SI7812162A8 - Process for determining alpha-amilase - Google Patents

Description

A technical problem

There was a need to find a new, technologically advanced process for the determination of «/ - amylase in solution as a body fluid outside the human or animal body, with enzymatic cleavage of the d-amylase substrate, at favorable lag-phase values and achievable maximum activity, without expensive and complicated apparatus and possible automation for industrial process execution.

The state of the art

Determination of serum α-amylase concentration is an important clinical parameter for pancreatic function. Conventional commercial reagents for the determination of α-amylase are warranted. predominantly on a system in which α-amylase degrades starch and the resulting degraded particles are determined in the visible or UV range, depending on whether they are used in the assay

- 2 as starch for amylase colored starch or native starch. The essential flaw in these procedures or. of the reagents is that the starch as a macromolecule cannot be sufficiently characterized and standardized so that the individual batches exhibit very different levels of metabolism and must always be operated at the same time as standard for measurements. Better results would require a more uniform substrate that gives reliable fission results.

A step forward In the direction of a more uniform substrate, the use of maltopentasis was implied. α-aailase cleaves it into maltotriose and maltose, maltotriose and maltose are converted by glucose to glucose, which can then be determined by any method, e.g. by the known hexokinase method.

In addition to maltopentasis, maltotetraose and maltohexaose have been proposed as a substrate (US patent 4.

879 265, * 000 042). However, tetraose achieved significantly worse results than pentase and hexaose worse than tetraose. Thus, the stochiometric reaction is also referred to as maltotetraose and -pentase, while the deviations from the stoichiometric reaction, which can hardly be tolerated, are found for hexaose.

The drawback of maltopentasis, which was also found in tetraosis, is that a significant blank value of reagents appears, ie that the measurement reaction is already underway, before the experiment to be determined is added. In addition, this blank value of reagents is not constant at higher substrate concentrations, but changes more than 25 minutes before the constancy of this side reaction is reached. It has also been shown that there are, in fact, no presumed different cleavages of pancreatic-a-amylase and saliva-a-amylase by maltopentasis, which actually do not exist at all (J. BC, 1970, 24 ^, 3917 to 3927; J. Biochem. 21 »P · XVIH ¹ 952) _e Description of the solution of a technical problem with implementation examples

The object of the invention is therefore to create a novel, technological process for the determination of cC-amylase, using a substrate that exhibits better Purity and Unity than known substrates, which is readily available and, with respect to serum free value, lag phase duration and achievable maximum activity, corresponds to I demand. In addition, simple measurement without expensive and complicated apparatus and suitability for rapid diagnostics such as test strips for process automation should be possible.

According to the invention, this task is solved by a threaded process for the determination of & - amylase by enzymatic cleavage of a substrate

-amylase and measurement of the cleavage product in which the mackerel solution is reacted with ° t - glucosidase and a compound of general formula I

f

- 4 in which R represents a glucosidic, phenylglucoside, mononitrophenylglucoside, dinitrophenylglucoside, sorbitol or gluconic acid group.

Surprisingly, maltoheptasis as an α-amylase substrate has been shown to have better properties, even though oligomaltoses already proposed for this purpose have shown a significant decrease in fitness from mal topentasis to maltohexosis, since they achieve significantly worse results than with pentaoza. · Therefore, it was expected that further extensions of the maltose oligosaccharide chain would cause errors that would no longer be tolerated. Surprisingly, however, we achieved better results than with pentase. Thus, the blank value of the reagents in 0.02 ml mackerel with the substrate was 75%, and with maltobeptasis only 15%, calculated on the final determination value in the normal range.

In addition, we have found that, instead of maltoheptasis, we can also use certain derivatives of maltoheptasis which, in the action of α-amylase, produce a derivatized cleavage product that can be determined to be particularly advantageous.

The process of the invention is particularly suitable for determining the cleavage products of α-glucosidase or maltosephosphorylase.

Determination by α-glucosidase and compounds of general formula I in which R is a glucoside produces the cleavage of maltoheptasis, maltotetraose and maltotriose

- 5 are vaccinated in glucose, and the latter is then measured in a known manner.

In the presence of α-glucosidase, the hexokinase process is particularly preferred for the measurement of glucose produced. The principle of this embodiment of the process of the invention can be given by the following equations:

maltoheptasis + HpO f ^am ^ ³ - ^aza > maltotriose + maltose * trause maltotriose + 2HpO g-glucosidase> 5 glucose maltotetraose + 3HpO ^a ~ g ^lucosidase 4 glucose glucose + 7 ATP 7 glucose-6-P glucose-6-Ρ + 7N ^{+ G 6 FDH} > 7 gluconate-6-P + 7NADH + 7H ⁺

Maltoheptasis derivatives, in particular the compounds of general formula I in which R does not represent a glucoside group, are also particularly suitable for this embodiment of the invention. In the action of both enzymes, α-amylase and α-glucosidase, the substituent, i.e., a phenyl group, a mononitrophenyl group, or a dinitrophenyl group, respectively. the final residue of sorbite or glucone acid is cleaved off and easily determined. Phenyl groups may be in the α- or β-position. in the case of the α-position, their cleavage is effected by the action of α-amylase and α-glucosidase itself, and the cleaved, substituted or unsubstituted phenols can then be readily determined by known color reactions. The invention is also applicable to substituents in position β. In this case, β-glucosidase is used in addition to α-glucosidases.

