JPH10262658A - Sarcosine oxidase crystal, and production and three-dimensional structure of the crystal - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain columnar crystals of sarcosine oxidase useful for diagnosis of kidney diseases, etc., in high purity and yield by crystallizing under specific conditions in which ammonium sulfate is added as a precipitant to a solution of purified sarcosine oxidase. SOLUTION: Sarcosine oxidase columnar crystals are obtained from a solution (protein concentration is preferably 10-50 mg/ml) of purified sarcosine oxidase deviated from Bacillus sp. NS-129 by adding ammonium sulfate (concentration is preferably 1.9-2.2M) as a precipitant using a hanging drop method. The obtained crystals has such a high quality that its three dimensional structure can be elucidated up to the level of side chains by X-ray crystal structure analysis. The space group of the crystal is P4, 2, 2, and the lattice constant is (a)=(b)=177.0Å, (c)=72.6Å and α=β=γ=90 deg.. The sizes of the crystals are up to 0.1×0.2×1.5mm.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a sarcosine oxidase which is very useful information for improving sarcosine oxidase used as an enzyme for diagnosis of kidney disease in the field of clinical medicine by an arbitrary mutation operation. The present invention relates to a sarcosine oxidase crystal required to know its three-dimensional structure, a method for producing the crystal, and a three-dimensional structure of the sarcosine oxidase.

[0002]

2. Description of the Related Art Conventionally, in the field of clinical medicine, creatinine or creatine in serum and urine has been measured by an enzyme assay, and based on the measured values, for example, diseases of renal function, muscular diseases, etc. A diagnosis has been made. The principle of the quantification method is as shown in the following reaction formula, and sarcosine oxidase is used for the quantification. In addition, the parenthesis in the following formula means the enzyme used for each reaction.

Creatinine + H ₂ O → creatine (creatinine / amide hydrolase) Creatine + H ₂ O → sarcosine + urea (creatine / amidinohydrolase) sarcosine + H ₂ O + O ₂ → glycine + formaldehyde + H ₂ O ₂ (sarcosine oxidase)

Conventionally, sarcosine oxidase has been used in the genus Corynebacterium [J. Biochem. , 89
Vol., P. 599, 1981], genus Bacillus (see JP-A-8-238087 and JP-B-1-34035), genus Cylindrocarbon (see JP-A-56-927).
No. 90), Pseudomonas sp.
No. 3379), the genus Arthrobacter (Japanese Unexamined Patent Application Publication No.
It is known that it is produced by microorganisms such as Streptomyces (see JP-A-4-94688). However, those enzymes have high Km values for sarcosine,
Alternatively, it has a drawback that its substrate specificity is inferior, for example, it acts not only on sarcosine but also on ethyl glycine and proline to some extent, and is not always satisfactory. This is because, for example, the above-mentioned ethyl glycine and proline are compounds that can be present in serum due to the origin of medication during treatment or the like, and sarcosine oxidase acts on these as well, resulting in a positive measurement error. Give
This is because it causes a problem in diagnosis.

[0005] Under such circumstances, in order to solve the above-mentioned drawbacks, attempts have been made to improve enzymes by mutation of the gene encoding sarcosine oxidase. However,
Since the three-dimensional structure of the sarcosine oxidase protein or a closely related enzyme protein with high homology is unknown, it is impossible to improve the enzyme by site-directed mutation targeting residues near the active center. Is
We had to rely on uncertain improvements that depended only on probability, using the random mutation method.

[0006]

As described above, enzymatic quantification is used for quantifying creatinine or creatine, but the amount of creatinine or creatine contained in serum or urine is extremely low. Since it is so small that it also acts on substances from the dosage, the measurements are unreliable. Therefore, in the quantification, an enzymatic measurement method with high sensitivity and high substrate specificity is expected.

[0007] In order to solve the above-mentioned problems, there are various problems in a method of improving an enzyme by a random mutation method used to obtain an improved enzyme. That is, K
It is very difficult to select strains having subtle parameter differences such as m value and substrate specificity from a large number of mutants obtained by random mutagenesis. There are countless mutations that can never be obtained (for example, the substitution of phenylalanine encoded by TTT with methionine encoded by ATG requires simultaneous two-base substitution, It is hardly possible to see it).

