KR20110117166A - Fast reaction kinetics of enzymes having low activity in dry chemistry layers - Google Patents

Abstract

The present invention relates to methods for measuring analytes and suitable diagnostic elements therefor.

Description

FAST REACTION KINETICS OF ENZYMES HAVING LOW ACTIVITY IN DRY CHEMISTRY LAYERS}

The present invention relates to methods for measuring analytes and suitable diagnostic elements therefor.

Diagnostic elements are an important component of clinically relevant assays. In this regard, the first focus is on the determination of analytes, for example metabolites or substrates, which are measured indirectly or directly, for example with the aid of enzymes specific for the analyte. In this case, the analyte is quantified after conversion with the help of the enzyme-coenzyme complex. In this process, the analyte to be measured is contacted with a suitable enzyme, coenzyme and optionally a mediator and the coenzyme is physicochemically changed, for example, oxidized or reduced by enzymatic reaction. If a mediator is additionally used, the mediator typically transfers the electrons released during the reaction of the analyte from the reduced coenzyme to the optical indicator or conductive component of the electrode, such that the reaction is measured photometrically or electrochemically, for example. Can be. Calibration yields a direct relationship between the measurement and the concentration of the analyte to be measured.

Diagnostic elements known from the prior art are characterized by a limited shelf life and the specific requirements on the environment (eg cold or dry storage) to allow this shelf life to be achieved. Thus, in certain forms of application, for example, tests run by the end user themselves, such as blood sugar self-monitoring, may be misleading due to false, inconspicuous inaccurate storage of the measurement system that is hardly noticeable by the consumer. Outcomes can occur, which can lead to the wrong treatment of each disease. The erroneous result is primarily because the enzymes, coenzymes and mediators used in these diagnostic elements are generally sensitive to moisture and heat and are inactivated over time.

A known measure used to increase the stability of diagnostic elements is to use stable enzymes, for example enzymes from thermophilic microorganisms. In addition, enzymes can be stabilized by chemical modification, in particular crosslinking. In addition, enzyme stabilizers such as trehalose, polyvinyl pyrrolidone and serum albumin may also be added, or the enzyme may be sealed in the polymer network, for example by photopolymerization.

Another method of stabilizing enzymes is by mutations that are introduced site-specific or non-site-specifically. In this regard, the use of recombinant techniques, which specifically affect the properties of the corresponding enzyme by targeted changes in the DNA coding for the enzyme, has proved particularly suitable.

Baik et al. (Appl. Environ. Microbiol (2005), 71, 3285) isolated and isolated three mutants of glucose dehydrogenase from Bacillus megaterium comprising amino acid substitutions E170K, Q252L or E170K / Q252L. The analysis is described. Mutants E170K and Q252L have low stability only at low salt concentrations and high pH values, and double mutants show significantly increased stability under test conditions due to enhanced interactions at the dimer-dimer interface.

Vazquez-Figueroa et al. (ChemBioChem (2007), 8, 2295) describe the location of glucose dehydrogenase from Bacillus subtilis, Bacillus thuringiensis and Bacillus licheniformis. The development of thermostable glucose dehydrogenases, including introducing amino acid substitutions at 170 and 252, is disclosed. In this connection, it is mentioned that the mutations E170K and Q252L, individually as well as in combination, result in the stabilization of glucose dehydrogenase from Bacillus subtilis.

However, when genetically modified stabilizing enzymes are used compared to wild type variants, there is a problem that they have lower substrate turnover values per unit of time since they typically have considerably lower activity than the corresponding wild type enzymes. Given the fact that enzymes with high specific activity are preferably used for clinical and diagnostic chemistry such as blood glucose measurements, the use of stabilizing enzymes is often an unacceptable substitute for the use of natural enzymes.

Another difficulty is that high enzymatic activity, which correlates with high substrate turnover per unit of time, is usually only achieved with each natural coenzyme in each case. When artificial coenzymes are used in place of natural coenzymes, enzyme activity usually decreases significantly and the rate of conversion to the substrate also decreases.

Accordingly, it is an object of the present invention to provide a stable diagnostic element which eliminates some or more of the disadvantages of the prior art, in particular for measuring glucose. In particular, the diagnostic element should ensure high stability of the substrate as well as the high stability of the coenzyme as well as the high conversion rate of the substrate.

This object is achieved according to the invention by an analyte measurement method comprising the following steps:

(a) contacting a sample containing the analyte with a diagnostic element comprising a dry reagent layer containing:

(i) mutated dehydrogenase specific for the analyte and

(ii) artificial coenzymes, and

(b) determining the presence and / or amount of the analyte.

Surprisingly, mutated dehydrogenases with extremely low activity in the presence of artificial coenzymes in cuvette tests show a faster reaction rate in diagnostic elements with a dry reagent layer as in test strips, and at least of natural coenzyme (wild type coenzyme) It has been found within the scope of the present invention to yield as many turns as there are in the presence. This is presumably due to the fact that at high concentrations of the component, other factors besides the enzyme activity influence the rate of deflection decisively, in which the complex formation between enzymes, coenzymes, reduced coenzymes, analytes and oxidized analytes is affected. It appears particularly decisive.