For dinitrophenyl residues, both nitro groups may be in any position, e.g. as 2,4-, 2,6- or 3,5-substituents.

Nitrophenols or. dinitrophenols resulting from the cleavage of nitro-containing substituents are self-colored compounds which can be optically

we determine. If phenol is cleaved alone, this can be determined by known methods, e.g. by metabolism with a nucleophilic agent such as 3-methyl-6-sulfonyl-benzthiazolone-hydrazone- (2) (HSK) in the presence of monophenol oxidase. This reaction produces a red dye that can be measured.

When sorbitol is cleaved, e.g. by sorbitdehydrogenase oxidized to fructose; at the same time the present RAD is reduced to NADH. The formation of the latter can be readily determined in a known manner in a UV spectrophotometer. if this is not available, the metabolism with tetrazolium salt, such as e.g. INT (2- (p-iodophenyl) -3- (p-nitrophenyl) 5-phenyl-tetrazolium chloride) is also colored in the presence of a diaphorase or other electron carrier.

-? formazan, which can be measured in the visible range.

In an analogous manner, the released gluconic acid can be determined by known methods, e.g. with gluconate kinase, 6-phosphogluconic kielin dehydrogenase and NADP, as well as optionally the tetrazolium salt and electron transporter.

In general, pH values between 5 and 9 are suitable for carrying out the process of the invention. · Preferably, we operate at pfl values between 7 and 7 "5" because we obtain the best results and the shortest reaction times. when used in the invention of the nitrophenyl compound, the range of suitable pH values is slightly narrower and generally lies between pH 6 and pH 8.5.

Suitable as buffers are those that are effective in the range of major activity of the enzymes used. Preferred are phosphate, HEPES (N- (2-hydroxyethyl) -piperazine-N-2ethanesulfonic acid) and glycylglycine. Preferred concentrations are between 10 and 200 mM / l.

α-glucosidase is generally used in amounts between 0.1 and 5000 U / ml. A particular advantage of the process of the invention is that relatively large amounts of this enzyme can be used such that it is a step that determines the rate of α-amylase cleavage. Of course, even greater amounts of this enzyme can be used, but this does not bring us any further benefits any more.

Compounds of general formula I used in the invention are generally used in the assay in amounts of between 0.1 and 250 mM / Ι. Quantities between 0.5 and 100 mM / l are preferred.

The saturation of the α-amylase substrate with maltoheptasis lies at a concentration of 8 to 10 mM. Therefore, a minimum concentration of 8 mM maltoheptasis is reasonably used if we are to operate under substrate saturation conditions, which happens regularly.

An activated α-amylase agent is conveniently added to the pool. Such activating agents are known. Sodium chloride or potassium chloride are preferred.

In a further preferred embodiment of the process of the invention, the cleavage products of the maltosephosphorylase metabolite are formed to form glucose-1-phosphate, which is then determined in a known manner. In a particularly preferred embodiment, glucose-1-phosphate is determined by conversion to glucose-6-phosphate by β-phosphoglucose-mutase, oxidation of the resulting glucose-6-phosphate by NAD in the presence of glucose-6-phosphate-dehydrogenase upon gluconate formation -6-phosphate and NADH, the NADH formation being easily monitored photometrically. The measurement signal can be further enhanced by further oxidation with NAD in the presence of 6-phosphogluconate dehydrogenase to form ribulose-5-phosphate and a further NADH molecule.

The principle of this embodiment is given by the following equations:

maltoheptasis + 1 ^ 0 β · ³¹¹¹ * - ³²³ ^ maltotriase + maltotetraose -> maltose maltose + PO ^ 5- “Sl ^ phosphorylase ^ _{glucose +} β-ginkgo with e-1-P β-glucose-lP β-PGluM, ^ of glucose-6-Ρ glucose-6-Ρ + NAD ⁺ gluconate-6-Ρ + NADH + H ⁺ gluconate-6 ~ P + NAD ⁺ ribulose - ^ - ^ + NADH + H ⁺

An advantage of this embodiment of the invention is that maltosephosphorylase is more specific than α-glucosidase and therefore does not interfere with endogenous glucose.

With respect to the buffer, what has been stated in the same manner as the embodiment of the process of the invention according to the α-glucosidase method is applied.

The α-amylase determination reagent comprises an α-amylase substrate and a system for determining one of the cleavage products formed by α-amylase from the amylase substrate, the substrate being

- 10 α-amylase substrates, characterized in that the substrate consists of a compound of general formula I.

The preferred system for determining the products of cepit ve is the α-glucosidase system containing α-glucosidase, alkali chloride and buffer. If the substrate consists of maltoheptasis itself, hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH), as well as NAD, ATP and magnesium, are required as enzymes.