An object of the present invention is to improve sarcosine oxidase, which is used as an enzyme for diagnosing kidney disease in the field of clinical medicine, by any mutation, and to obtain sarcosine oxidase which is very useful information. High-quality sarcosine required to know the three-dimensional structure
An object of the present invention is to provide a columnar crystal of oxidase, a method for producing the columnar crystal, and a three-dimensional structure of sarcosine oxidase obtained by X-ray crystal structure analysis using the columnar crystal.

[0009] The three-dimensional structure of sarcosine oxidase is extremely useful since a useful mutant sarcosine oxidase can be efficiently produced by performing site-specific mutation based on the three-dimensional structure.

[0010]

Means for Solving the Problems The inventors of the present invention have a heat-stable and relatively high substrate specificity for sarcosine, which is suitable for a highly sensitive enzymatic assay of creatinine or creatine, etc. As a result of intensive studies using a heat-resistant sarcosine oxidase N described in Japanese Patent Publication No. 1-34035 as a material to obtain a high-quality crystal of the enzyme capable of elucidating the three-dimensional structure of the enzyme, about 2M was obtained.
By using ammonium sulfate as a precipitant,
We succeeded in obtaining columnar crystals of the enzyme. Then, X-rays are applied to the columnar crystal to obtain a resolution of about 2.7.
It was confirmed that X-ray diffraction intensity data of Å could be obtained.

Further, the present inventors have proposed that the sarcosine
By performing X-ray crystal structure analysis on the columnar crystal of oxidase N, it was demonstrated that the three-dimensional structure could be clarified, and the present invention was completed based on these findings.

That is, the present invention is a columnar crystal of sarcosine oxidase characterized by having a grade capable of elucidating its three-dimensional structure to the level of a side chain by X-ray crystal structure analysis. Examples of the sarcosine oxidase include Bacillus sp.
And those derived from NS-129.

In the above columnar crystal, the space group of the crystal is P4 ₁ 2 ₁ 2 and the lattice constant is a = b = 177.0 °,
c = 72.6 ° and α = β = γ = 90 °. The size of the crystal is about 0.2 × 0.2 × 1.5 m
m.

Further, the present invention provides a method for preparing a purified sarcosine oxidase solution using ammonium sulfate as a precipitant,
A method for producing a columnar crystal of sarcosine oxidase, characterized by crystallizing by a hanging drop method.

Further, the present invention relates to a three-dimensional structure of sarcosine oxidase obtained by performing an X-ray crystal structure analysis using the columnar crystal of sarcosine oxidase.

By using the three-dimensional structure of the sarcosine oxidase obtained based on the columnar crystals of the sarcosine oxidase of the present invention thus obtained, the arrangement of the residues at the active center becomes clear, thereby improving the structure. The residues to be mutated to obtain a type of enzyme can be considerably narrowed down. In other words, instead of relying on stochastic mutation operations by random mutation, a specific residue is replaced with an arbitrary amino acid by site-specific mutation, and a small number of obtained mutants are compared in detail, and the substrate specificity is determined. It has become possible to select such excellent mutants.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
First, as a material for obtaining the sarcosine oxidase columnar crystals of the present invention, microorganisms having a sarcosine oxidase producing ability, for example, Corynebacterium, Bacillus, Cylindrocarbon, Pseudomonas, Arthrobacter, Strepto Any sarcosine oxidase produced by a strain belonging to the genus Myces or the like can be used. Among them, Bacillus
SP (Bacillus sp.) NS-129
Sarcosine oxidase N (hereinafter referred to as SON, see Japanese Patent Publication No. 1-334035) derived from [Microtechnical Research Institute No. 671 (FERMBP-671)] is preferably used. The main physicochemical properties of the above-mentioned SON are shown below.

A. Action: As shown in the following reaction formula, this enzyme is an enzyme that catalyzes the reaction of oxidizing and decomposing sarcosine to produce glycine, formaldehyde, and hydrogen peroxide. Sarcosine + H ₂ O + O ₂ → glycine + formaldehyde + H ₂ O ₂

B. Substrate specificity: The relative activity (%) when the absorbance at a wavelength of 510 nm obtained by performing an enzymatic reaction using N-methylglycine (sarcosine) as a substrate and another substance as a substrate is as follows: is there.
The data of the following relative activities (%) are described in
This is an excerpt of data on the SON described in No. 15281.

