KR101604624B1 - Fast reaction kinetics of enzymes having low activity in dry chemistry layers - Google Patents

Abstract

The present invention relates to a method for analyte determination and a suitable diagnostic element therefor.

Description

[0002] FAST REACTION KINETICS OF ENZYMES HAVING LOW ACTIVITY IN DRY CHEMISTRY LAYERS [0003]

The present invention relates to a method for analyte determination and a suitable diagnostic element therefor.

Diagnostic factors are an important component of clinically relevant assays. In this regard, the first focus is on the determination of analytes such as metabolites or substrates, for example indirectly or directly, with the help of enzymes specific for the analyte. In this case, the analyte is quantified after conversion with the help of enzyme-coenzyme complex. In this process, the analyte to be assayed is brought into contact with suitable enzymes, coenzymes and optionally mediators, and the coenzymes are physicochemically changed, for example oxidized or reduced, by an 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 the conductive component of the electrode, and the reaction is measured, for example, photometrically or electrochemically . In calibration, a direct relationship between the measured value and the concentration of the analyte to be measured is calculated.

Diagnostic elements known from the prior art are characterized by a limited shelf life and the specific requirements for the environment in which this shelf life is to be achieved (for example cooling or drying storage). Thus, for certain types of applications, for example, tests performed by the end-user himself, such as blood glucose self-monitoring, the false, inconspicuous and incorrect storage of a measurement system, Results may occur, which may lead to erroneous treatment of each disease. Incorrect results are primarily because enzymes, coenzymes, and mediators used in these diagnostic elements are generally sensitive to moisture and heat and are inactivated over time.

Known methods used to increase the stability of diagnostic elements include the use of enzymes from stable enzymes, for example, thermophilic microorganisms. In addition, enzymes can be stabilized by chemical modification, especially by cross-linking. In addition, enzyme stabilizers such as trehalose, polyvinylpyrrolidone 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 the enzyme is by site-specific or non-site-specifically introduced mutations. In this regard, the use of recombinant techniques that specifically affect the properties of the corresponding enzymes by targeted changes in DNA coding for the enzymes has proven to be particularly suitable.

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

Vazquez-Figueroa et al. (ChemBioChem (2007), 8, 2295) reported the position of the glucose dehydrogenase enzyme from Bacillus subtilis, Bacillus thuringiensis and Bacillus licheniformis , 170, and 252, which are incorporated herein by reference in their entirety. In this connection it is mentioned that the mutations E170K and Q252L individually and in combination cause stabilization of the glucose dehydrogenase from Bacillus subtilis.

However, when genetically modified stabilizing enzymes are used compared to wild-type variants, they have a lower substrate turnover value per unit of time, since they usually have quite low activity than the corresponding wild-type enzyme. The use of stabilizing enzymes is often an unacceptable alternative to the use of natural enzymes, given the fact that enzymes with high specific activity are preferably used in clinical and diagnostic chemistries such as blood glucose measurements.

Another difficulty is that high enzyme activity correlated with high substrate turnover per unit of time is usually only achieved with each natural coenzyme in each case. When an artificial coenzyme is used instead of a natural coenzyme, the enzymatic activity is usually greatly reduced and the conversion rate to the substrate is also reduced accordingly.

Therefore, the object underlying the present invention is to provide a stable diagnostic element, especially for measuring glucose, which overcomes some of the disadvantages of the prior art. In particular, the diagnostic element must ensure the high stability of the coenzyme as well as the enzyme while ensuring a high substrate conversion rate.

This object is achieved according to the invention by means of an analytical measuring method comprising the following steps:

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

(i) a mutated dehydrogenase specific for the analyte and

(ii) an artificial coenzyme, and

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

Surprisingly, the mutagenized dehydrogenase, which has extremely low activity in the presence of artificial coenzyme in the cuvette test, exhibits a faster reaction rate in the diagnostic element with a dry reagent layer as in the test strip, It has been found within the scope of the present invention to produce as many conversions as are present. The reason for this is presumably because other factors other than the enzyme activity at a higher concentration of the component are crucial to the rate of turnover and in this regard the complex formation state between the enzyme, the coenzyme, the reduced coenzyme, the analyte and the oxidized analyte It appears to be particularly crucial.