Preferably, the reagent is based on the aglucosidase system from x 105 to 3 x 10 * U / l α-glucosidase,

10 ⁵ to 5 x 10 * HK,

10 ⁵ to 5 x 10 * G6PDH,

0.5 to 8 mM / Ι NAD,

0.5 to 5 mM / Ι ATP, up to 3 mM / Ι Mg ²⁺ to 100 mM / Ι NaCI or KC1, up to 200 mM / Ι buffer, pH 6.2 to 7 8 8, and up to 100 mM / Ι maltoheptosis or a multiple or fragment thereof, in the dry or dissolved form.

In another preferred embodiment, the reagent consists of α-glucosidase, KC1 or NaCI, buffer and sub2 6 strata. In particular, this reagent contains 10 to 5 ^x 10 U / l α-glucosidase, 1 to 100 mM / Ι NaCl or KC1, 10 to 250 mM / 1 buffer, pH 5 to 9, and 0.1 to 250 mM / mal of the maltoheptoose derivative with general fcrmula I calculated on the concentration in the assay. The reagent may be present in dry, especially lyophilized form, or in solution form, as a mixture of all the ingredients or separately.

In a further embodiment, the reagent of the above type further comprises β-glucosidase or / and phenoloxidase and HSK.

According to a further embodiment of the invention, the reagent contains, in addition to α-glucosidase, NaCl or KC1, buffer and substrate, sorbitdehydrogenase and NAD or gluconate kinase, ATP, 6-phosphogluconic acid dehydrogenase and NADP, as well as optionally tetrazolium salt and diaphoresis. phenazinmetesulfate (PMS).

A preferred reagent of this type contains 1 x 10 to 3 χ 10 ⁶ U / l α-glucosidase, 2 x 10 ^ to 5 * 10 ^ U / l sorbitdehydrogenase, 1 x 10 ^y to 5 x 10 U / l hexokinase, 0.5 up to 50 mM / Ι ATP, 10 to 500 U / l diaphoresis (Chlostridium Kluyveri), 0.01 to 0.5 mM / Ι tetrazolium salts, 0.1 to 10 mM / Ι NAD,

0.2 to 5 mM / Ι MgC ^, 0.5 to 20 mM / Ι maltoheptite, 1 to 100 mM / Ι NaCl or KC1, and 10 to 250 mM / Ι buffer, pH 5.5 to 8.5.

In the present case, a non-ionic surfactant in an amount of between 5 and 50 mM / l is present.

- 12 Also, the preferred embodiment contains 100 to 5 * 10 ⁶ U / l α-glucosidase, 10 to 10 ^ U / l 6-phosphogluconate debidrogenase, 20 to 2 x 10 ^ U / l gluconate kinase, 0.5 up to 25 mM / Ι ATP, 0.05 to 1.0 mM / Ι NADP, 1.0 to 20 mM / 1 maltoheptagluconic acid, 0.5 to 5 mM / Ι MgClg, 1 to 100 mM / Ι NaCl, and 10 to 250 mM / fer buffer, pH 5 »5 to 8.5.

Further reagent contains

0.1 to 250 mM / l of α-nitrophenyl maltoheptaozid or dinitrophic 1-maltoheptao wall, x 10 ^ to 2.5 x 10 ⁶ U / l of α-glucosidase, up to 100 mM / Ι of sodium chloride or potassium chloride and 10 up to 250 mM / Ι phosphate buffer, pH 7.0 to 8.0.

Another embodiment of the reagent comprises:

0.1 to 250 mM / Ι α-phenylmaltoheptaozide, χ 10 ² to 1.5 x 10θ U / l α-glucosidase, to 10 ^ U / l monophenol oxidase,

0.1 to 10 mMl / 1 HSK, up to 100 mM / Ι NaCl or ZC1, and up to 250 mM / Ι buffer.

Another preferred system for determining cleavage products is a maltosephosphorylase system consisting essentially of maltosephosphorylase, β-phosphoglucomutase (PFMG), glucose6-phosphate-dehydrogenase (G6PDH), glucose-1,6-diphosphate

- 13 (G1, 6DP), NAD, buffer, maltoheptosis and, as appropriate

6-phosphogluconate dehydrogenase (6PGDH).

A particularly preferred reagent with this proofing system consists of

0.5 x 10 * to 20 x 10 * U / l mortar | zephosphorylase, χ 10 ² to 1 x 10 ⁴ β-FGluM, x 10 to 3 x 10 U / l glucose-6-f osf at-dehydrogenase,

0.5 to 10 mM / Ι NAD,

0.001 to 1 mM / Ι of glucose-1,6-diphosphate,

0.5 to 100 mM / Ι buffer, pH 6.0 to 7.5, up to 50 mM / Ι maltoheptose, and optionally 2 4 x 10 to 1 x 10 U / l 6-phosphogluconate dehydrogenase.

