JP5751571B2 - Novel protein with bilirubin oxidation activity - Google Patents

Description

  The present invention relates to bilirubin oxidase, a gene encoding the enzyme, a method for producing the enzyme, and use of the enzyme.

  Blood bilirubin concentration is measured as a diagnostic standard for various liver disease diagnosis and jaundice differentiation. Methods for measuring serum bilirubin are broadly classified into enzyme methods using enzymes such as bilirubin oxidase, chemical methods using diazo reagents and vanadate ions (Patent Document 1), and methods using high-performance liquid chromatography. .

  Among the methods for measuring serum bilirubin, in the enzyme method, methods using bilirubin oxidase, ascorbate oxidase, laccase, or tyrosinase have been reported (Patent Documents 2, 3, 4, 5, etc.). As the bilirubin oxidase used for the measurement, it is desirable to use an enzyme having high stability. Therefore, various additives have been devised in order to stabilize the enzyme (Patent Documents 6, 7, etc.). In addition, thermostable bilirubin oxidase has been reported (Patent Document 8).

  Therefore, ascorbine is more stable than bilirubin oxidase that is practically used, such as bilirubin oxidase derived from Suehiro moth (Patent Document 9) and bilirubin oxidase derived from the genus Milosecium (Patent Document 10). A method for measuring bilirubin in a sample using acid oxidase has also been developed (Patent Document 11). The bilirubin oxidase activity of the ascorbate oxidase retains 100% activity at 60 ° C. for 30 minutes, but is completely deactivated at 80 ° C. for 30 minutes.

  The thermostable bilirubin oxidase exhibits a residual activity of 90% after heat treatment at 80 ° C. for 10 minutes and about 50% at 90 ° C. for 10 minutes (Patent Document 8). Furthermore, the endospore coat component (Endospore coat component, CotA) of bacteria belonging to Bacillus subtilis has a known gene sequence and amino acid sequence and is known to have laccase activity (Non-patent Document 1). ). A thermostable bilirubin oxidase is also known that exhibits a residual activity of 90% after heat treatment at 80 ° C. for 30 minutes and about 60% at 90 ° C. for 30 minutes (Patent Document 12).

JP 05-018978 A JP 59-17999 A Japanese Patent Laid-Open No. 02-238897 Japanese Patent Laid-Open No. 11-72497 JP 2000-166595 A Japanese Patent Laid-Open No. 08-66196 Japanese Patent Laid-Open No. 10-42869 Japanese Patent Application Laid-Open No. 61-209587 JP 59-135886 A JP-A-57-159487 JP 09-178755 A Japanese Patent Laid-Open No. 18-68003

F. Kunst et al., The complete genome sequence of the Gram-positive bacterium Bacillus subtilis, Nature, 1997, 390, 249-256

  An object of the present invention is to provide a bilirubin oxidase reagent, a gene thereof, a production method thereof, and a method for quantitatively measuring bilirubin in order to provide a more useful bilirubin oxidase reagent than currently known bilirubin oxidase reagents. It is to provide.

  As a result of intensive studies to solve the above problems, we succeeded in isolating genes encoding novel oxidases having bilirubin oxidation activity from metagenomic libraries, and the proteins encoded by these genes In addition to obtaining knowledge that the protein has an oxidizing action, excellent heat resistance, and high stability, and found that bilirubin in a test sample can be quantified very accurately, the present invention has been completed. It was.

That is, the present invention is as follows.
1) (a) consisting of the amino acid sequence shown in SEQ ID NO: 2 or (b) a sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2 A protein comprising and having bilirubin oxidation activity.

2) A gene encoding the protein described in 1) above.

3) The DNA according to any one of the following (a) to (c).
(A) DNA consisting of the base sequence shown in SEQ ID NO: 1.
(B) a DNA that hybridizes under stringent conditions with a DNA having the base sequence shown in SEQ ID NO: 1 or a base sequence complementary thereto and that encodes a protein having bilirubin oxidation activity.
(C) a gene that includes a sequence in which one or several bases are deleted, substituted, or added in the base sequence represented by SEQ ID NO: 1 and encodes a protein having bilirubin oxidation activity.

4) A recombinant vector into which the gene described in 2) or 3) above has been introduced.