In this regard, the method according to the invention, in a preferred embodiment, the rate of conversion of the analyte in the diagnostic elements described herein is equal to the rate of conversion of the analyte in the corresponding diagnostic element comprising the corresponding wild type coenzyme instead of the artificial coenzyme. Or larger. The rate of conversion of the analyte in the diagnostic element used according to the invention is preferably at least 20%, more preferably at least 50%, most preferably 100 compared to the rate of analyte in the diagnostic element comprising wild type coenzymes. Increase by at least%, for example from 100% to 200%.

As used herein, the term “mutated dehydrogenase” or “dehydrogenase mutant” has a modified amino acid sequence compared to wild type dehydrogenase and differs from one or more amino acids from the same number of amino acids, ie wild type dehydrogenase. By genetically modified variant of natural dehydrogenase (wild type dehydrogenase) with amino acids.

Mutated dehydrogenases can be obtained by mutations from wild-type dehydrogenases derived from any biological source, and in the sense of the present invention the term "biological source" refers to prokaryotes such as bacteria as well as eukaryotes such as mammals and Other animals. The introduction of the mutation (s) can occur position-specifically or non-position-specifically, preferably position-specifically, using recombinant methods known in the art, so that it is natural for each requirement and condition. One or more amino acid substitutions occur within the amino acid sequence of the dehydrogenase.

The dehydrogenase mutants obtained in this way and used in the method according to the invention preferably have increased thermal and / or hydrolytic stability compared to the corresponding wild type dehydrogenase. Examples of such mutants are described, among others, in Baik (Appl. Environ. Microbiol. (2005), 71, 3285), Vazquez-Figueroa (ChemBioChem (2007), 8, 2295) as well as WO 2005/045016 A2. These disclosures are expressly incorporated herein by reference.

Mutated dehydrogenases in the sense of the present invention particularly preferably have reduced specific enzymatic activity compared to the corresponding wild type dehydrogenase. The term “specific enzyme activity” (represented as U / mg enzyme) as used in this application means the amount of substrate that is converted under predefined conditions per 1 mg of enzyme and per minute. On the other hand, the term “lyophilisate activity” refers to the amount of substrate which is converted under predetermined conditions per 1 mg and per minute of lyophilisate comprising an enzyme in combination with an auxiliary substance.

The mutated dehydrogenase used in the method according to the invention is preferably nicotinamide adenine dinucleotide (NAD / NADH) -dependent or nicotinamide adenine dinucleotide phosphate (NADP / NADPH) -dependent mutated dehydrogenase, which is Preferably mutated alcohol dehydrogenase (EC 1.1.1.1; EC 1.1.1.2), mutated L-amino acid dehydrogenase (1.4.1.5), mutated glucose dehydrogenase (EC 1.1.1.47), mutation Glucose-6-phosphate dehydrogenase (EC 1.1.1.49), mutated glycerol dehydrogenase (EC 1.1.1.6), mutated 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), mutated lactate Dehydrogenase (EC 1.1.1.27; EC1.1.1.28), mutated maleate dehydrogenase (EC 1.1.1.37), and mutated sorbitol dehydrogenase. Mutated dehydrogenases are particularly preferably mutated glucose dehydrogenases (EC 1.1.1.47).

When a mutated glucose dehydrogenase is used within the scope of the present invention, it may comprise amino acid (s) modified at any position in its amino acid sequence, as compared to the corresponding wild type glucose dehydrogenase. . The mutated glucose dehydrogenase preferably comprises a mutation at one or more of positions 170 and 252 of the amino acid sequence of the wild type glucose dehydrogenase, with mutants having mutations at positions 170 and 252 particularly preferred. It has been proven that mutated glucose dehydrogenases do not contain additional mutations in addition to these mutations.

The mutations at positions 170 and / or 252 can basically comprise any amino acid substitution that results in an increase in stabilization, eg, thermal and / or hydrolytic stability of the wild type dehydrogenase. Mutations at position 170 preferably include amino acid substitutions of arginine or lysine to glutamic acid, in particular amino acid substitutions of lysine to glutamic acid, while amino acid substitutions of leucine to lysine for position 252 are preferred.

The wild type glucose dehydrogenase used to prepare the mutants of the above-mentioned glucose dehydrogenase is preferably derived from bacteria and glucose dehydrogenation from Bacillus megaterium, Bacillus subtilis or Bacillus thuringiensis, in particular Bacillus subtilis. Enzymes are particularly preferably used. In the most preferred embodiment, the mutated glucose dehydrogenase GlucDH_E170K_K252L having the amino acid sequence represented by SEQ ID NO: 1 obtained by mutation of wild type glucose dehydrogenase from Bacillus subtilis is within the scope of the method according to the invention. Used.