The reagent may be present in dry or dissolved form or may be impregnated on a sheet carrier, e.g. foil, absorbent paper or the like. In this case, it consists of at least three layers, respectively. layers, the first layer having a substrate, the second layer being used as a barrier layer and the third layer having a system for determining the fission products. If such a multilayer reagent material suitable for a simple rapid α-amylase test is brought into contact with the amylase-containing liquid mackerel, the α-amylase cleaves the substrate and the cleavage products diffuse through the intermediate layer into the third layer containing

- 14 Determination System «In this case, a sensible staining reaction is used, in order to arrest the α-amylase concentration visually by the resulting discoloration, if the cleavage products are not already stained.

According to the invention, the maltotheptase derivatives used can be prepared by various methods. In the case of phenylated derivatives, both chemical and enzymatic methods can be used. Chemical synthesis is based in principle on the metabolism of peracetylated maltoheptasis with the corresponding phenol in the presence of the Friedel-Crafts catalyst. This method is suitable for both phenol alone and mononitrophenol and dinitrophenol. Alternatively, it is also possible to first prepare a phenyl derivative and then nitrate it, e.g. according to the procedure described in Bula. Chem. Soc. Japan 34. (1961) 781.

This method is particularly suitable for the mononitro derivative, where it is possible to separate the resulting o- and p-nitrophenyl derivatives.

Preferably, this reaction is carried out by melting or boiling at reflux in a non-polar solvent with ZnC ^, SnCl ^ or TiCl ^ as a Friedel-Crafts catalyst. After the introduction of phenol or. the nitrophenol is cleaved in a known manner by a protecting group, e.g. with sodium methylate, ammonia, KOH or barium methoxide, each in methanol solution, with aqueous barium hydroxide solution, etc.

-15Enzymatic preparation of phenyl derivatives is carried out by transglucosidation of phenylglucoside or. of corresponding nitriranth phenylglucosides with α-cyclodextrin, amylase or soluble starch in the presence of a specific microbial transferase. Preferably, a rabbit transferase from Bacillus macerans is used. Therefore, the known amylase of Bacillus macerans microbe (EC 2.4.1.19. DSM 24; isolation: JA de Pinto, LL Campbell, Biochemistry 2 »(1968) 114 can be used for transglucosidation; transfer reaction: Methods and Carbohydr. Chemistry, Vol. II. 54?), Which, in addition to its hydrolytic and cyclizing effect, also exhibits glucose1transferential efficacy.

The compound of formula I in which R represents the sorbitol moiety can be prepared from maltoheptasis by reduction with sodium boron hydride (NaBH 4) under favorable conditions.

I

A compound _of formula I in which R represents a gluconic acid group can finally be prepared chemically or enzymatically by known methods for the preparation of gluconic acid from maltoheptosis, e.g. by oxidation with bromine (Methods in Carbohydr. Chemistry, Vol. II, 15) ·

As mentioned above, the invention not only creates a rapid and specific method for the determination of α-amylase, but particularly the complete or far-reaching removal of the lag-16 phase, which is particularly important when using the procedure in assays. Furthermore, the process of the invention can be carried out in many embodiments without complicated evaluation apparatus and is therefore particularly suitable for rapid diagnostics and optical sighting. At the same time, various embodiments of the process of the invention can also be performed with UV measuring devices. Further advantages are the strict proportionality and the fact that the chemically related substances contained in the blood do not cause any disturbance.

The substrates used in the invention can be obtained easily and with great purity. A simple process for the preparation of maltoheptasis is described in German patent application P 27 41 191.2. The process is suitable for the determination of α-amylase in biological fluids, such as serum, heparin plasma, urine and the like, or in other liquid or solid materials.

The following examples illustrate the invention.

-17 EXAMPLE 1

Maltogenic method (α-glucosidase system)

The reagent mixture containing α-glucosidase, G6PDH, HK, NAD, ATP, maltoheptasis, Mg ²⁺ , NaCI and phosphate buffer was dissolved in 2.0 ml of distilled water. The stability of the reagent at room temperature is about 1 hour, with peratures below 8 ° C for 6 hours.

The resulting solution has the following concentrations

reagents: phosphate buffer 50 mM / dL; NaCI 50 mM / l Mg ²⁺ 2 mM / l maltoheptaoza 10 mM / l ATP 1.2 mM / l NAD 2 mM / l HK > 2 U / l G6PDH ar 2 U / l α-glucosidase ar io u / l.

I dissolve! 0.02 ml of mackerel serum was added at 25 ° C, transferred to a cuvette with a layer thickness of 1 cm, and then the extinction was determined in the photometer at Hg 365 nm, 340 nm or Hg 334 nm. After 10 minutes, read the extinction and then repeat the reading at intervals of 1 minute five times.

-18Ιζ of the extinction differences found per minute (Δε / min), calculate the mean, subtract the empty reagent value and insert the corrected value into the calculation. The calculation is as follows:

U / l 25 ° C = 4244 x PE 365 nm / min = 2290 x0E 340 nm / min ^e 2335 χΔε 334 nm / min

FIG. 1 shows the results of a series of human serum dilutions with saline obtained by this method.