5) A transformant transformed with the recombinant vector described in 4) above.

6) A method for producing a protein having bilirubin oxidation activity, comprising culturing the transformant according to 5) above and collecting a protein having bilirubin oxidation activity from the obtained culture.

7) A reagent for quantifying bilirubin which contains at least the protein described in 1) as an active ingredient.

8) A method for quantitatively measuring bilirubin in a sample, comprising contacting a certain amount of the protein according to 1) above with a test sample containing bilirubin and measuring the absorbance.

According to the present invention, a novel protein having extremely high heat resistance and excellent bilirubin oxidation activity can be provided.
The change in absorbance based on the oxidation of bilirubin by the protein accurately reflects the bilirubin concentration in the test sample. According to the present invention, a very useful reagent for quantifying bilirubin can be provided, and various liver disease diagnosis or jaundice differentiation can be provided. This greatly contributes to medical examinations.

The graph which shows the optimum pH of the bilirubin oxidase of this invention. The graph which shows the heat resistance of the bilirubin oxidase of this invention. The graph which shows the relationship between the light absorbency change accompanying bilirubin oxidation of the bilirubin oxidase of this invention, and a bilirubin density | concentration.

The present invention is described in detail below.
In this specification, when it is simply described as “bilirubin”, it is meant to include all of direct bilirubin, indirect bilirubin, and total bilirubin (total of direct bilirubin and indirect bilirubin).

The test sample that can be used in the present invention is not particularly limited as long as it contains bilirubin, and examples thereof include biological samples such as plasma, serum, and urine. In this specification, bilirubin oxidation activity refers to the catalytic action of an oxidation reaction using bilirubin as a substrate.
(1) Bilirubin oxidase The protein having bilirubin oxidase activity of the present invention (hereinafter referred to as bilirubin oxidase) has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and exhibits high stability.
The bilirubin oxidase of the present invention is an enzyme that catalyzes the reaction represented by the following formula 1 using bilirubin as a substrate.

  The properties of the bilirubin oxidase of the present invention and known bilirubin oxidase are compared and shown in Table 1. The bilirubin oxidase of the present invention is a novel enzyme having different properties from those of known enzymes that catalyze the same reaction, has extremely good heat resistance, has a wide optimum pH, and is more stable than known enzymes. Indicates. Therefore, no stabilizer is required.

さらに、本発明においては、上記配列番号２に示されるアミノ酸配列において、1若しくは数個のアミノ酸に欠失、置換、挿入若しくは付加等の変異が生じたものであっても、当該タンパク質がビリルビン酸化活性を有する限り、本発明のビリルビン酸化酵素遺伝子に含まれる。

Furthermore, in the present invention, even if one or several amino acids have a mutation such as deletion, substitution, insertion or addition in the amino acid sequence shown in SEQ ID NO: 2, the protein is not subjected to bilirubin oxidation. As long as it has activity, it is included in the bilirubin oxidase gene of the present invention.

(2) Gene The gene of the protein having bilirubin oxidation activity of the present invention is obtained from a metagenomic library constructed by extracting the DNA of a complex microorganism from activated sludge. That is, the metagenomic library is composed of a clone containing an Escherichia coli EPI300 strain as a host and a fosmid vector as a vector and containing DNA having an average length of about 30 kbp. The bilirubin oxidase gene of the present invention The metagenomic library was cultured in a medium in which 2,4-Dinitrophenol-containing LB (Luria-bertani) agar medium was further layered on LB (Luria-bertani) agar medium. It was obtained from

The present invention is based on the fact that the gene thus obtained was expressed, the protein expressed by the gene was confirmed to have an oxidative activity for bilirubin, and the gene was identified as a gene encoding bilirubin oxidase. for the gene, using substantially the B ilirubin- o xidizing p rotein, it was named bop.
The gene of the present invention will be further described below.

  The base sequence of the bilirubin oxidase gene (bop gene) of the present invention is represented by SEQ ID NO: 1 in the sequence listing, but is not limited thereto, and may be any one that encodes the amino acid sequence represented by SEQ ID NO: 2, In the amino acid sequence shown in SEQ ID NO: 2, even if one or several amino acids have mutations such as deletion, substitution, insertion or addition, as long as the protein has bilirubin oxidation activity, the present invention Of bilirubin oxidase gene.