According to the invention, the diagnostic elements described herein also include artificial coenzymes in addition to the mutated dehydrogenase specific for the analyte. Artificial coenzymes within the meaning of the present invention are chemically modified relative to natural coenzymes and at atmospheric pressure compared to natural coenzymes, in particular at temperatures ranging from 0 ° C. to 50 ° C., especially acids and bases and / or nucleophiles (pH 4 to pH 10) nucleophile) is a coenzyme that has high stability against alcohols or amines and therefore can exhibit its activity over longer periods of time than natural coenzymes under the same environmental conditions.

Artificial coenzymes preferably have a high hydrolytic stability over natural coenzymes and are particularly preferably completely resistant to hydrolysis under test conditions. Artificial coenzymes may have reduced binding constants for dehydrogenases compared to natural coenzymes, for example the binding constant is reduced by more than two times.

Preferred examples of artificial coenzymes that can be used within the scope of the method according to the invention are artificial NAD (P) / NAD (P) H compounds, ie natural nicotinamide adenine dinucleotide (NAD / NADH) or natural nicotinamide adenine dinucleotide Is a chemical derivative of phosphate (NADP / NADPH) or a compound of formula (I)

If the artificial coenzyme is an artificial NAD (P) / NAD (P) H compound, the artificial NAD (P) / NAD (P) H compound is preferably phosphorus through a linear or cyclic organic moiety, in particular a cyclic organic moiety. -Containing moieties include, for example, 3-pyridine carbonyl or 3-pyridine thiocarbonyl moieties linked to phosphate moieties without glycosidic linkages.

Artificial coenzymes are particularly preferably selected from compounds of the formula (II) or salts thereof or optionally reduced forms thereof:

[In the meal,

A = adenine or an analog thereof,

T = each occurrence independently represents O, S,

U = in each case independently ^{_{OH, SH, BH 3 -,}} BCNH 2 - represents,

V = in each case independently represents two groups forming an OH or phosphate group, or a cyclic phosphate group,

W = COOR, CON (R) ₂ , COR, CSN (R) ₂ , where R independently in each occurrence represents H or C ₁ -C ₂ alkyl,

X ¹ , X ² Each occurrence independently represents O, CH ₂ , CHCH ₃ , C (CH ₃ ) ₂ , NH, NCH ₃ ,

Y = NH, S, O, CH ₂ ,

Z = linear or cyclic organic residue,

Provided that Z and pyridine residues are not linked by glycosidic bonds.

In the compound of formula (II), Z is preferably a linear residue of 4-6, preferably 4 carbon atoms, in which one or two carbon atoms are optionally replaced with one or more heteroatoms selected from O, S and N , A residue comprising a heteroatom selected from O, S and N, and optionally a cyclic group having 5 or 6 carbon atoms optionally containing one or more substituents, and a residue CR ⁴ ₂ which binds to the cyclic group and X ² R ⁴ in each occurrence is independently H, F, Cl, CH ₃ ).

Z is particularly preferably a saturated or unsaturated carbocyclic or heterocyclic 5-membered ring, in particular a compound of formula (III)

[In the meal,

A single or double bond may be present between R ^{5 ′} and R ^{5 ″} ,

R ⁴ = Independently in each occurrence represent H, F, Cl, CH ₃ ,

R ⁵ = CR ⁴ ₂ ,

^"When present between and R ^5", R ^{5 'is} a single bond R ⁵ = O, S, NH, NC ₁ -C ₂ alkyl, CR4 ₂ , CHOH, CHOCH ₃ , R ^{5 "} = CR ⁴ ₂ , CHOH, CHOCH ₃ ,

When a double bond is present between R ^{5 '} and R ^{5 "} , then R ^5' = R ^{5 "} = CR ⁴

R ⁶ , R ^{6 '} = Each occurrence independently represents CH or CCH ₃ ].

In a preferred embodiment, the compounds according to the invention comprise adenine or adenine analogs such as C ₈ -substituted and N ₆ -substituted adenine, deaza variants such as 7-deaza, aza variants such as 8-aza or combinations such as 8- to aza or 7-aza, or carboxamide, and see for example, it includes a click analogs formate azithromycin, having the 7-aza variant is halogen, C _{1 -6} alkynyl, C _{1 -6} alkenyl or C _{1 -6} 7 position by alkyl It may be substituted in.

In a further preferred embodiment, the compound is a ribose for example 2-methoxydeoxyribose, 2'-fluorodeoxyribose, hexitol, altritol or polycyclic analogs such as bicyclic sugars, LNA sugars and tricyclo sugars Instead, including adenosine analogs.

In particular, (a) a phosphate oxygen is also a child sorted theoretical in a compound of formula (II) (isotronically), for example S ^- or BH ₃ ^- O ^- by that, by NH, NCH _3, or CH ₂ O = O can be replaced by = S. In the compounds of the formula (II) according to the invention, W is preferably CONH ₂ or COCH ₃ .

