II, Nfl I, Nfl II, Nfl III, Ngi III, Ngo I, Ngo II, Nla I, Nla II, Nla III, Nla IV, Nla SI, Nla SII, Nmi I, Nmu I, Nmu DI, Nmu EI, Nmu EII, Nmu FI, Nmu SI, Noc I, Nop I, Nop II, Not I, Nov I , Nov II, Nru I, Nsi I, Nsi Al, Nsi HI, Nsp (7524) I, Nsp (7524) II, Nsp (7524) III, Nsp (7524) IV, Nsp (7524) V, Nsp BI, Nsp BII, Nsp HI, Nsp HII, Nsu I, Nsu DI, Nun I, Nun II, Oxa I, Oxa II, Pae R7, Pai I, Pal I, Pan I, Pfa I, Pfl I, Pgl I, Pgl II, Pma I, Ppu I, Psp I, Pst I, Pvu I, Pvu II, Rhe I, Rhp I, Rhp II, Rhs I, Rle I, Rlu I, Rme I, Rrb I, Rrh I, Rrh II , Rro I, Rsa I, Rsh I, Rsp I, Rsr I, Sac I, Sac II, Sac III, Sal I, Sal II, Sal PI, Sau I, Sau 3AI, Sau 96I, Sbo I, Sca I, Sci NI, Scr FI, Scu I, Sdu I, Sdy I, Sex I, Sex II, Sfa I, Sfa GUI, Sfa NI, Sfi I, Sfl I, Sfr I, Sga I, Sgo I, Sgr I, Shy I, Shy TI, Sin I, Sin MI, Sin MII, Ska I, Ska II, Sla I, Slu I, Sma I, Sna I, Sna BI, Sod I, Sod II, Spa I (Xho I), Spa I (Sph I), Sph I, Ssa I, Ssp I, Sst I, Sst II, Sst III, Sst IV, Stu I, Taq I, Taq II, Taq XI, Tfl I, Tgi I, Tha I, Tte I, Tth 111I , Tth IIIII, Tth IIIIII, Tth HB8I, Ttn I, Ttr I, Vha I, Xam I, Xba I, Xho I, Xho II, Xma I, Xma II, Xma III, Xmn I, Xni I, Xor I, Xor II, Xpa I and analogous and isoschizomere enzymes.

For all restriction endonucleases investigated so far in the process according to the invention an increase of the usual Enzyme activity detected. An inhibition of the reaction through the additional component was never observed. The extent the increase in activity depends on the respective enzyme, whereby also the manufacturer plays a role; same enzymes different manufacturers are by the invention Additional component activated or stabilized differently. This indicates that the additional component  presumably capable of contaminating the enzyme and / or DNA preparation to bind and complex.

Further suitable enzymes for the process according to the invention are, for example: ligases, such as T4 DNA ligase, E. coli. DNA Ligase, etc .; RNAses such as RNAse A, RNAse P, RNAse H; kinases, like the DNA polynucleotide kinase I, RNA polynucleotide kinase, T4 polynucleotide kinase, DNA and RNA polymerases, in particular Template-dependent polymerases, such as DNA polymerase I, Klenow fragment of DNA polymerase I, Taq DNA polymerase, genetically modified DNA polymerases, such as Sequenase (Trademark), Etc.; but also template independent polymerases, such as terminal ones transferases; Exonucleases, such as Bal 31 nuclease, DNAses from pancreas; or topoisomerases, such as the DNA Topoisomerase I, etc.

For the process according to the invention are also additional components suitable, that from dried milk powder, thus dried and defatted milk. Suitable dry milk preparations are, for example, all commercial milk powder.

Also particularly suitable are additional components from milk protein. These are for the enzyme reaction process protease, DNAsen and RNAsen-free milk protein fractions are preferred.

The targeted search for effective additives from the milk of showed that especially the caseins - α- and β-casein - the milk protein fraction highly effective and activity-enhancing while lysozyme and lactalbumin have little effect have.

The enzyme activity in the reaction solution is improved, by the additional component according to the invention to an aqueous  added to the buffer solution with enzyme and nucleotide substrate becomes. For the increase and stabilization of enzyme activity it is irrelevant whether the addition of the invention component later or simultaneously with the enzyme in the reaction mixtures is given or whether the additional components te from the beginning in the reaction mixture or in the reaction buffer is present.