If we divide the reagent into two parts, one of which contains maltoheptasis and the other a mixture of all other reagents, the stability of the solutions called them can be increased. For the reagent mixture, the temperature at temperatures up to 8 ° C is 50 hours, at room temperature about 8 hours. The stability of the solution of maltoheptasis is 6 weeks, respectively. about 1 week.

EXAMPLE 2

Determination with the maltose_f ₀ system of sophylase

Two reagents were prepared consisting of an amylase substrate and a maltose phosphorylase system. One reagent contained a maltoheptoase substrate, the other a soluble starch suitable for the art. The concentration of reagents after dissolution in water was as follows:

invention comparison

phosphate buffer 20 mM; pH 6.5 20 mM; pH 6.5 NAD 2 mM 2 mM maltoheptaoza 10 mM - soluble starch - 5 mg / ml glucose-1,6-diphosphate traces traces maltose phosphorylase (microorg.) 3 U / ml 3 U / ml β-phosphoglucomutase (microorg.) 1 U / ml 1 U / ml G6PDH (Leuconostoc mesent) 9 U / ml 9 U / ml 6PGDH (Leuconostoc 1 U / ml 1 U / ml

mesent)

The resulting solution was incubated at 30 ° C, mackerel solution was added and the difference in extinction at Hg 334 nm was determined in the photometer. After a 10-minute preincubation, the difference in extinction was measured over a 10-minute period. The following formula was then applied to 2.0 ml of reagent and 0.10 ml of mackerel; -οπ ^{= ΔΕ / π1η x 1699 U / 1}

- 20 When using five different human sera, the following reagents were determined with the above reagents:

serum invention starch 1 57.4 U / l 15.3 U / l 2 61.2 U / l 27.3 U / l 3 95.1 U / l 39.1 U / l 4 114 U / l 44.2 U / l 5 374 U / l 161 U / l

EXAMPLE 3

Preparation of α-phenylmaltoheptaozide

a) Tridecosacetyl-p-D-maltoheptasis

57 »6 g (50 mM) of maltoheptoase and 41 g (500 mM) of refreshing sodium acetate were suspended in 543 ml (5 * 75 mol) of acetic anhydride and stirred vigorously at 100 ° C for 4 hours. After being cooled to about 60 ° C, they are stirred in about 1 L of ice water and stirred overnight at 4 ° C, leaving a tough, semi-crystalline mass precipitated · After decanting the supernatant, mix the residue again with ice water. The product crystallizes completely. The product is separated, washed and dried.

Yield 80.7 g (76% of theory) colorless crystals [a] p ^T »+ 137,5 ⁰ (c« 1,15 chloroform), m.p. - 150 ⁰ (sharp,

- 21 The mother liquor (decantate) was evaporated in vacuo to dryness and the residue was also crushed with ice water and crystallized.

Yield 22.5 g

Ca] p ^T - 156 ⁰ (c - 1, chloroform), m.p. 150 ° C.

Total yield 105 g »97% of theory.

The product may be recrystallized from ethanol without recrystallizing again after melting and dryness. The product is therefore optically uniquely configured at the C ^ atom.

b) Dodecosacetyl-α-phenyl-D-maltoheptaozide

9.54 g (4.5 mM) of tridecosacetyl-PD-maltoheptoose, 0.61 g (4.5 mM) of freshly melted ZnClg and 4.25 g (4-5 mM) of distilled phenol were stirred at 2.5 h for moisture at 100 ° C. It was then warmly dissolved in ethyl acetate and washed with 2 x 100 ml HgO, 5 x 100 ml 1 n NaOH, 100 ml 1 n acetic acid and 100 ml saturated sodium chloride solution. Initially, the brown solution turns light yellow. After drying over MgSO4, evaporate to dryness and take up in warm methanol (5θ ml). The syrupy material, which is crystallized from ethanol, is recovered overnight.

Yield: 8.7 g (90% of theory), m.p. 155 to 165 ° θ (dec.) [A] p ^T + 147 ⁰ (c »8 in chloroform).

- 22 c) Phenyl-a-D-maltoheptaozid

Dissolve 10.8 g (5 mM) of peracetyl-1-phenyl-aD-maltoheptaozid in warm 200 ml of absolute methanol and add dioxane at room temperature while stirring 50 ml of 0.1 n sodium methylate and stir for 16 hours at room temperature. temperature. After 20 minutes, the product begins to secrete semi-crystalline "Add 200 ml acetone to the sun" cooled to 4 ° C and aspirated. Dissolve the product in water (160 ml), decolourise with activated carbon and batch the cation exchange salts (Dowex 50 H ⁺ ).

Yield: 5.6 g (91% of theory) of a colorless lyophilisate Dx] p ^T = + 176 ⁰ (c »10 in E ₂ 0).

The product also contains some free maltoheptasis and - after HPLC analysis (detection at 254 nm) - 2 more UV-positive impurities. They are separated by chromatography on cross-linked dextran (Sephadex LH20) with water as eluant.