In this regard, the method according to the invention, in a preferred embodiment, is characterized in that the rate of turnover of the analyte in the diagnostic element described herein is equal to the rate of turnover of the analyte in the corresponding diagnostic element comprising the corresponding wild-type coenzyme in place of the co- Or greater. The turnover rate of the analyte in the diagnostic element used according to the invention is preferably at least 20%, more preferably at least 50%, most preferably at least 100 %, For example from 100% to 200%.

As used herein, the term "mutated dehydrogenase" or "dehydrogenase mutant" refers to a protein having a modified amino acid sequence relative to a wild-type dehydrogenase and having the same number of amino acids, Quot; means a genetically modified variant of a natural dehydrogenase (wild-type dehydrogenase) having an amino acid.

The mutagenized dehydrogenase can be obtained by mutagenesis from a wild-type dehydrogenase from any biological source, and in the sense of the present invention the term "biological source" includes prokaryotes such as bacteria as well as eukaryotes such as mammals and Other animals include. The introduction of the mutation (s) can be site-specific or non-site-specific, preferably site-specific, using recombinant methods known in the art and can be naturally occurring Resulting in one or more amino acid substitutions within the amino acid sequence of the dehydrogenase.

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

The mutated dehydrogenase in the sense of the present invention particularly preferably has a reduced specific enzyme activity relative to the corresponding wild-type dehydrogenase. The term "specific enzyme activity" (expressed as U / mg enzyme) as used in the present application means the amount of substrate converted per mg of enzyme and under predefined conditions per minute. On the other hand, the term "lyophilizate activity" means the amount of substrate converted per mg of lyophilisate containing the enzyme in combination with the auxiliary substance and under predetermined conditions per minute.

The mutated dehydrogenase used in the method according to the invention is preferably a nicotinamide adenine dinucleotide (NAD / NADH) -dependent or nicotinamide adenine dinucleotide phosphate (NADP / NADPH) -dependent mutagenized dehydrogenase, 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), mutagenized (EC 1.1.1.49), mutated glycerol dehydrogenase (EC 1.1.1.6), mutated 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30), mutated lactate Is selected from the dehydrogenase (EC 1.1.1.27; EC 1.1.1.28), the mutated maleate dehydrogenase (EC 1.1.1.37) and the mutated sorbitol dehydrogenase. The mutated dehydrogenase is particularly preferably a mutated glucose dehydrogenase (EC 1.1.1.47).

When a mutated glucose dehydrogenase is used within the scope of the present invention, it may contain amino acid (s) modified at any position in its amino acid sequence, basically 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 position 170 and position 252 being particularly preferred. It has been proved that mutated glucose dehydrogenases do not contain additional mutations in addition to these mutations.

Mutations at positions 170 and / or 252 may basically include any amino acid substitutions that result in stabilization, for example, an increase in heat and / or hydrolytic stability of the wild type dehydrogenase. The mutation at position 170 preferably comprises amino acid substitution of glutamic acid with arginine or lysine, in particular amino acid substitution of glutamic acid with lysine, while amino acid substitution of lysine with leucine with respect to position 252 is preferred.

The wild-type glucose dehydrogenase used to prepare the mutants of the above-mentioned glucose dehydrogenase is preferably derived from a bacterium and is selected from the group consisting of Bacillus megaterium, Bacillus subtilis or Bacillus thuringiensis, especially glucose dehydrogenase from Bacillus subtilis Enzymes are particularly preferably used. In a most preferred embodiment, the mutated glucoside dehydrogenase GlucDH_E170K_K252L having the amino acid sequence as shown in SEQ ID NO: 1 obtained by mutation of the wild-type glucose dehydrogenase from Bacillus subtilis is within the scope of the process according to the invention Is used.

In accordance with the present invention, the diagnostic elements described herein also include an artificial coenzyme in addition to the mutated dehydrogenase specific to the analyte. The artificial coenzymes within the meaning of the present invention are chemically modified as compared to natural coenzymes and are characterized in that they have an acid and base and / or nucleophile (s) in water, in particular in the range of from 0 ° C to 50 ° C, nucleophiles, such as alcohols or amines, and are therefore capable of exhibiting their activity over a longer period of time than the natural coenzyme under the same environmental conditions.

The artificial coenzyme preferably has a higher hydrolytic stability than the natural coenzyme, and it is particularly preferred that it has complete resistance to hydrolysis under the test conditions. Artificial coenzymes can have reduced binding constants for dehydrogenases compared to natural coenzymes, for example the binding constant is reduced to more than two-fold.