Substrate Relative activity (%) N-methyl-glycine (sarcosine) 100 N-ethyl-glycine 4.4 N-methyl-L-valine 7.7 N-methyl-L-leucine 25.4 N-methyl -L-isoleucine 7.2 L-proline 0.5

C. Km value: The Km value (Michaelis constant) of the present enzyme for sarcosine is 4.7 mM at 37 ° C. and pH 7.7 (phosphate buffer).

D. Optimum pH and stable pH range: The optimum pH of this enzyme is pH 6.7 when sarcosine is used as a substrate.
１10.0. The stable pH range is pH 6.5.
１11.5.

E. Range of suitable working temperature: The range of suitable working temperature of the present enzyme is 45 to 60 ° C. f. Thermal stability: 98% of enzyme activity is maintained at 55 ° C. for 10 minutes, and 75% of residual activity is shown at 60 ° C. for 10 minutes.

G. Molecular weight: The molecular weight of this enzyme is about 49000 as measured by column gel filtration using Sephadex G-150. The molecular weight calculated based on the amino acid sequence of the present enzyme is about 43,000.

H. Flavin enzyme protein: flavin adenine dinucleotide (hereinafter referred to as FAD) is covalently bound to the enzyme protein, and the binding ratio is 1: 1.

In producing the columnar crystals of sarcosine oxidase of the present invention, the purified sarcosine oxidase to be used as the raw material is, for example, a commercially available sarcosine oxidase (for example, "Sarcosine oxidase SOD-TE" manufactured by Kikkoman Corporation). Alternatively, those obtained by culturing a microorganism having the ability to produce sarcosine oxidase described above and purifying it by a conventional method (for example, see Japanese Patent Publication No. 1-34035) are used. Alternatively, clone only the gene encoding sarcosine oxidase,
A product obtained by expressing a large amount in Escherichia coli and purifying the same is used. Hereinafter, preparation of a DNA containing a gene encoding sarcosine oxidase,
Oxidase of Escherichia coli
li) expression and purification.

Examples of the donor microorganism for the gene encoding sarcosine oxidase include microorganisms having sarcosine oxidase-producing ability, such as sarcosine oxidase.
When the oxidase is SON, Bacillus sp. NS-129 strain and the like can be mentioned.

Then, a microorganism having sarcosine oxidase-producing ability is inoculated into an appropriate medium by a conventional method and cultured to obtain a culture. This culture is, for example,
10,000 r. p. m. And centrifuged for 10 to 15 minutes to obtain the cells.

Next, using the cells, for example, agricultural biological chemistry [Agr
ic. Biol. Chem. 55, 12
59 to 1263, 1991).
Cloning the sarcosine oxidase gene,
The sarcosine oxidase can be expressed in a large amount in Escherichia coli.

Next, the DNA containing the sarcosine oxidase gene obtained as described above was
Escherichia (Esc) containing recombinant DNA inserted into NA and having the ability to produce sarcosine oxidase.
In order to produce sarcosine oxidase using a strain belonging to the genus herichia), this strain may be cultured by a conventional solid culture method, but preferably by a liquid culture method.

Further, as a medium for culturing the above strain,
For example, one or more nitrogen sources, such as yeast extract, peptone, meat extract, corn steep liquor or soybean or wheat koji leachate, may contain potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, magnesium chloride, One or more of inorganic salts such as ferric iron, ferric sulfate and manganese sulfate are added, and if necessary, saccharide raw materials, vitamins and the like are appropriately added.

The initial pH of the medium depends on the strain used, but it is usually appropriate to adjust the pH to 7 to 9. The cultivation is preferably carried out at 30 to 42 ° C., preferably at about 37 ° C. for 4 to 24 hours, preferably 6 to 8 hours, by aeration-agitation deep culture, shaking culture, static culture, or the like.

After completion of the cultivation, sarcosine oxidase can be collected from the culture using conventional enzyme collecting means. For example, the cells may be subjected to ultrasonic treatment, trituration, or the like according to a conventional method, or the present enzyme may be extracted using a lytic enzyme such as lysozyme, or may be shaken or left in the presence of toluene or the like for self-treatment. The enzyme is digested and the enzyme is excreted outside the cells. This solution is filtered,
The solid portion is removed by centrifugation or the like, and if necessary, nucleic acid is removed with streptomycin sulfate, protamine sulfate or manganese sulfate, and then ammonium sulfate, alcohol,
Acetone or the like is added for fractionation, and the precipitate is collected, dialyzed against water, and dried under vacuum to obtain a crude enzyme preparation.