  For example, 1-10, preferably 1-5 amino acids may be deleted in the amino acid sequence shown in SEQ ID NO: 2, and 1-10 in the amino acid sequence shown in SEQ ID NO: 2, preferably 1 -5 amino acids may be added or inserted, or 1-10, preferably 1-5 amino acids in the amino acid sequence shown in SEQ ID NO: 2 are substituted with other amino acids, Included in the bilirubin oxidase gene of the present invention.

  A mutant of the gene of the present invention having the above function or activity can be synthesized by site-directed mutagenesis. The method for introducing mutations into a gene is not limited, but a commercially available mutation introduction kit using site-directed mutagenesis (for example, Mutan-K (TAKARA) or Mutan-G (TAKARA)) is used. Can be used.

Whether or not the recombinant protein produced by these mutated genes has enzyme activity can be confirmed by measuring the function and activity as described above by reacting the mutated protein with bilirubin.
On the other hand, in the present invention, it hybridizes under stringent conditions with DNA comprising the base sequence of the bilirubin oxidase gene (bop gene) (SEQ ID NO: 1) or DNA having a sequence complementary to the DNA, and A gene encoding a protein having each function or activity is also included in the bilirubin oxidase gene of the present invention as long as it is a gene encoding a protein having bilirubin oxidation activity. Stringent conditions refer to conditions in which specific hybrids are formed and non-specific hybrids are not formed. For example, high homology (identit y good Mashiku 90% or higher) refers to conditions under which a DNA having a hybridizing. Such “stringent conditions” are, for example, heating in a solution of 2 × SSC, 0.1% SDS and 50% formamide at 25 ° C., and then in a solution of 0.1 × SSC, 0.1% SDS at 68 ° C. The conditions for washing with
However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and the same stringency can be realized by appropriately selecting these factors.

In the present invention, one or several bases are deleted or substituted in the base sequence (SEQ ID NO: 1) of the bilirubin oxidase gene (bop gene) as long as it encodes a protein having bilirubin oxidation activity. Also included are genes with inserted or added sequences.
That is, 1-10 amino acids, preferably 1-5 amino acids may be deleted in the amino acid sequence shown in SEQ ID NO: 2, and 1-10 in the amino acid sequence shown in SEQ ID NO: 2, preferably 1 -5 amino acids may be added or inserted. Furthermore, the bilirubin oxidase gene of the present invention includes those in which 1-10, preferably 1-5, amino acids in the amino acid sequence shown in SEQ ID NO: 2 are substituted with other amino acids.

(3) Recombinant vector and host The recombinant vector of the present invention can be obtained by linking a bilirubin oxidase gene to an appropriate vector.

The vector used in the present invention is not particularly limited as long as it can be replicated in a host, such as a plasmid, a cosmid, or a bacteriophage. The host is not particularly limited as long as the vector functions, and various bacteria such as Escherichia coli, actinomycetes, and the like can be used.
The direction and order of the gene to be introduced into the vector may be arbitrarily selected and sequenced as long as the gene is expressed and the expressed protein can function as an enzyme.

(4) Transformant containing the recombinant vector of the present invention:
The transformant of the present invention is produced by introducing the recombinant vector into the host and performing transformation.
In the case of producing a transformant, it is carried out by introducing the above recombinant Bacter into a host, such as a known electroporation method, protoplast method, calcium chloride method (spheroplast method), etc. Can be used.
In addition, when producing a transformant, bilirubin oxidase production ability is improved by selecting and using a vector, a host, a combination of an induction substrate that induces high expression, a promoter, an operator, an enhancer, etc. The transformant which has can be produced.
For example, when Escherichia coli BL21 strain is used as the host, pET19b vector containing the start codon ATG and RBS upstream and T7 / T7lac promoter upstream is used as the vector, and the resulting bilirubin oxidase gene is introduced. The body shows good bilirubin oxidase productivity.
It is possible to produce a microorganism that highly expresses a bilirubin oxidase gene by using a combination of not only this example but also a host, a vector, an expression control system and the like so as to function well.