R ⁵ is preferably CH ₂ in the group of formula (III). It is also preferred that R ^{5 '} is selected from CH ₂ , CHOH and NH. In a particularly preferred embodiment, R ^{5 ′} and R ^{5 ″} are in each case CHOH. In a further preferred embodiment, R ^{5 ′} is NH and R ^{5 ″} is CH ₂ . R ⁴ = H, R ⁵ = CH ₂ , R ^{5 '} = R ^{5 "} = CHOH and R ⁶ = R ^6' Most strongly preferred are compounds of formula (III), wherein = CH.

In the most strongly preferred embodiment, the artificial coenzyme is the compound carbaNAD known from the literature (J.T. Slama, Biochemistry (1988), 27, 183 and Biochemistry (1989), 28, 7688). Other stable coenzymes that can be used according to the invention are in the publications of WO 98/33936, WO 01/49247, WO 2007/012494, US 5,801,006, US 11 / 460,366 and Blackburn et al. (Chem. Comm. (1996), 2765). The disclosures of which are expressly incorporated herein by reference.

The diagnostic element used in the method according to the invention can be any diagnostic element which comprises a dry reagent layer containing mutated dehydrogenase and artificial coenzyme and can be wetted by the analyte containing sample. In addition to mutated dehydrogenases and artificial coenzymes, the reagent layer may optionally contain additional reagents used for qualitative detection or quantitative determination of analytes, such as, for example, suitable mediators and / or suitable auxiliaries and / or additives. have.

Diagnostic elements to which the analyte can be applied in the form of aqueous or non-aqueous solutions are preferably used within the scope of the present invention. In a particularly preferred embodiment of the invention, the diagnostic element is a test tape, a test disc, a test pad, a test strip, a test strip drum, or a diagnostic element mentioned in WO 2005/084530 A2, which is expressly referenced herein. The diagnostic element described in this application includes in each case one or more test sites that can be contacted with an analyte containing sample and capable of qualitative and / or quantitative determination of the analyte using suitable means.

The term "test tape" as used herein refers to more than one individual test site, preferably at least 10 individual test sites, more preferably at least 25 individual test sites, most preferably at least 50 individual test sites. Means a tape-shaped diagnostic element usually containing. The individual test sites are preferably arranged at a distance of several mm to several cm each, for example less than 2.5 cm from each other, and the test tape records the distance traveled during tape movement and / or for continuous test sites for calibration. The marker site may optionally be included in between. Such test tapes are for example described in EP 1739 432 A1, the disclosure of which is expressly incorporated by reference.

As used herein, the term "test disk" means a disk-shaped diagnostic element that may include one or more individual test sites, eg, 10 or more individual test sites. In one embodiment, the test disc is a thin layer of test chemistry that may be wetted by the sample and may be used for analyte measurement by applying an analyte sample thereon, whereby a larger or smaller size of the test disc site may be wetted by the sample. For example, with a layer having a thickness of about 20 μm. Non-wet areas of the test disc that can be partially or fully wetted due to the passage of moisture through the test chemical layer are then available for further measurement of the analyte.

The method according to the invention can be used to determine any biological or chemical substance that can be detected photochemically or electrochemically. The analyte is preferably malic acid, alcohol, ammonium, ascorbic acid, cholesterol, cysteine, glucose, glucose-6-phosphate, glutathione, glycerol, urea, 3-hydroxybutyrate, lactic acid, 5'-nucleotidase, peptide , Pyruvate, salicylate and triglyceride, and glucose is particularly preferred. In this regard, the analyte may originate from any source, but preferably includes, but is not limited to, whole blood, plasma, serum, lymph, bile, cerebrospinal fluid, extracellular tissue fluid, urine as well as glandular secretions such as saliva or sweat Included in The presence and / or amount of the analyte in the sample from whole blood, plasma, serum or extracellular tissue fluid is preferably determined by the diagnostic elements described herein.

Qualitative and / or quantitative determination of analytes may occur in any manner. For this purpose, any method for detecting enzymatic reactions which produce measurable signals which are known in the prior art and which can be analyzed or which can be read manually or using suitable means can be used basically. Within the scope of the present invention, electrochemical techniques are preferably used, as well as optical measurement methods including, for example, measurements of absorption, fluorescence, circular polarization dichroism (CD), optical rotational dispersion (ORD), refractive index, and the like. The presence and / or amount of the analyte is particularly preferably determined photometrically or by fluorometry, for example by indirectly by a fluorometrically measurable change of the artificial coenzyme.

In a further aspect, the present invention relates to a diagnostic element for measuring an analyte comprising a dry reagent layer containing:

(a) mutated dehydrogenase specific for the analyte, and

(b) artificial coenzymes.

With regard to preferred embodiments of diagnostic elements as well as mutated dehydrogenases or artificial coenzymes contained therein, reference is made to embodiments related to the detailed description of the methods of the invention.