EXAMPLE 4

In the right p-ni tropheni1-a-D-maltoheptaozid

a) Peracetyl-β-nitrophenyl-α-D-maltoheptaozide

13.6 g (20 mM) of peracetyl-maltoheptasis prepared according to Example 1 (a) are dissolved in 11.9 g (100 mM)

- 23 p-nitrophenol in 90 ml of absolute benzene and 10.4 g - 4.5 ml (40 mM) of SnCl2 are added while stirring and while removing the moisture. The volume is precipitated, which dissolves again when heated. · Boil for 1 hour under reflux. When cooled, a tough mass is eliminated which, after the addition of 80 ml of ethyl acetate, is again converted into solution. By stirring the solution in 180 ml of 2 n solution ^ 2 ^ 3 ^, tin oxide hydrate is removed. This is decided by adding some activated charcoal. The aqueous phase was decided and the organic phase was washed thoroughly, finally with saturated NaCl solution. · After drying and evaporating the solution, a resinous product was obtained which crystallized from ethanol. Yield: 6.2 g (41% of theory), m.p. = 100 ° C (decomposition) [a] p ^ = + 131 ⁰ (c »1 in chloroform).

The product is also obtained by performing the nozzle in chloroform or with TiCl ^ instead of SnCl ^.

b) p-nitrophenyl-α-D-maltoheptaozide

5.5 g (7 mM) of peracetyl-p-nitrophenyl-maltohept * · ozide were suspended in 100 ml of absolute methanol and 5 ml of about 1 n sodium methylate solution was added. The starting material becomes honey-like and slowly dissolves, after a while crystalline precipitation of the deacetylated product begins, which is complete after stirring overnight. Suction off, rinse with methanol and dry.

- 24 Yield 2.8 g (86% of theory) [<χ] ξ ^Τ - + 124 ⁰ (c - 0.6 in HgO).

The product was purified on cross-linked dextran (Sephadex LH20) with water as eluant. 1.2 g (45%) of the compound described above are obtained which are inactive in the enzymatic assay. The pool of this was also obtained by the nitration of α-phenylmaltoheptaozid from Example 1 with Bula nitric acid. Chem. Soc. Japan 34 (1961) 718 ·

Applying dinitrophenol instead of mononitrophenol gives the corresponding dinitrophenyl compounds.

EXAMPLE 5 p-Nitrophenyl-α-maltooligosaccharides by enzymatic synthesis with amylase from Bacillus macerans (E.C.2.4.1.19 from Bac. Mac.

DSM 24).

Amylase from Bacillus macerans also has transfer properties, in addition to their hydrolitic and cyclizing effects. glycosyls that can be utilized for the synthesis of oligosaccharides and oligosaccharide derivatives (Methods and Carbohydrate Chemistry II, 347). For the synthesis of p-nitrophenyl oligosaccharides, this procedure was optimized as follows:

Mix 680 mg of amylase from Bacillus macerans (EC2.4.1.19 from Bac. Mac. DSM 24) ((lyophilisate) (0.46 U / mg weight, protein content in weight 28.5%, without p-nitrophenyl cleavage activity) -aD-glucoside),

- 25 400 mg α-D-β-nitrophenylglucoside,

3.5 g α-cyclodextrin, and ml Soerensen phosphate buffer, pH 6.2; 0,01 M. Incubate the incubator for 24 hours at 37 ° C. · For purification, the α- and the resulting β-cyclodextrin are first separated via a tetravioletene inclusion compound. After chromatography on dextran excess (Sephadex LH20), 50 mg of p-nitrophenyl-maltoheptaozid lyophilisate is obtained, which is very active in the amylase assay.

EXAMPLE 6

Preparation of Maltoheptaite g Maltoheptoase was dissolved in 5θ ^m l of HgO, 2 g of NaBH 4 was added in portions and stirred for 75 minutes at room temperature (test-reduced sugar negative). Chromatography on a cation exchanger (Lowex 50 H ⁺ ) removes Na ⁺ (pH value of the solution after passing through the exchanger 3 »5) evaporated to remove boric acid as methyl ester. The solution, which is ultimately neutral, is lyophilized.

Yield: 9 g (90% of theory), without reducing sugars Content (enzymatically determined) of glucose 86.1% of sorbitol 89 »0% water 8.5%.

- 26 EXAMPLE 7

Preparation of maltoheptonic acid

11.5 g (0.01 Bol) of maltoheptoase and 6 g of Ba-benzoate were placed in 150 ml of water. While stirring and cooling, 1 ml of bromine was added and stirred for an additional 36 hours. After the expulsion of excess bromine with nitrogen, 4 ml of 4 n HpSO 4 was added and some activated carbon was filtered and the filtrate was extracted with chloroform to remove benzoic acid. 3 2 2 g AggCOCO was added to the aqueous solution and stirred (pH: neutral). The insoluble salts were filtered off and the clear solution was poured over 20 ml of Amberlite JR H ⁺ . The acidic eluate was immediately neutralized with NaOH and lyophilized.