(5) Production of bilirubin oxidase
In the present invention, the target bilirubin oxidase can be obtained by culturing a transformed microorganism into which the bilirubin oxidase gene has been introduced, and collecting it from the culture. “Culture” means any of culture supernatant, cultured cells, or disrupted cells. The method of culturing the microorganism of the present invention in a medium is performed according to a usual method used for culturing microorganisms. As a medium for culturing microorganisms such as actinomycetes and bacteria, any medium that contains a carbon source, nitrogen source, inorganic salts, etc. that can be assimilated by the microorganism and that can efficiently culture microorganisms can be used. Any of synthetic media may be used.

  In the case of the recombinant Escherichia coli shown above, it is cultured in a nutrient medium usually used in this research field, and if necessary, an inducer such as IPTG is added to highly express the enzyme in a nutrient medium in which Escherichia coli can grow. be able to. For example, under normal culture conditions, pre-cultured for 8-12 hours at 37 ° C in an LB medium (1.0% peptone, 0.5% dry yeast extract, 1.0% NaCl) supplemented with antibiotics. Using the cells as inoculum, inoculate a new LB medium at a volume ratio of 1-5%, main culture at 37 ° C for 2-4 hours, add IPTG, and further culture at 30 ° C for 2-4 hours.

  After the culture, when the target bilirubin oxidase is produced in the cells, the enzyme protein is extracted by disrupting the cells. When the target enzyme protein is produced outside the cells, the culture solution is used as it is, or the cells are removed by centrifugation or the like. Thereafter, by using general biochemical methods used for protein isolation and purification, such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, From this, bilirubin oxidase can be isolated and purified. Whether or not the target bilirubin oxidase is obtained can be confirmed by SDS-PAGE or the like.

(6) Quantification of bilirubin The bilirubin oxidase of the present invention oxidizes bilirubin in a sample by the action of bilirubin oxidase, and bilirubin that changes by the oxidation brings about a change in absorbance of the sample solution.
According to the present invention, the amount of bilirubin in a sample can be measured by measuring this optical change. The wavelength used for measuring the absorbance is 400 nm to 450 nm. Therefore, the bilirubin oxidase of the present invention is useful as a reagent for quantifying bilirubin in a sample.
The pyrilvin oxidase of the present invention may be combined with other materials used for bilirubin quantification as a reagent kit for quantification of bilirubin, and the kit may be a kit containing an enzyme expressed from a bop gene having bilirubin oxidative activity. Although it will not specifically limit, For example, the kit which combined the reagent with the bilirubin measuring reagent etc. is mentioned.

  These reagents and kits can be provided in the form of a liquid product, a frozen product of the liquid product, a lyophilized product of the liquid product, or a dried product of the liquid product (by heat drying and / or air drying and / or vacuum drying). Liquid products, liquid frozen products, and liquid freeze-dried products are preferred, liquid products and liquid freeze-dried products are more preferred, and liquid products are most preferred. In another embodiment, a frozen liquid product may be preferable. As yet another aspect, lyophilization of a liquid product may be preferred. Further, as described above, the pyrilrubin oxidase of the present invention has high stability, and a reagent or kit can be constructed without adding a stabilizer.

  Examples of the present invention are shown below, but the present invention is not limited to these examples.

Example 1
First, the base sequence of fosmid DNA was determined from the metagenomic clone E371, and the bop gene (SEQ ID NO: 1) was amplified by PCR using it as a template. The pET19b vector (Novagen)
Introduced. Primer sequences used for PCR are as follows. 24DNPoxi F-3 (5'-GGAATTCCATATGACAAAACTAACGCGACGC-3 '(SEQ ID NO: 3)) and 24DNPoxi R-3 (5'-CGGGATCCTCAGGCCCGAATCTCGAAATTA-3' (SEQ ID NO: 4)). The pET19b vector contains ATG as the start codon, RBS upstream, and T7 / T7lac promoter upstream. Expression can be expected. The constructed plasmid was transformed into Escherichia coli / BL21-E375 strain as a host.