Nevertheless, reaction mixtures are preferred in which the additional component according to the invention in an amount of 1 to 10 000 mg / L, preferably from 30 to 3000 mg / L, and be especially preferably from 50 to 300 mg / L. The amount the additional component can be both the enzyme and the sub strat. Generally, however, are physiologically similar Be rich preferred because under these conditions usually the Maximum activity of the enzymes is. Preferred Concentration of the additional component in the reaction solution in the DNA Restriction: 1-1000 mg casein / L, with 10-100 mg casein / L are particularly preferred.

The abovementioned additional component is inventively ver applies to the activity of the enzymes in the reaction solution too improve and stabilize. The activity-improving Addition is particularly advantageous in the enzymati treatment of plasmid DNA, phage DNA and geno mix DNA from prokaryotes and eukaryotes. It is the Degree of purification of the DNA irrelevant, d. h., the additional components te is for both commercially available DNA and for self-isolated DNA suitable.

The additional component used according to the invention becomes the reaction solution advantageously in the form of a ready to use or multiply concentrated buffer solution. The more Special concentration of the buffer solution is more advantageous wise between 2fach and 25fach, whereby 3fach, 5fach and  10 times more preferred. For solubility reasons it is preferred that the additional component and the buffers / Salt solution stored in separate containers and shipped who the. In general, however, are ready to use or Mehrfachkonzen trated solutions of buffer, salt and additional component before Trains t. It goes without saying that all solutions sterilized or sterilized by filtration and RNase-free Herge be presented.

When preparing the ready-made or Mehrkonkon centered buffer / reaction solution may be advantageous still weak, the enzyme reaction non-interfering detergents add, so that the additional component of the invention or the milk proteins during multiple freezing and Auf Do not thaw the multiply concentrated reaction solution fall. For the same reason, it may also be advantageous, the ready to use or multiply concentrated buffer / reac antifreeze, such as glycerol, glycol, etc. add.

The advantageous effects of the invention used Zusatzkom Components will now be described below with reference to the examples Reference to the figures described in more detail. Show it:

Figure 1 shows the ethidium bromide stained restriction fragments of lambda DNA after Hin dIII digestion in the presence and absence of the additional component used in the invention and after gel electrophoretic separation on a 0.6% agarose gel.

Fig. 2 shows the ethidium bromide stained restriction fragments of lambda DNA by Eco RI digestion in the presence and absence of the additional component used in the invention and by gel electrophoretic separation on a 0.6% agarose gel.

FIG. 3 shows the activities of Hin dIII in the digestion of lambda DNA in the presence and absence of the additional component used according to the invention; FIG.

Figure 4 illustrates the activity of Eco RI in the digestion of lambda DNA in the presence and absence of the additional component used in the invention.

Figure 5 shows the activities at the Pst I digestion of lambda DNA in the presence and absence of the additional component used in the invention.

Fig. 6 shows the activities of Bam HI in the digestion of lambda DNA in the presence and absence of the additional component used in the invention.

Example 1:

5 × 1 μg lambda DNA was added to 50 μl each of 1 × reaction buffer 100 mM NaCl; 10 mM Tris-HCl, pH 8.0 (37 ° C); 5 mM MgCl ₂ , 1 mM β-mercaptoethanol) with 100 μg / ml casein at 37 ° C. for one hour with following amounts of restriction enzyme Hin dIII: 0.125 U, 0.25 U, 0.5 U, 1.0 U and 2 U, digested. Thereafter, the reaction was stopped by addition of EDTA (ethylenediaminetetra acetate disodium salt) - the final concentration in the reaction solution was 25 mM EDTA - and the reaction was stored on ice until analyzed at 0 ° C. The reaction solution was then treated with bromophenol blue colored 30% sucrose solution and a 20 μl aliquot of the stained solution on a 0.6% ethididium bromide agarose gel with TAE running buffer: 40 mM Tris-acetate, pH 8.2; 1mM EDTA; charged and elec trophoretisch (100 V, 2-3 h) separated. The DNA bands in the agarose gel were stained during separation by the present Ethididumbromid (1 ug / ml) and the gel photographed under UV light (254 nm) with a corresponding UV filter photo.