Yield: 8 g (72% of theory)

Content (enzymatically determined) of glucose 85% of gluconic acid 80% of water 9,3% ·

EXAMPLE 8

Evidence of α-amylase with phenyl-α-maltoheptaozid as α-amylase substrate splits phenyl-α-maltoheptaozide into phenyl-α-maltotriide or -tetray, which is metabolised by α-glucosidase to phenol and glucose. The released phenol is coupled via mono-f e no 1-oxidase oxidatively with the nucleophilic reagent 3-methyl-6-sulfonyl-benzthiazolone-hydrazone- (2) (HSK) in

- 27 a red dye whose rate of production is proportional to the activity of the amylase in mackerel and can be monitored photomically.

Test principle:

1. a) phenyl-a-maltoheptaozid + Η ^ Ο phenyl-a-Dtri- (tetra) -glucopyranoside + maltotetra- (tri) -ose

b) Phenyl-a-D-tri- (tetra) -glucopyranoside + 3 (4) HgO> phenol + 5 (4) glucose

e) Phenol + HSK + Og / _{barrel + 2} ^ 0

Measuring conditions:

° C, measuring wavelength 492 nm, 1 cm in veto.

i

The composition of the reagent is shown in the following tables.

TABLE

Reagent_Concentration in test

phosphate buffer, pH 7.4 0.05 M / l sodium chloride 0.05 M / l phenyl-a-D-maltoheptaozid 10 mM / 1 HSK 1 mM / 1 mono f enol-ox idase 1 U / ml α-glucosidase 10 U / ml

test volume: 2.0 ml

- 28 Instead of phosphate buffer, glycine, glycylglycine, hepes, tris, tris, tra and other buffers have also proved useful.

EXAMPLE 9

Determination of α-amylase by p-nitrophenyl-maltoheptaozide

Test principle:

p-nitrophenyl-a-maltoheptaozid gr ^amylase > (glucose) _n + p-nitrophenyl-a- (glucose) _m g ^{c ,} S ^onion ? I ^< I ^a ?. ^a » _n glucose + p-nitrophenol.

Following the cleavage of maltoheptaozid, the cleavage products are broken down into glucose and p-nitrophenol. The p-nitrophenolate anion is colored yellow in the alkaline solution and can therefore be directly optically measured.

Test system:

Reaction mixture: 50 mM / Ι phosphate buffer, pH 7.4 mM / Ι sodium chloride up to 50 U / ml α-glucosidase or. 10 mM / Ι p-nitrophenyl-α-maltoheptaozide

Test nozzle:

ml of reaction mixture + 50 / Ul maceration (serum) temperature: 50 ° C wave. length: 405 nm

- 29 Start with serum after measuring temperature is reached (10 minutes preincubation).

EXAMPLE 10

Determination of α-amylase by maltoheptite

Test principle:

maltoheptait + HgO ^<x “Sgl. ^lase T> maltotriite + maltotetraose maltotriite + ^ 0. ^. rg ^^^ idaza> sorbit + maltose sorbit + NAD ⁺ ~ ^DH > fructose + NADH + H ⁺

NADH + INT + H ^{+ diaphorase} > formazan + NAD ⁺

SDH = sorbit dehydrogenase.

Test system:

The reaction nozzle ml Test concentration Na- / K-FO, ₊ -buffer 0.02 M / l 1.92 12.8 mM / Ι P0 ₄ + Triton Χ100 2 ml / 100 ml + NaCl 10 mM / Ι, pH 6.9 6.4 mM / l MgCl ₂ 0.3 M / l 0.01 1.0 mM / l maltoheptite 100 mM / 1 0.10 3.3 mM / l NAD c = 40 mg / ml 0.05 1.0 mM / l INT c - 0.6 mg / ml 0.20 0.09 mM / l diaphragm (Clostr.Kluyveri) 0.05 167 U / l

mg / ml ⁺

- 30 Reaction nozzle ml Concentration in test

ATP c «50 mg / ml 0.10 3.29 mM / l hexokinase 280 U / ml ⁺ 0.05 4666 U / l SDH 180 U / ml ⁺ 0.30 18000 U / l α-glucosidase 1120 U / ml ⁺ 0.20 74670 U / l mackerel (serum) 0.02

+ in test buffer

Starting with the addition of mackerel measurements is made at 4-92 nm; temperature: 25 ° 0.