The crude enzyme preparation is further subjected to a conventional method of enzyme isolation and purification, for example, (1) column chromatography of DEAE-cellulose, (2) ammonium sulfate fractionation, (3)
QAE-Sephadex column chromatography,
(4) TSK-GEL butyl-Toyopearl 650C
Hydrophobic chromatography of [Toyo Soda Co., Ltd.]
(5) A highly purified sarcosine oxidase standard can be obtained by combining a method such as gel filtration with Sephadex or other methods as necessary. It is used as a material in chemical conversion.

The physicochemical properties of the recombinant-derived purified sarcosine oxidase obtained by the above-mentioned purification means are exactly the same as the physicochemical properties of the above-mentioned strain. Next, the crystallization of sarcosine oxidase will be described.

Generally, crystallization of a protein is not easy. Although the optimum conditions for crystallization can be found relatively easily, in the case of many proteins, a crystal that can be subjected to X-ray crystal structure analysis can be obtained only after examining various conditions. In addition, not a few proteins do not crystallize under any conditions. Unless a protein crystal is obtained, X-ray crystal structure analysis cannot be performed. Therefore, the three-dimensional structure of the protein cannot be clarified from X-ray crystal structure analysis.

At present, as another method for determining the three-dimensional structure of a protein, NMR (nuclear magnetic) is used.
resonance, nuclear magnetic resonance). In NMR, it is not necessary to crystallize a sample, and data can be collected in the state of a protein solution. But,
It is virtually possible to determine the structure by NMR with a molecular weight of 1
Limited to no more than 10,000 compounds. Therefore, at present, most proteins, including sarcosine oxidase having a large molecular weight, have to rely on X-ray crystal structure analysis.

Further, at the time of X-ray crystal structure analysis, not all protein crystals can be analyzed. Although there are differences depending on the capability of the X-ray diffractometer, the conditions of the protein crystals that can be subjected to the measurement are preferably large in size, preferably 0.1 mm square or more,
High transparency and high unity are required. In any case, it is important to obtain high-resolution diffraction intensity data when X-rays are applied. In particular, if a structural analysis is performed including the direction of the side chain of each amino acid constituting a protein, a resolution of at least about 3 ° is required.

In order to crystallize the sarcosine oxidase of the present invention, it is preferable that sarcosine oxidase, which is a material for crystallization described above, be prepared in a state of being concentrated in a thin buffer solution. The pH of the solution is desirably within the stable pH range of the enzyme. Therefore, if necessary, for example, the buffer is replaced with a 5-20 mM Tris-HCl buffer (pH 7.5-8.5), and the sarcosine oxidase concentration is reduced to about 10-100 mg / ml. The buffer may be any buffer as long as it is weakly alkaline. Also, buffer replacement,
Concentration and the like can be performed by ultrafiltration. Then, a suitable precipitant for crystallization is searched by adding various precipitants to the concentrate.

Various precipitants can be used for the crystallization of sarcosine oxidase of the present invention, and examples thereof include inorganic salts such as ammonium sulfate, polyethylene glycols, and organic solvents such as ethanol. Hampton Research
As a result of examining various precipitating agents using “Crystal Screen” manufactured by Research Inc., when ammonium sulfate was used as a precipitating agent under weakly alkaline conditions, the columnar crystals aimed at by the present invention were found to be formed. It was found that it could be obtained. When the concentration of ammonium sulfate is less than 1.9 M, crystallization becomes difficult. When the concentration exceeds 2.2 M, rounded crystals increase, and many small crystals are formed. Columnar crystals are difficult to obtain. For example, as a result of further study on the case of SON, the condition for obtaining the highest quality SON columnar crystal aimed at by the present invention is that the concentration of ammonium sulfate is preferably 1.9 to 2.2M, and 2.0 to 2.1M.
Is particularly preferable, and the SON concentration is 10 to 50 m.
g / ml, preferably 30 mg / ml, and any buffer for crystallization may be used as long as it is a weakly alkaline buffer. For example, a 100 mM Tris-HCl buffer (pH 8.5) is suitable. Any temperature may be used as long as it is used, and the temperature for crystallization may be any ordinary temperature, but it is preferable to leave it at about 20 ° C. for 2 days or more.