See FIG. 1: Lanes 7-11 show Hin dIII restrictions of the lambda DNA, which were carried out in the presence of the additional component used according to the invention:

track Units (U) Hin dIII / μg lambda DNA 7 | 2.0 U 8th 1.0 U 9 0,5 U 10 0.25 U

The experiment shows that in the presence of Zu used according to the invention already sufficient 0.5 units Hin dIII to 1 μg lambda DNA to spal completely within one hour th; The activity of Hin dIII is thus compared to standard conditions - according to the definition of the enzyme unit - about doubled.

Comparative Example 1

5 × 1 μg lambda DNA were analogously to Example 1 in each case 50 ul 1 × reaction buffer 100 mM NaCl; 10 mM Tris-HCl, pH 8.0 (37 ° C); 5 mM MgCl ₂ , 1 mM β-mercaptoethanol) without additional component for one hour at 37 ° C digested with restriction enzyme Hin dIII and analyzed as above.

See Figure 1: Lanes 1-5 show various Hin dIII restrictions of lambda DNA in which no additional component was present in the reaction solution.

track Units (U) Hin dIII / μg lambda DNA 1 | 2.0 U 2 1.0 U 3 0,5 U 4 0.25 U 5 0.125 U

It was found a common enzyme activity.

Example 2:

5 × 1 μg lambda DNA were analogously to Example 1 in each case 50 ul 1 × reaction buffer 100 mM NaCl; 50 mM Tris-HCl, pH 7.5 (37 ° C); 10 mM MgCl ₂ , 1 mM β-dithioerythritol) with 100 ug / ml casein at 37 ° C for one hour digested with various amounts of restriction enzyme Eco RI and as above (see Fig. 2) analysis Siert. Lanes 7-12 show the corresponding Eco RI Re strictions of lambda DNA with the additional component in the reaction solution:

track Units (U) Eco RI / μg lambda DNA 7 | 2.0 U 8th 1.0 U 9 0,5 U 10 0.25 U 11 0.125 U 12 0.0625 U

The experiment shows that in the presence of Zu used according to the invention 0.25 units of Eco RI are sufficient, by 1 μg Completely split lambda DNA within one hour; the activity of Eco RI is thus over Standardbedingun gen - according to the definition of the enzyme unit - about four kindled.  

Comparative Example 2

5 × 1 μg lambda DNA were analogously to Example 2 in each case 50 μ1 × reaction buffer 100 mM NaCl; 50 mM Tris-HCl, pH 7.5 (37 ° C); 10 mM MgCl ₂ , 1 mM β-dithioerythritol) without additional component used according to the invention were digested at 37 ° C. for one hour with various amounts of restriction enzyme Eco RI and analyzed as described above (see FIG. 2). Lanes 1-6 show the corresponding Eco RI restrictions of the lambda DNA without additional component in the reaction solution:

track Units (U) Eco RI / μg lambda DNA 1 | 2.0 U 2 1.0 U 3 0,5 U 4 0.25 U 5 0.125 U 6 0.0625 U

Example 3:

5 × 1 μg lambda DNA were analogously to Example 1 in each case 50 ul 1 × reaction buffer 100 mM NaCl; 10 mM Tris-HCl, pH 8.0 (37 ° C); 5 mM MgCl ₂ , 1 mM β-mercaptoethanol) with 100 μg / ml casein at 37 ° C for one hour with various amounts of restriction enzyme Bam HI: 0.125 U; 0.25 U; 0.5 U; 1.0 U and 2.0 U; digested and analyzed as above (see Fig. 5).

The experiment shows that in the presence of the invention used to set component already 0.5 units Bam HI sufficient to completely cleave 1 ug lambda DNA within one hour; the activity of Bam HI was thus approximately doubled by the additional component (see Fig. 5).

Comparative Example 3.

5 × 1 μg lambda DNA were analogously to Example 3 in each case 50 μl 1 × reaction buffer 100 mM NaCl; 10 mM Tris-HCl, pH 8.0 (37 ° C); 5 mM MgCl ₂ , 1 mM β-mercaptoethanol) without additional component used according to the invention at 37 ° C. for one hour with various amounts of restriction enzyme Bam HI: 0.125 U; 0.25 U; 0.5 U; 1.0 U and 2.0 U; digested and analyzed as above. It was found a common enzyme activity (see Fig. 5).