EXAMPLE 11

Determination of α-amylase with maltoheptagluconic acid

Test principle:

maltoheptagluconic acid + HgO ^{a , am} il ^{& za} ^, maltottiogluconic acid + maltotetraose maltottiogluconic acid + Hg ⁰ gluconic acid + maltose gluconic acid + ATP g ¹ - ^ukoDat ~ ^kina2a > gluconic acids-6-M. + ADP-glucose F ♦ HADP * ribulose-5-Ρ + NADPH + COg + H ⁺

- 51 Test system:

Reaction nozzle ml Concentration in test

Na- / K-FO ₄ buffer 0.02 M / l + NaCl 10 mM / Ι, pH 6.9 2.19 14.6 mM / Ι phospho 7.3 mM / Ι NaCI MgClg 1 M / l 0.01 5.5 mM / l maltoheptagluconic acid 150 mM / 1 0.10 5 mM / l NADP c «10 mg / ml 0.10 0.38 mM / l ATP c 50 mg / ml 0.10 2.7 mM / l gluconate kinase 40 U / ml 0.03 1066 U / l 6-phosphogluconic acid-dehydrogenase 24 U / ml 0.10 800 U / l α-glucosidase 1120 U / ml 0.30 11.2 x 10 * U / l mackerel (serum) 0.02

I

Start with the addition of mackerel: measurement is made at 340 nm or 3θ5 nm, ° c, temperature: test volume:

3,00 ml

An indication of the best known applicant _? Utilizing the claimed invention p-nitrophenyl-α-maltooligosaccharides by enzymatic synthesis with amylase from Bacillus macerans (EC2.4.1.19 from Bac. mac.

DSM 24).

Amylase from Bacillus macerans has, in addition to its liidrolytic and cyclizing effect, glycosyl transfer properties that can be exploited for the synthesis of oligosaccharides and oligosaccharide derivatives (Methods and Carbohydrate Chemistry II, 547). For the synthesis of p-nitrophenyl oligosaccharides, this procedure was optimized as follows:

Mix 680 mg of amylase from Bacillus macerans (EC2.4.1.19 from Bac. Mac. DSM 24) ((lyophilisate) (0.46 U / mg weight, protein content in weight 28.5%, without p-nitrophenyl cleavage activity) -aD-glucoside),

400 mg of α-D-β-nitrophenylglucoside,

5.5 g α-cyclodextrin, and ml Soerensen phosphate buffer, pH 6.2; 0,01 M. The incubation was incubated for 24 hours at 57. For purification, the α- and β-cyclodextrin obtained were first separated via a tetrachlorethylene inclusion compound. After chromatography on dextran excess (Sephadex LH20), 50 mg of p-nitrophenyl-maltobeptaozid lyophilisate is obtained, which is very active in the amylase assay.

Determination of »-ητη-ΐ and ft7.fi by p-nitrophenyl-maltoheptaozide

Test principle:

p-nitrophenyl-a-maltoheptaozid - (glucose) _n + p-nitrophenyl-a- (glucose) _m ^a ~ g ^lucosidase » _n glucose + p-nitrophenol.

Following the cleavage of maltoheptaozid, the cleavage products are broken down into glucose and p-nitrophenol. p-nitrophenolate anion It is colored yellow in alkaline solution and can therefore be directly optically measured.

Test system:

Reaction mixture: 50 mM / l phosphate buffer, pH 7.4 mM / l sodium chloride to 50 U / ml α-glucosidase or. M0 mM / 1 p-nitrophenyl-α-maltoheptaozide

Test nozzle:

ml of reaction mixture + 50 / Ul maceration (serum} temperature: 50 ° C wavelength: 405 nm

Start with serum after reaching measuring temperature (10 minutes preincubation).

Claims (11)

PATENT APPLICATIONS A method for the determination of ^ - amylase by enzymatic cleavage of a d-amylase substrate, characterized in that the mackerel solution is digested with ct-glucosidase and a compound of the general formula I in which it represents R is a glucoside, phenylglucoside, mononitrophenylglucoside, dinitrophenylglucoside, sorbitol group or gluconic acid group ^ and the ROH compound formed is then measured. Method according to claim%, characterized in that we operate at pH values between 5 and 9. Process according to claim 1 or 2, characterized in that Ο, Ί up to 250 mM / 1 of the compounds of formula I are used. - rf Process according to one of the preceding claims, characterized in that an additional β-glucosidase is used for the substituent at position β in the R group sugar residue. Process according to claim 1, characterized in that the cleavage products are digested with maltosephosphorylase to form glucose-1-phosphate and determined. Method according to claim 5, characterized in that, to determine the resulting glucose-1-phosphate, it is metabolized by phosphoglucomutase to glucose-6-phosphate and thereby reduced NAD in the presence of glucose-6-phosphate dehydrogenase in NADH that we measure. Process according to one of the preceding claims, characterized in that in the case where R represents a phenylglucoside group, the cleaved phenol is determined by 5-methyl-6sulfonyl-benzthiazolone-hydrazone- (2) in the presence of monophenoloxidase. Process according to one of Claims 1 to 6, characterized in that in case R represents a sorbitol group, the sorbitol cleaved is determined by sorbitol dehydrogenase and NAD. Process according to one of Claims 1 to 6, characterized in that in case R represents a group of gluconic acid, the cleaved gluconic acid is determined by gluconate kinase, 6-phosphogluconic acid dehydrogenase and NADP. - 3 ¢ - A process according to claim 8 or 9, characterized in that, upon reduction of NAD, the resulting NADH is reacted with this trazolium salt in the presence of an electron carrier into formazan and determined. A method according to claim 10, characterized in that the diaphorase or electron transporter is used as the electron carrier. fe nazinmetosulfate.