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

When the artificial coenzyme is an artificial NAD (P) / NAD (P) H compound, the artificial NAD (P) / NAD (P) H compound is preferably introduced via a linear or cyclic organic moiety, - containing residues such as 3-pyridinecarbonyl or 3-pyridinethiocarbonyl residues which are linked without a glycosidic bond to the phosphate moiety.

The artificial coenzyme is particularly preferably selected from compounds of the formula (II) or salts thereof or optionally in its reduced form:

[Wherein,

A = adenine or an analogue thereof,

T = independently represent O, S in each case,

U = independently for each occurrence represents OH, SH, BH ₃ ^- , BCNH ₂ ^-

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

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

X ¹ , X ² = In each case independently selected from _{O, CH 2, CHCH 3,} C (CH 3) 2, represent NH, NCH _3,

Y = NH, S, O, CH ₂ ,

Z = a linear or cyclic organic residue,

Provided that the Z and pyridine moieties are not connected by a glycosidic bond.

In compounds of formula (II), Z is preferably a linear moiety of 4-6 carbon atoms, preferably 4 carbon atoms, in which one or two carbon atoms are optionally replaced by one or more heteroatoms selected from O, S and N , O, S, and heteroatoms selected from N, and optionally a moiety CR ⁴ ₂ (formula bonded to the residue, and the cyclic group, and X ^2, which comprises a cyclic having 5 or 6 containing at least one substituent optionally of R ⁴ is in each case independently H, F, Cl, CH ₃ Im).

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

[Wherein,

A single or double bond may be present between R ^{5 '} and R ^{5 "}

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

R ⁵ = CR < ^{4 &} _gt ; ₂ ,

When a single bond is present between R ^{5 '} and R ^{5 "} , R ^5' And = O, S, NH, and 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 "} , R ^5' = R ^{5 "} = CR ⁴ ,

R ⁶ , R ^{6 '} = In each case independently represents a CH or CCH _3].

In a preferred embodiment, the compounds according to the invention are selected from the group consisting of adenine or adenine analogues such as C ₈ -substituted and N ₆ -substituted adenines, deaza variants such as 7-deaza, aza variants 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 Lt; / RTI >

In a further preferred embodiment, the compound is, for example, 2-methoxydeoxyribose, 2'-fluorodeoxyribose, hexitol, altritol or a polycyclic analog such as a bicycline sugar, LNA sugar and tricyclo sugar, But instead include 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 formula (II) according to the invention, W is preferably CONH ₂ or COCH ₃ .

R ⁵ is preferably the CH ₂ group in the general 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 '} 0.0 > = CH < / RTI > is most strongly preferred.

In the most strongly preferred embodiment, the artificial coenzyme is the compound carbaNAD known from the literature (JT Slam, Biochemistry (1988), 27, 183 and Biochemistry (1989), 28, 7688). Other stable coenzymes that can be used in accordance with the present invention are those described in 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 hereby expressly incorporated 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 a mutated dehydrogenase and an artificial coenzyme and can be wetted by the analyte containing sample. In addition to the mutagenized dehydrogenase and artificial coenzyme, the reagent layer may optionally contain additional reagents, such as, for example, suitable agents, as well as suitable adjuvants and / or additives, used in the qualitative detection or quantitation of analytes have.

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

The term "test strip" as used herein refers to one or more individual test sites, preferably at least 10 individual test sites, more preferably at least 25 individual test sites, and most preferably at least 50 individual test sites Type diagnostic element that normally includes a tape-type diagnostic element. The individual test sites are preferably arranged at a distance of several millimeters to several centimeters each, for example at a distance of less than 2.5 cm from each other, and the test tape records the distance traveled during tape movement and / And optionally a marker site between the marker regions. Such test tapes are described, for example, in EP 1739 432 A1, the disclosures of which are expressly incorporated by reference.

As used herein, the term "test disc" means a disc-shaped diagnostic element that can include one or more individual test sites, for example, 10 or more individual test sites. In one embodiment, the test disc may have a test disc portion with a larger or smaller size test disc region that can be wetted by the sample, depending on the volume of the sample, by applying the analyte sample thereon, , For example a layer having a thickness of about 20 [mu] m. The non-wetted area of the test disc, which can be partially or completely wetted by the passage of moisture through the test chemical layer, is then available for further measurement of the analyte.