Example 4:

5 × 1 μg lambda DNA were analogously to Example 1 in each case 50 ul 1 × reaction buffer 100 mM NaCl; 50 mM Tris-HCl, pH 7.5 (37 ° C); 10 mM MgCl ₂ , 1 mM β-dithioerythritol) with 100 μg / ml casein; at 37 ° C for one hour with various amounts of restriction enzyme Pst I: 0.125 U; 0.25 U; 0.5 U; 1.0 U and 2.0 U; digested and analyzed as above (see Fig. 6).

The experiment shows that in the presence of the invention used to set component already 0.5 units of Pst I to completely cleave 1 ug lambda DNA within one hour; the activity of Pst I was thus approximately doubled by the additional component (see Fig. 6).

Comparative Example 4

5 × 1 ug lambda DNA were analogously to Example 4 in each case 50 ul 1 × reaction buffer 100 mM NaCl; 50 mM Tris-HCl, pH 7.5 (37 ° C); 10 mM MgCl ₂ , 1 mM β-dithioerythritol) without additional component used according to the invention at 37 ° C. for one hour with various amounts of restriction enzyme Pst I: 0.125 U; 0.25 U; 0.5 U; 1.0 U and 2.0 U; digested and analyzed as above. It was found a common enzyme activity ( Fig. 6).

Comparison of enzyme activities in the presence and absence the additional component used according to the invention:

From the respective photographs of the various DNA re strictures in the presence and absence of the erfindungsge according to additional components used (see Examples and Vergleichbei 1-4), the strength of the lambda DNA fragments was determined and from this the proportion of digested and undigested Lambda DNA in the various restriction experiments be Right.

The results are shown in Figures 3 to 6. On the abscissa, the enzyme units are applied and on the ordinate the percentage of digested after one hour Lambda-DNA. The broken line represents restrictions in the presence of the additional component used according to the invention (100 μg / ml Casein); the solid line for the associated Comparative Examples without the additional component.

The diagram in FIG. 3 compares the activities of Hin dIII in the presence (Example 1) and absence (Comparative example 1) of the additional component used according to the invention.

The diagram in FIG. 4 compares the activities of Eco RI in the presence (Example 2) and absence (Comparative Example 2) of the additional component used according to the invention.

The diagram in FIG. 5 compares the activities of Pst I in the presence (Example 3) and absence (Comparative Example 3) of the additional component used according to the invention (the associated photo is not shown).

The diagram in FIG. 6 compares the activities of Bam HI in the presence (Example 4) and absence (Comparative Example 4) of the additional component used according to the invention (the associated photo is not shown).

The quantitative determination of the DNA fragments shows that ins especially at low enzyme concentrations (see: Diagrams 3-4) a particularly high activation and / or stabilization the enzymes occurs. It can also be seen from the diagrams that at all concentrations one - mostly proportional - he increased enzyme activity occurs, so that the beneficial We kung of the additional component probably not alone on one Stabilization of the enzymes is based, but at least partially also on a positive allosteric effect on enzyme and / or substrate.

Claims (8)

1. Enzyme reaction process with an enzyme activity-improving additive component, wherein the enzyme nucleotide-containing molecules, in particular polynucleic acids, as substrate, characterized in that an aqueous reaction solution with enzyme and substrate as an additional component milk, dry milk powder, milk protein or protease and DNAsen- and RNAsen- free milk protein fraction is added. 2. Enzyme reaction method according to claim 1, characterized characterized in that as an additional component casein is added. 3. Enzyme reaction method according to claim 1 or 2, characterized in that the additional component in in an amount of 1 to 10 000 mg / L is added. 4. Enzyme reaction method according to claim 1 or 2, characterized in that the additional component in an amount of 30 to 3000 mg / L is added. 5. Enzyme reaction method according to claim 1 or 2, characterized in that the additional component in in an amount of 50 to 300 mg / L is added. 6. Enzyme reaction method according to one of claims 1 to 5, characterized in that the additional component as a solution in a ready-to-use or multiply concentrated enzyme buffer is added.   7. Enzyme reaction method according to one of claims 1 to 6, characterized in that the additional component along with weak, not disturbing the reaction Detergents is added. 8. Enzyme reaction method according to one of claims 1 to 6, characterized in that the additional component is added together with antifreeze.