The invention is further illustrated by the following figures and examples:

Figure 1: 0.0 mg / dl, 35.2 mg / dl, 54.2 mg / dl, 146.6 mg / dl, 249.0 mg / dl, 338.6 mg / dl and 553.6 mg / dl as a coenzyme in a glucose concentration of (displayed from top to bottom) NAD Kinetics of wild type glucose dehydrogenase from Bacillus subtilis in the presence of / NADH.
Figure 1A: enzyme activity 1556.2 kU / 100 g by weight.
Figure 1B: enzyme activity 1004.0 kU / 100 g by weight.
Figure 1C: enzymatic activity 502.0 kU / 100 g by weight.
Figure 1D: enzymatic activity 251.0 kU / 100 g by weight.
Figure 1E: Enzymes 25.10 kU / 100 g by weight.
Figure 2: In the presence of carbaNAD / carbaNADH as a coenzyme at glucose concentrations of 0.0 mg / dl, 34.4 mg / dl, 141.2 mg / dl, 236.6 mg / dl, 333.8 mg / dl and 525.8 mg / dl (shown from top to bottom). Reaction rate of glucose dehydrogenase double mutant GlucDH_E170K_K252L obtained by mutation of wild type glucose dehydrogenase from Bacillus subtilis. Enzyme activity: 4.60 kU / 100 g mass.
Figure 3: Fluorescence spectra of complex glucose dehydrogenase (GlucDH) / NADH before and after titration with gluconolactone.
Figure 4: Fluorescence spectra of complex glucose dehydrogenase (GlucDH) / NADH before and after titration with glucose.
Figure 5: the wild-type glucose dehydrogenase, and the presence of the reaction rate of glucose conversion of NADH at various glucose concentrations.
Figure 5A: Reaction rate in the absence of additional added gluconolactone at glucose concentrations of 77.0 mg / dl, 207.0 mg / dl, 300.0 mg / dl and 505.0 mg / dl (shown from top to bottom).
Figure 5B: Reaction rate in the presence of additionally added gluconolactone at glucose concentrations of 96.2 mg / dl, 274.0 mg / dl, 399.0 mg / dl and 600.0 mg / dl (shown from top to bottom).
Figure 6: Representation of the amino acid sequence of glucose dehydrogenase double mutant GlucDH_E170K_K252L.

Example

Example 1 Preparation of a Double Mutant of Glucose Dehydrogenase (GlucDH_E170K_K252L) from Bacillus subtilis with Amino Acid Substitutions E170K and K252L

In order to produce a stabilized enzyme compared to natural dehydrogenase, the nucleic acid sequence of glucose dehydrogenase from Bacillus subtilis was introduced in plasmid pKK177 (cloned via EcoRI and HindIII). Mutations E170K and K252L were introduced by position-specific mutagenesis first at position 170 of the amino acid sequence of wild type glucose dehydrogenase, followed by position-specific mutagenesis at position 252 of the sequence. Each mutagenesis step was performed using primers specifically designed as part of a PCR reaction.

The obtained PCR product was transformed into Escherichia coli XL1blue MRF ′. Cells were plated, plasmid containing clones were incubated overnight and enzyme activity was measured before and after temperature stress (stress test: 30 min at 50 ° C.). The results are shown in Table 1.

Residual activity of wild type glucose dehydrogenase and mutants GlucDH_E170K and GlucDH_E170K_K252L from Bacillus subtilis after stress (tested on Escherichia coli XL1blue MRF ') Residual Activity After Stress (%) Wild-type Glucose Dehydrogenase 23 Mutant GlucDH_E170K 80 Mutant GlucDH_E170K_K252L 130

Positive clones were tested two additional times prior to sequencing. The double mutant GlucDH_E170K_K252L obtained in this manner was transformed into p.S. 520 as a helper plasmid in the preparation strain Escherichia coli NM522.

Example 2: Purification of Double Mutant GlucDH_E170K_K252L

Each 10 g biomass was taken up in 50 mL 30 mM potassium phosphate buffer pH 6.5 and ground at about 800 bar. After separation of cell debris, at a load of <40 mg protein per ml of column volume, DEAE Sepharo using a linear gradient of buffer B (buffer A + 500 mM NaCl) in buffer A (30 mM potassium phosphate buffer pH 6.5) Chromatography was performed on GE Healthcare Company. Fractions showing glucose dehydrogenase activity were combined and adjusted to conductivity 230 mS / cm with ammonium sulfide (Aldrich Company).

After centrifugation, the clear supernatant was chromatographically separated on phenyl Sepharose FF (GE Healthcare Company) at a maximum load of 10 mg protein per ml of column volume. Elution was carried out using a linear gradient of pure buffer A in buffer A adjusted to conductivity 230 μmS / cm with ammonium sulfide. Fractions were tested for their enzymatic activity, combined, rebuffered to a concentration of about 50 mg / ml in 60 mM potassium phosphate buffer pH 6.5, concentrated and lyophilized.

Example 3: Determination of activity in cuvette test of wild type glucose dehydrogenase and double mutant GlucDH_E170K_K252L from Bacillus subtilis

Both enzymes were tested for specific or lyophilic activity in the presence of NAD / NADH or carbaNAD / carbaNADH of the double mutant GlucDH_E170K_K252L generated in Example 1 as well as wild type glucose dehydrogenase from Bacillus subtilis. Glucose dehydrogenase activity test was performed.