The method according to the present invention can be used to measure any biological or chemical substance that can be photochemically or electrochemically detected. The analyte is preferably selected from the group consisting of malic acid, alcohol, ammonium, ascorbic acid, cholesterol, cysteine, glucose, glucose-6-phosphate, glutathione, glycerol, urea, 3-hydroxybutyrate, lactic acid, 5'-nucleotidase, , Pyruvate, salicylate and triglyceride, with glucose being particularly preferred. In this regard, the analyte may originate from any source, but preferably includes whole blood, plasma, serum, lymph, bile, cerebrospinal fluid, extracellular fluid, urine as well as pre- . The presence and / or amount of the analyte in the sample from whole blood, plasma, serum or extracellular fluid is preferably measured by the diagnostic elements described herein.

Qualitative and / or quantitative measurement of the analyte can occur in any manner. For this purpose, any method for detecting an enzymatic reaction that produces a measurable signal that is known in the prior art and can be analyzed, or passively, or read using suitable means, can be used basically. Within the scope of the present invention, electrochemical techniques as well as optical measurement methods including measurements such as, for example, absorption, fluorescence, circularly polarized different color (CD), optical rotation dispersion (ORD), refractive index and the like are preferably used. The presence and / or amount of the analyte is particularly preferably measured photometrically or fluorometrically, for example indirectly by fluorescence measurable changes in the coenzyme.

In a further aspect, the invention relates to a diagnostic element for analyte measurement comprising a dry reagent layer comprising:

(a) an analyte-specific mutagenized dehydrogenase, and

(b) Artificial coenzyme.

With respect to the preferred embodiment of the mutagenized dehydrogenase or the artificial co-enzyme contained therein as well as the diagnostic element, an embodiment related to the detailed description of the method of the present invention is referred to.

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

Figure 1: NAD as a coenzyme at a glucose concentration of 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 / / Reaction rate 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 mass.
Figure 1C: Enzyme activity 502.0 kU / 100 g mass.
Figure 1D: Enzyme activity 251.0 kU / 100 g mass.
Figure 1E: Enzyme activity 25.10 kU / 100 g mass.
Figure 2: In the presence of carbaNAD / carbaNADH as a coenzyme at a glucose concentration 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 / Reaction Rate of Glucose Dehydrogenase Double Mutant GlucDH_E170K_K252L Obtained by Mutagenization of Wild Type Glucose Dehydrogenase from Bacillus subtilis. Enzyme activity: 4.60 kU / 100 g mass.
Figure 3: Fluorescence spectrum of conjugated glucose dehydrogenase (GlucDH) / NADH before and after titration with gluconolactone.
Figure 4: Fluorescence spectrum of conjugated glucose dehydrogenase (GlucDH) / NADH before and after titration with glucose.
Figure 5: Reaction rate of glucose conversion in the presence of wild-type glucose dehydrogenase and NADH at various glucose concentrations.
Figure 5A: Rate of reaction in the absence of the additionally added gluconolactone at a glucose concentration of 77.0 mg / dl, 207.0 mg / dl, 300.0 mg / dl and 505.0 mg / dl (shown above).
Figure 5B: Rate of reaction in the presence of additional added gluconolactone at a glucose concentration of 96.2 mg / dl, 274.0 mg / dl, 399.0 mg / dl and 600.0 mg / dl (shown above).
Figure 6: Marking of the amino acid sequence of the 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

To produce a stabilized enzyme relative to the native dehydrogenase, the nucleic acid sequence of the glucose dehydrogenase from Bacillus subtilis was introduced in the plasmid pKK177 (cloned via EcoRI and HindIII). The mutations E170K and K252L were introduced by position-specific mutagenesis first at position 170 of the amino acid sequence of the wild-type glucose dehydrogenase, followed by position-specific mutagenesis at position 252 of the above sequence. Each mutagenesis step was carried out using a primer specifically designed as part of the PCR reaction.

The obtained PCR product was transformed into Escherichia coli XL1blue MRF '. Cells were plated, plasmid-containing clones were cultured overnight, and enzyme activity was measured before and after thermal stress (stress test: 50 占 폚 for 30 minutes). The results are shown in Table 1.