<Culture of transformed Escherichia coli and preparation of cell extract>
Inoculate Escherichia coli / BL21-E375 with the prepared plasmid into LB medium containing 50 μg / ml ampicillin, and add 1 mM IPTG, a T7 promoter inducer, when the absorbance at 600 nm of the culture becomes 0.6 did. Thereafter, the cells are further cultured at 37 ° C. for 4 hours, collected by centrifugation (150,000 G, 1 minute, 4 ° C.), suspended in 10 mM Tris-HCl buffer solution (pH 8.5) containing 25 ppm of copper chloride, and ultrapure. After disrupting the cells using a sonicator, the cells were centrifuged (15,000 G, 5 minutes, 4 ° C.), and the supernatant was obtained to obtain a cell extract.

<Purification of enzyme showing bilirubin oxidation activity>
Escherichia coli / BL21-E375 introduced with the prepared plasmid was inoculated into LB medium containing 50 μg / ml ampicillin and cultured at 37 ° C. overnight. The culture broth was diluted 60: 1 into LB liquid medium supplemented with antibiotics, and 1 mM IPTG as a T7 promoter inducer was added when the absorbance at 600 nm of the culture broth became 0.6. Thereafter, the cells are further cultured at 37 ° C. for 4 hours, collected by centrifugation (4,000 G, 15 minutes, 4 ° C.), and the pellet is recovered. Next, the pellet is suspended in 1 ml Lysis Buffer (50 mM NaH ₂ PO4, 300 mM NaCl, 10 mM Imidazole, pH 8.0) containing 1 mg lysozyme, and microbial cells on ice are destroyed with a sonicator after ice cooling for 30 minutes. To do. Collect the supernatant after centrifugation (10,000 G, 20 min, 4 ° C.), transfer 20 μl supernatant to a new tube and store at −20 ° C. On the other hand, add 600 μl Lysis Buffer to Ni-NTA spin column, centrifuge (700 G, 2 minutes), and add sample to Ni-NTA spin column. After centrifugation (700 G, 2 minutes), add 600 μl Wash Buffer to the Ni-NTA spin column, centrifuge again (700 G, 2 minutes), and transfer the column to a new tube. Further, 200 μl Elution Buffer was added to Ni-NTA spin column and centrifuged, and the solution eluted from the column was used as the enzyme solution.

Example 2
The enzyme solution obtained in Example 1 was used to test the enzymatic properties of the enzyme. In the test, the following method for measuring bilirubin oxidation activity was used.
<Method for measuring bilirubin oxidation activity>
Add 100 μl of bilirubin solution diluted with 50 mM Britton & Robinson buffer (pH 7.5) to a 1.5 ml tube, add 0.5 μl of 100 mM CuSO ₂ solution, and leave at 37 ° C. for 5 minutes or longer. After adding the enzyme solution, measure 440 nm for 2 minutes with an absorptiometer. Enzyme activity was measured by monitoring the time course of decrease in absorbance at 440 nm at 37 ° C. (As). The same procedure is performed using the inactivated enzyme solution as a blind test, and the absorbance difference is measured (Ab). Enzyme activity was determined from As-Ab.
Unless otherwise specified, reagents used in this experiment were commercially available, such as those manufactured by Wako Pure Chemical Industries, Kokusai Kagaku, and Sigma Aldrich.

<Dependence of bilirubin oxidation activity on copper ion concentration>
A 15 μM-1.0 mM CuSO ₄ solution was added to a reaction solution containing 100 mM sodium acetate buffer (pH 4.5) and 1 mM substrate (ABTS), and the activity was measured with a total of 200 μl of the reaction solution. The enzyme was reacted by adding 2 μl of the enzyme extracted with QIAGEN Ni-NTA Spin Kit, and the absorbance at 418 nm was measured for 2 minutes. As a result, it was revealed that the activity of this enzyme depends on the copper ion concentration.
In the experiment conducted at the time of examining the following properties, 0.5 mM copper sulfate at which the concentration of copper ions necessary for activity almost reached saturation was added to the activity measurement solution.