Preparation of Reagent Solutions

Tris buffer (0.1 M, pH 8.5; 0.2 M NaCl)

11.68 g NaCl (Sigma-Aldrich Company) and 12.11 g Tris (Sigma-Aldrich Company) were dissolved in about 900 mL secondary distilled water, adjusted to pH 8.5 with 1 N HCl and charged up to 1000 mL with secondary distilled water.

Dilution buffer (3.8 mM NAD; 0.1 M Tris, pH 8.5; 0.2 M NaCl)

250 mg NAD (Roche Company) was dissolved in 100 ml Tris buffer (0.1 M, pH 8.5; 0.2 M NaCl).

Glucose solution

2 g D (+) glucose monohydrate (Sigma-Aldrich Company) was dissolved in 10 mL secondary distilled water. After adjusting for 2 hours at room temperature and adjusting the mutarotation equilibrium, the solution was ready for use.

NAD solution (15 mM)

10 mg NAD (Roche Company) was dissolved in 1 ml secondary distilled water.

carbaNAD solution (15 mM)

10 mg carbaNAD (Roche Company) was dissolved in 1 ml secondary distilled water.

Sample manufacturing

To prepare for the measurement, 10 mg of the enzyme to be tested was dissolved in 1 ml dilution buffer and kept at room temperature for 60 minutes to allow for reconstitution. This was then diluted to 0.12-0.23 U / ml with dilution buffer.

Measurement procedure

To perform the measurements, pipette 1.35 ml Tris buffer, 0.1 ml glucose solution and 0.05 ml NAD solution or 0.05 ml carbaNAD solution (incubated at 25 ° C., respectively) into a plastic cuvette, mix together and incubate at 25 ° C. in a cuvette carriage It was.

After the absorbance of the solution no longer changed (blank value), 0.025 mL sample was introduced into the cuvette to initiate the reaction and the absorbance of the sample was monitored for a period of 5 minutes.

Measuring wavelength: 340 nm

Test volume: 1.525 ml

Path length: 1 cm

Temperature: 25 ℃

Rating range: 1-5 minutes

evaluation

The following formula was used to assess the activity in aqueous solutions of each enzyme:

Activity = (1.525 x ΔA / min x dilution factor) / (ε ₃₄₀ x 0.025 x 1) U / mL

[In the meal,

ΔA = A _t -A ₀ = slope of change in absorbance over time

ε ₃₄₀ = 6.3 [1 x mmol ^-1 x cm ^-1 ].

The measurement results are shown in Table 2.

Activity of Wild-type Glucose Dehydrogenase (WT-GlucDH) and Double Mutant GlucDH_E170K_K252L from Bacillus subtilis WT-GlucDH GlucDH_E170K_K252L NAD U / mg lyophilizate 203 167 U / mg Enzyme 484 270 Km mM 0.08 0.07 V _max (U / mg lyophilisate) 122 144 carbaNAD U / mg lyophilizate 3.4 2.3 U / mg Enzyme 8.2 3.7 % U / mg Lyophilized for NAD 1.7% 1.4% Km mM 0.3 1.4 V _max (U / mg lyophilisate) 3 13

As shown in Table 2, the activity of the system WT-glucDH / carbaNAD (8.2 U / mg enzyme) under standardized conditions in the cuvette was two orders of magnitude lower than the activity of the system WT-GlucDH / NAD (484 U / mg enzyme). Likewise, it was found that the activity of the double mutant GlucDH_E170K_K252L in the presence of artificial coenzyme carbaNAD is about two orders of magnitude lower (3.7 U / mg enzyme) than in the presence of natural coenzyme NAD (270 U / mg enzyme).

Example 4: Determination of reaction rate in dry reagent layer of wild type glucose dehydrogenase and double mutant GlucDH_E170K_K252L from Bacillus subtilis (WT-GlucDH)

A variety of test strips were prepared comprising the natural glucose dehydrogenase from Bacillus subtilis (WT-GlucDH) or the double mutant GlucDH_E170K_K252L prepared in Example 1 together with NAD / NADH or carbaNAD / carbaNADH as coenzymes to speed up the reaction of the enzyme. Was measured.

Specifically, 18.4 g 1 M phosphate buffer pH 7.0, 1.4 g Gantrez S97 (International Specialty Products Company), 2.94 g 16% NaOH solution, 0.34 g Mega 8 (Sigma-Aldrich Company), 0.039 g Geropon T77 (Rhone-Poulenc Company ) And 1.90 g polyvinyl pyrrolidone 25000 (Fluka Company) were prepared first for this purpose.