The residual activity of the wild-type glucose dehydrogenase and the mutants GlucDH_E170K and GlucDH_E170K_K252L from Bacillus subtilis after the stress (tested in Escherichia coli XL1blue MRF ') Residual activity after stress (%) Wild-type glucose dehydrogenase 23 Mutant GlucDH_E170K 80 Mutant GlucDH_E170K_K252L 130

Two positive clones were tested before sequencing. The double mutant GlucDH_E170K_K252L thus obtained was transformed into the producing Escherichia coli NM522 strain using pUBS-520 as a helper plasmid.

Example 2: Purification of the double mutant GlucDH_E170K_K252L

Each of 10 g of biomass was taken in 50 ml of 30 mM potassium phosphate buffer pH 6.5 and milled at about 800 bar. After separation of the cellular debris, a linear gradient of buffer B (buffer A + 500 mM NaCl) in buffer A (30 mM potassium phosphate buffer, pH 6.5) was added to DEAE sepharose Chromatography was performed on a GE Healthcare Company. The fractions exhibiting glucose dehydrogenase activity were combined and adjusted to a conductivity of 230 mS / cm with ammonium sulphide (Aldrich Company).

After centrifugation, the clear supernatant was chromatographically separated on phenyl Sepharose FF (GE Healthcare Company) at a maximum loading 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 a conductivity of 230 mS / cm with ammonium sulphide. The fractions were tested for their enzymatic activity, combined and re-buffered to a concentration of about 50 mg / ml in 60 mM potassium phosphate buffer, pH 6.5, concentrated and lyophilized.

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

In order to examine the specific activity or lyophilizate activity of NAD / NADH or carbaNAD / carbaNADH of the double mutant GlucDH_E170K_K252L produced in Example 1 as well as the wild-type glucose dehydrogenase from Bacillus subtilis, Glucose dehydrogenase activity test was performed.

Preparation of reagent solution

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 filled 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 adjustment of the settling time and the mutation equilibrium at room temperature for 2 hours, 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 preparation

To prepare for the assay, 10 mg of the enzyme to be tested was dissolved in 1 ml dilution buffer and maintained at room temperature for 60 minutes to allow reconstitution. Thereafter, it was diluted with a dilution buffer to 0.12 to 0.23 U / ml.

Measurement procedure

1.35 ml of Tris buffer, 0.1 ml of glucose solution and 0.05 ml of NAD solution or 0.05 ml of carbaNAD solution (incubated at 25 [deg.] C respectively) were pipetted into a plastic cuvette, mixed together and incubated at 25 [deg.] C in a cuvette carriage Respectively.

After the absorbance of the solution no longer changes (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.

Measured wavelength: 340 nm

Test volume: 1.525 ml

Path length: 1 cm

Temperature: 25 ℃

Scope: 1 - 5 minutes

evaluation

Using the following equation, the activity of each enzyme in an aqueous solution was evaluated:

Activity = (1.525 x DELTA A / min x dilution factor) / (epsilon ₃₄₀ x 0.025 x 1) U / ml

[Wherein,

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

竜₃₄₀ = 6.3 [1 x mmol ^-1 x cm ^-1 ].

The measurement results are shown in Table 2.

The activity of the wild-type glucose dehydrogenase (WT-GlucDH) and the double mutant GlucDH_E170K_K252L from Bacillus subtilis WT-GlucDH GlucDH_E170K_K252L NAD U / mg lyophilisate 203 167 U / mg enzyme 484 270 Km mM 0.08 0.07 V _max (U / mg lyophilisate) 122 144 carbaNAD U / mg lyophilisate 3.4 2.3 U / mg enzyme 8.2 3.7 % U / mg lyophilisate 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 the standardization conditions in the cuvette was two orders of magnitude lower than the activity of the system WT-GlucDH / NAD (484 U / mg enzyme). Similarly, the activity of the double mutant GlucDH_E170K_K252L in the presence of artificial co-enzyme carbaNAD was found to be 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: Measurement of reaction rate of wild-type glucose dehydrogenase and double mutant GlucDH_E170K_K252L from Bacillus subtilis (WT-GlucDH) in a dry reagent layer

Various test strips containing NAD / NADH or carbaNAD / carbaNADH as a coenzyme were prepared by using natural glucose dehydrogenase from Bacillus subtilis (WT-GlucDH) or the double mutant GlucDH_E170K_K252L prepared in Example 1, Were measured.