<Examination of optimal pH for bilirubin oxidation activity>
The optimum pH for Wako Pure Chemical Industries was determined. The optimum pH of the enzyme activity was measured in the range of pH 2.5-9.5. 50 mM Britton & Robinson buffer (50 mM boric acid, 50 mM acetic acid, 50 mM phosphoric acid mixed buffer, pH adjusted with sodium hydroxide), 0.5 mM copper sulfate, 50 mM bilirubin solution 1 μl was added and the absorbance at 440 nm was measured for 2 minutes. Enzymes extracted with QIAGEN Ni-NTA Spin Kit were used. As a result, as shown in FIG. 1, the enzyme was found to have an optimum pH around pH 6-11.

<Thermal stability of bilirubin oxidation activity>
5 μl of the enzyme solution was heated at each temperature for 30 minutes and allowed to stand in ice water. The enzyme activity was measured with a reaction solution containing 0.1 M potassium phosphate buffer (pH 7.5), 0.5 mM copper sulfate, and 0.025 mg / ml bilirubin. As shown in FIG. 2, it was found that even when the enzyme was heated at 90 ° C., it maintained the same activity as when it was not heat-treated.

<Bilirubin oxidation activity at high temperature>
A 50 mM Britton & Robinson buffer solution (pH 7.5) in which the enzyme solution was dissolved was kept warm at 90 ° C. or 80 ° C., and a part of the solution was taken at regular intervals and left in ice water. 0.5 μl of 100 mM CuSO ₄ solution was added to 100 μl of 50 mM Britton & Robinson buffer (pH 7.5) containing 50 mM bilirubin, and finally the above enzyme solution was added, and the enzyme activity was measured at an absorbance of 440 nm. 5 μl of enzyme solution was added. A change with time of the activity as shown in FIG. 2 was obtained. From this graph, it was found that this enzyme is stable at 90 ° C. for more than 100 minutes.

<Bilirubin oxidation specific activity>
1 μl of enzyme solution was added to an activity measurement solution containing 50 mM Britton & Robinson buffer (pH 7.5), 0.5 mM copper sulfate, and 50 mM bilirubin solution, and the absorbance at 440 nm was measured for 2 minutes. The amount was 2.5 μmol / min / mg based on bilirubin manufactured by Wako Pure Chemical Industries, Ltd.

<Molecular weight>
The measured value by SDS-PAGE was a molecular weight of 59,000. The molecular weight calculated from the primary amino acid sequence of SEQ ID NO: 2 is 58,485.

Example 3
<Quantification of bilirubin using bilirubin oxidase>
Bilirubin from Wako Pure Chemical Industries, Ltd. was diluted with 0.2 M NaOH to prepare 10, 50, and 100 mM bilirubin standard solutions for calibration curves. 1 μl of the enzyme solution obtained in Example 1 was added to an activity measurement solution containing 50 mM Britton & Robinson buffer (pH 7.5), 0.5 mM copper sulfate, and bilirubin solution, and the absorbance at 440 nm was measured for 2 minutes. As a result, as shown in FIG. 3, the amount of change in bilirubin concentration and absorbance at a wavelength of 440 nm was plotted linearly at R ² = 0.9932, and bilirubin could be quantified by measuring the absorbance change at a wavelength of 440 nm. Moreover, the quantitative results were extremely accurate.

FERM P-21900

SEQ ID NO: 1: DNA base sequence of bop gene

Claims (8)

  (A) consisting of the amino acid sequence shown in SEQ ID NO: 2, or (b) including the sequence in which one or several amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2, And a protein having bilirubin oxidation activity.   A gene encoding the protein according to claim 1. The DNA according to any one of the following (a) to (c).
(A) DNA consisting of the base sequence shown in SEQ ID NO: 1.
(B) a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence of the nucleotide sequence complementary to SEQ ID NO: 1, and encodes a protein having the bilirubin oxidative activity DNA.
(C) DNA encoding a protein comprising a sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 and having bilirubin oxidation activity. A vector comprising the gene according to claim 2 or the DNA according to claim 3.   A transformant comprising the vector according to claim 4.   A method for producing bilirubin oxidase, comprising culturing the transformant according to claim 5 and collecting bilirubin oxidase from the resulting culture.   A reagent for quantifying bilirubin, comprising at least the protein according to claim 1 as an active ingredient.   A method for quantitatively measuring bilirubin in a sample, comprising contacting a certain amount of the protein according to claim 1 with a test sample containing bilirubin and measuring the absorbance.

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