The partial solution was then stored in the refrigerator overnight as a partial solution 2 consisting of 0.50 g sodium chloride, 21.3 g secondary distilled water, 4.43 g Transpafill (Evonik Company) and 2.95 g Propiofan (BASF Company) (17.4 g 1 M phosphate) Buffer pH 7.0, 0.5 g sodium chloride, 14.35 g 2 M dipotassium hydrogen phosphate), and the respective amounts of dehydrogenase, coenzyme and optionally bovine serum albumin (BSA; Roche Company) are in each case shown in Table 3 below. It was. Enzymatically inactive bovine serum albumin was added to the formulation when a reduced amount of natural dehydrogenase was used to keep the matrix properties of the test strip as constant as possible.

Dehydrogenase and Coenzyme Contents in Test Strips Used enzyme Enzyme Mass (g) Lyophilized Activity (U / mg) Coenzyme Coenzyme Mass (g) BSA Mass (g) kU / 100 g mass WT-GlucDH 6.2 251 NAD 7.36 0 1556.2 WT-GlucDH 4.0 251 NAD 7.36 2.2 1004.0 WT-GlucDH 2.0 251 NAD 7.36 4.2 502.0 WT-GlucDH 1.0 251 NAD 7.36 5.2 251.0 WT-GlucDH 0.1 251 NAD 7.36 6.1 25.10 GlucDH Double Mutant 2.0 2.3 carbaNAD 2.1 0 4.60

The test strip obtained in this manner was measured in a laboratory measuring instrument (Roche Company, Inc.), which includes an excitation LED (375 nm) and a conventional detector (BPW34 blue-enhanced). The sample material applied was blood containing adjusted glucose values. The measurement results are shown in FIGS. 1 and 2.

As shown in FIGS. 1A-1E, the reaction rate of glucose conversion by wild type glucose dehydrogenase in the presence of NAD worsened with decreasing enzyme content and thus enzymatic activity in the test strip. Thus, it was predicted that the reaction rate of glucose conversion by the double mutant GlucDH_E170K_K252L in the presence of carbaNAD yielded even worse results due to the significantly lower enzymatic activity of only 4.60 kU / 100 g mass (see Table 3).

2 shows the reaction rate of the mutated glucose dehydrogenase obtained according to Example 1 in the presence of carbaNAD as coenzyme. As shown in FIG. 2, there was no decrease in the reaction rate predicted by the reduced enzyme activity. Rather, the double mutants showed a better reaction rate in the presence of carbaNAD than all formulations listed in Table 3 including the corresponding wild type glucose dehydrogenase and natural coenzyme NAD.

Considering the results described in Examples 3 and 4, this is not the activity of the enzyme, but the state of complex formation between enzymes, coenzymes, reducing coenzymes, glucose and gluconolactone, which are critical for the rate of glucose conversion in the dry reagent layer. The state of complex formation is clearly better in the case of the mutant prepared in Example 1 with carbaNAD / carbaNADH than for the wild type enzyme with native NAD / NADH.

Example 5 Detection of Triple Complexes Composed of Glucose Dehydrogenase, NADH and Glucose or Glunolactone

In order to confirm the presence of a triple complex consisting of enzymes, reducing coenzymes and glucose or gluconolactone, experiments on binding of analytes based on the fluorescence properties of NADH were carried out in cuvette tests.

For this purpose, 1 mg NADH (Roche Company) was dissolved in 1 ml phosphate buffer and the relevant fluorescence spectra were recorded. Thereafter, 10 mg of wild-type glucose dehydrogenase (GlucDH) from Bacillus subtilis was added to form the complex GlucDH-NADH, which is known from the literature, the complex having a significantly longer lifespan of NADH (0.4 in free state). 3 ns compared to ns), the mobile luminescence maximum value was calculated at 450 nm (see FIG. 3). Titration of the complex with gluconolactone simultaneously shifts the luminescence maximum to 427 nm, reducing fluorescence, indicating the presence of a novel complex that is a triple complex GlucDH-NADH-gluconolactone (see FIG. 3). .

The decrease in fluorescence, which also corresponds to shortening of life, is presumably due to rapid energy depletion by redox pair NADH-gluconolactone. When titrating the double complex GlucDH-NADH with glucose, an ineffective triple GlucDH-NADH-glucose complex is formed, which, due to the lack of a reducible species, can not lower the energy in a certain way, thus at the same emission maximum There is a tendency to show longer lifetime and higher intensity (see FIG. 4).

If the cleavage of the triple complex GlucDH-NADH-gluconolactone and thus the re-availability of the enzyme is critical to the rate of conversion of glucose to gloconolactone in the dry reagent layer, an additional gluconolactone (gluconolacto formed in the reaction) In addition to this, the addition of gluconolactone will slow down the conversion of glucose.

This assumption was confirmed in the measurement of the reaction rate at which blood was applied to the dry reagent layer according to Example 4 of the present application in the absence or presence of gluconolactone (see FIG. 5A). FIG. 5B shows significant deceleration of conversion compared to the sample measured in FIG. 5A.