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

The partial solution was then added to a partial solution 2 consisting of 0.50 g of sodium chloride, 21.3 g of secondary distilled water, 4.43 g of Transpafill (Evonik Company) and 2.95 g of Propiofan (BASF Company) as well as 17.5 g of 1 M phosphate Buffer pH 7.0, 0.5 g sodium chloride, 14.35 g 2 M potassium dihydrogen phosphate), and the amounts of dehydrogenase, coenzyme and optionally bovine serum albumin (BSA; Roche Company) Respectively. Enzymatically inactive bovine serum albumin was added to the formulation when using a reduced amount of natural dehydrogenase to keep the matrix properties of the test strip as constant as possible.

Amount of dehydrogenase and coenzyme in test strips used enzyme Enzyme mass (g) The lyophilisate activity (U / mg) Coenzyme Coarse 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 strips obtained in this way were measured in a laboratory measuring instrument (Roche Company, self-manufactured) comprising an excitation LED (375 nm) and a conventional detector (BPW34 blue-enhanced). The sample material applied was blood containing a calibrated glucose value. The measurement results are shown in Figs. 1 and 2. Fig.

As shown in Figures 1A-1E, the rate of conversion of glucose conversion by wild-type glucose dehydrogenase in the presence of NAD was deteriorated with decreased enzyme content in the test strip and consequently decreased enzyme activity. It was therefore predicted that the reaction rate of glucose conversion by the double mutant GlucDH_E170K_K252L in the presence of carbaNAD produced even worse results due to the significantly lower enzyme activity of only 4.60 kU / 100 g mass (see Table 3).

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

In view of the results described in Examples 3 and 4, this is not the activity of the enzyme, but the state of complex formation between the enzyme, coenzyme, reducing coenzyme, glucose and gluconolactone, which is crucial to the rate of forwarding of glucose in the dry reagent layer And the state of complex formation is clearly better in the case of the mutants prepared in Example 1 with carbaNAD / carbaNADH than in the wild type enzyme with natural NAD / NADH.

Example 5: Detection of triple complex consisting of glucose dehydrogenase, NADH and glucose or gluconolactone

In order to confirm the existence of a triple complex consisting of enzyme, reduced coenzyme and glucose or gluconolactone, experiments on the binding of analytes based on the fluorescence properties of NADH were carried out in the cuvette test.

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 known complex GlucDH-NADH in the literature, which formed a significantly longer lifetime of NADH (0.4 3 ns compared to ns), the maximum value of mobile emission at 450 nm was calculated (see FIG. 3). Titration of the complex with gluconolactone shifts the emission maxima at 427 nm, while reducing fluorescence, indicating the presence of a novel complex which is a triple complex GlucDH-NADH-gluconolactone (see Figure 3) .

Reduction in lifetime and corresponding fluorescence reduction is presumably due to rapid energy depletion by the redox pair NADH-gluconolactone. When a double complex GlucDH-NADH is titrated with glucose, an inefficient trivalent GlucDH-NADH-glucose complex is formed, which, due to the lack of reducible species, can not lower the energy in a particular way, Tend to exhibit longer lifetime and higher intensity (see FIG. 4).

If the cleavage of the triple complex GlucDH-NADH-gluconolactone and subsequent re-utilization of the enzyme is critical to the rate of conversion of glucose to gluconolactone in the dry reagent layer, additional gluconolactone (gluconolactone formed in the reaction , Addition of gluconolactone will slow the conversion of glucose, since it will inhibit the additional enzyme complex.

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

<110> F. Hoffmann - La Roche AG          Roche Diagnostics GmbH <120> Schnelle Reaktionskinetic von Enzymen mit niedriger Activity? 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> <223> Mutant of glucose dehydrogenase from Bacillus subtilis <220> <221> MUTAGEN <170> <220> <221> MUTAGEN <222> <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 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 Ser Ser 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 (17)

A method for assaying a sample,
Wherein the analyte is glucose, comprising the steps of:
(a) contacting a sample containing an analyte with a diagnostic element comprising a dry reagent layer comprising:
(i) from Bacillus megaterium, Bacillus subtilis or Bacillus thuringiensis as the mutated glucose dehydrogenase (EC 1.1.1.47) specific for the analyte, from Bacillus megaterium, Bacillus subtilis or Bacillus thuringiensis Of the wild-type glucose dehydrogenase, wherein the mutation at position 170, position 252, or both, of the amino acid sequence of the corresponding wild-type glucose dehydrogenase is obtained by mutagenesis of arginine or lysine of glutamic acid (NAD / NADH) -dependent or nicotinamide adenine dinucleotide phosphate (NADP / NADPH) -dependent, wherein the mutation at position 252 comprises an amino acid substitution of lysine to leucine Mutated glucose dehydrogenase, and
(ii) an artificial coenzyme which is a compound of the following formula (II) or a salt thereof or a reduced form thereof:

[Wherein,
Aza adenine, 7-deaza-8-azaadenine, and the 7-deaza-8-azaadenine are halogen, C _1-6 alkenyl, Lt; RTI ID = 0.0 &gt; Ci- ₆ &lt; / RTI &gt; alkyl,
T = in each case independently O, or S,
U = independently for each occurrence represents OH, SH, BH ₃ ^- , or BCNH ₂ ^-
V = independently for each occurrence represents OH or phosphate group,
W is COOR, CON (R) ₂ , COR, or CSN (R) ₂ , wherein R in each occurrence independently represents H or C ₁ -C ₂ alkyl,
X ¹ and X ² each independently represent O, CH ₂ , CHCH ₃ , C (CH ₃ ) ₂ , NH, or NCH ₃ ,
Y = NH, S, O, or CH ₂ ,
Z = a linear or cyclic organic residue,
Provided that the Z and pyridine moieties are not connected by a glycosidic bond, and
(b) measuring the presence, quantity, or both of the analyte. 2. The method of claim 1, wherein the rate of turnover of the analyte in the diagnostic element is equal to or higher than the rate of turnover of the analyte in the corresponding diagnostic element, including the corresponding wild-type coenzyme, instead of the artificial coenzyme. 3. The method according to claim 1 or 2, wherein a mutated dehydrogenase having reduced specific enzyme activity is used compared to the corresponding wild type dehydrogenase. 2. The method of claim 1, wherein the mutated glucose dehydrogenase has the amino acid sequence set forth in SEQ ID NO: 1. 3. The method according to claim 1 or 2, wherein carbaNAD is used as an artificial coenzyme. The method according to claim 1 or 2, wherein the test tape, the test disc, the test pad, the test strip or the test strip drum are used as diagnostic elements. 3. The method according to claim 1 or 2, wherein the presence, quantity, or both of the analyte is measured photometrically or fluorometrically. As a diagnostic element for analyte measurement in a sample,
Wherein the analyte is glucose and comprises a dry reagent layer containing:
(a) Bacillus megaterium, Bacillus subtilis or Bacillus thuringiensis as the mutated glucose dehydrogenase (EC 1.1.1.47) specific for the analyte. Of the wild-type glucose dehydrogenase, wherein the mutation at position 170, position 252, or both, of the amino acid sequence of the corresponding wild-type glucose dehydrogenase is obtained by mutagenesis of arginine or lysine of glutamic acid (NAD / NADH) -dependent or nicotinamide adenine dinucleotide phosphate (NADP / NADPH) -dependent, wherein the mutation at position 252 comprises an amino acid substitution of lysine to leucine Mutated glucose dehydrogenase, and
(b) an artificial coenzyme which is a compound of the following formula (II) or a salt thereof or a reduced form thereof:

[Wherein,
Aza adenine, 7-deaza-8-azaadenine, and the 7-deaza-8-azaadenine are halogen, C _1-6 alkenyl, Lt; RTI ID = 0.0 &gt; Ci- ₆ &lt; / RTI &gt; alkyl,
T = in each case independently O, or S,
U = independently for each occurrence represents OH, SH, BH ₃ ^- , or BCNH ₂ ^-
V = independently for each occurrence represents OH or phosphate group,
W is COOR, CON (R) ₂ , COR, or CSN (R) ₂ , wherein R in each occurrence independently represents H or C ₁ -C ₂ alkyl,
X ¹ and X ² each independently represent O, CH ₂ , CHCH ₃ , C (CH ₃ ) ₂ , NH, or NCH ₃ ,
Y = NH, S, O, or CH ₂ ,
Z = a linear or cyclic organic residue,
Provided that the Z and pyridine moieties are not connected by a glycosidic bond. 9. The diagnostic element of claim 8, wherein the mutated glucose dehydrogenase has the amino acid sequence set forth in SEQ ID NO: 1. 3. The method of claim 2, wherein the rate of turnover of the analyte in the diagnostic element is 20% higher than the rate of turnover of the analyte in the corresponding diagnostic element, including the corresponding wild-type coenzyme, instead of the artificial coenzyme. delete delete delete delete delete delete delete

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