<110> F. Hoffmann-La Roche AG          Roche Diagnostics GmbH <120> Schnelle Reaktionskinetik von Enzymen mit niedriger Aktivit? in          Trockenschichten <130> 44103P WO <150> PCT / EP2009 / 001206 <151> 2009-02-19 <160> 1 <170> PatentIn version 3.3 <210> 1 <211> 261 <212> PRT <213> Artificial Sequence <220> Mutant of glucose dehydrogenase from Bacillus subtilis <220> <221> MUTAGEN <222> (170) <220> <221> MUTAGEN <222> (252) <400> 1 Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala   1 5 10 15 Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala              20 25 30 Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val          35 40 45 Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Gln Gly      50 55 60 Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile  65 70 75 80 Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Glu                  85 90 95 Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val             100 105 110 Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile         115 120 125 Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser     130 135 140 Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala 145 150 155 160 Ser Lys Gly Gly Ile Lys Leu Met Thr Lys Thr Leu Ala Leu Glu Tyr                 165 170 175 Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn             180 185 190 Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp         195 200 205 Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile     210 215 220 Ala Ala Val Ala Val Trp Leu Ala Ser Lys Glu Ser Ser Tyr Val Thr 225 230 235 240 Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe                 245 250 255 Gln Ala Gly Arg Gly             260

Claims (15)

Analyte measurement method comprising the following steps:
(a) contacting a sample containing the analyte with a diagnostic element comprising a dry reagent layer containing:
(i) mutated dehydrogenase specific for the analyte and
(ii) artificial coenzymes, and
(b) determining the presence and / or amount of the analyte. The method according to claim 1, characterized in that the rate of analyte conversion in the diagnostic element is equal to or higher than the rate of analyte in the corresponding diagnostic element comprising the corresponding wild type coenzyme instead of artificial coenzyme, in particular at least 20% higher. How to. The method according to claim 1 or 2, characterized in that a mutated dehydrogenase is used which has reduced specific enzymatic activity compared to the corresponding wild type dehydrogenase. The dehydrogenated mutant of claim 1, wherein the nicotinamide adenine dinucleotide (NAD / NADH) -dependent or nicotinamide adenine dinucleotide phosphate (NADP / NADPH) -dependent mutated dehydrogenase is mutated. It is used as an enzyme. 5. The method according to claim 1, wherein the mutated glucose dehydrogenase (EC 1.1.1.47) is used as the mutated dehydrogenase. 6. 6. The method of claim 5, wherein the mutated glucose dehydrogenase comprises a mutation at position 170 and / or position 252 of the amino acid sequence of the corresponding wild type glucose dehydrogenase, in particular at position 170 and position 252, wherein said position 170 Wherein the mutation in preferably comprises an amino acid substitution of arginine or lysine, in particular lysine to glutamic acid, and / or the mutation at position 252 preferably comprises an amino acid substitution of leucine to lysine. The method according to claim 5 or 6, wherein the mutated glucose dehydrogenase is derived from Bacillus megaterium, Bacillus subtilis or Bacillus thuringiensis, in particular Bacillus subtilis. It is characterized in that the glucose dehydrogenase obtained by the mutation of wild type glucose dehydrogenase of. 8. The method of any one of claims 5 to 7, wherein the mutated glucose dehydrogenase has the amino acid sequence shown in SEQ ID NO: 1. The artificial nicotinamide adenine dinucleotide (NAD / NADH) compound, the artificial nicotinamide adenine dinucleotide phosphate (NADP / NADPH) compound or the compound of formula (I) according to any one of claims 1 to 8 Method for use as coenzyme:

. 10. Process according to any one of claims 1 to 9, characterized in that the compound of formula (II) or a salt thereof or optionally a reduced form thereof is used as artificial coenzyme:

[In the meal,
A = adenine or an analog thereof,
T = each occurrence independently represents O, S,
U = in each case independently ^{_{OH, SH, BH 3 -,}} BCNH 2 - represents,
V = in each case independently represents two groups forming an OH or phosphate group, or a cyclic phosphate group,
W = COOR, CON (R) ₂ , COR, CSN (R) ₂ , wherein R independently in each occurrence represents H or C ₁ -C ₂ alkyl,
X ¹ , X ² Each occurrence independently represents O, CH ₂ , CHCH ₃ , C (CH ₃ ) ₂ , NH, NCH ₃ ,
Y = NH, S, O, CH ₂ ,
Z = linear or cyclic organic residue,
Provided that Z and pyridine residues are not linked by glycosidic bonds. The method according to any one of claims 1 to 10, wherein carbaNAD is used as an artificial coenzyme. The method according to any one of claims 1 to 11, wherein a test tape, a test disk, a test pad, a test strip or a test strip drum is used as a diagnostic element. The method according to any one of claims 1 to 12, characterized in that the presence and / or amount of the analyte is determined photometrically or fluorometrically. Diagnostic element for analyte measurement comprising a dry reagent layer containing:
(a) mutated dehydrogenase specific for the analyte, and
(b) artificial coenzymes. 15. The diagnostic element of claim 14, wherein the mutated dehydrogenase has the amino acid sequence shown in SEQ ID NO: 1.

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