The embodiment of crystallization under the above conditions includes a hanging drop method, a sitting drop method,
Any known method such as a dialysis method and a seeding method may be used, but a hanging drop method, which is a kind of vapor diffusion method, is preferably used.

This hanging drop method utilizes the principle that water diffuses from a low-concentration precipitant solution containing a protein to a high-concentration precipitant solution in a closed system.
This is the most commonly used crystallization method.

The appearance of the columnar crystal obtained as described above, the type and size of the unit cell, which is the minimum unit of the crystal, the number and symmetry of the molecules in the lattice, are specific to the crystal, and are the same. Different crystals may be obtained with different types of precipitants even for the above protein. However, if the protein is the same, the three-dimensional structure of the molecule is considered to be the same even if the appearance of the crystal is different. In addition, if the enzyme has a high homology, almost the same crystal may be obtained under the same conditions.

According to such a method, it is possible to obtain a columnar crystal of sarcosine oxidase of the present invention having such a quality that it can be subjected to X-ray crystal structure analysis to elucidate the three-dimensional structure of the enzyme to the side chain level.

X was added to the columnar crystals obtained as described above.
In order to obtain diffraction intensity data by irradiating a ray, R-AXIS (manufactured by Rigaku Corporation) or the like is used as an ordinary laboratory-level X-ray diffractometer. At a synchrotron radiation facility that can use, for example, a synchrotron radiation facility at the Institute of High Energy Physics in Tsukuba, a Weissenberg camera [(Weissenberg camera)
a); Sakabe, J .; Appl. Cryst. , Volume 16,
542, 1983, or Nucl. Instr. M
eth. 448, 1991, or East, J.M. Ap
pl. Cryst. Vol. 22, page 8, 1989]. A two-dimensional detector such as an X-ray film can be used as a detector used at that time, but an imaging plate (ima) which can be used repeatedly is preferable.
ging plate, manufactured by Fuji Film Co., Ltd.). And in reading the data from the detector,
Fuji Image Reader BA100 (manufactured by Fuji Film Co., Ltd.) is preferable, and the processing of the data is performed by WEIS (Higashi, T. (1989) J. Am.
Appl. Cryst. 22, 9 to 18).

From the pattern of the obtained X-ray diffraction intensity data, the crystallographic parameters can be almost specified at this time. For example, in the case of SON, the crystallographic parameters are changed to tetragonal [tetragonal]. And the space group is P4 ₁ 2 ₁ 2 and the lattice constant is a = b = 17
7.0 °, c = 72.6 °, α = β = γ = 90 °. The calculated solvent content of the crystal was 62.8%, which was estimated to include two SON molecules in the asymmetric unit. The resolution of the data was 2.7 °, which was at a level at which the three-dimensional structure including the direction of the side chain could be determined.

Next, in order to derive an electron density diagram of the molecules constituting the crystal based on the X-ray diffraction intensity data obtained as described above, the phase must be determined by a molecular replacement method or a heavy atom isomorphous replacement method. Is required, but when the structure of another protein molecule having a high homology with the molecule is not known, the heavy atom isomorphous substitution method is necessarily used. And
In order to perform the heavy atom isomorphous substitution method, two or more types of heavy atom isomorphous substitutions are required for the crystal.

The heavy atom reagent used in the search for the heavy atom isomorphous substituent may be any reagent containing any heavy metal atom such as HgCl ₂ , but it is essential that the reagent specifically binds to a specific site of the protein molecule of the crystal. Condition. For example, S
In the case of ON, a preferable heavy atom isomorphous substituent can be obtained by K ₂ PtCl ₄ or the like in addition to HgCl ₂ . It should be noted that various programs can be used in the process of determining the phase.
P4 (Collaborative Computat)
ionical Project, Number 4 (199
4) ActaCrystallog. sec. D. 5
0, 760 to 763).

Next, if a good initial phase is obtained by the heavy atom isomorphous substitution method, an electron density diagram can be drawn.
The electron density diagrams are shown in various DM (Density Mod).
By performing the information processing (one of the programs of the CCP4), it is possible to convert to a clearer electron density diagram.

When modeling the molecule based on the electron density diagram obtained as described above, various programs can be used. Preferably, for example, a program O (Program O) (Jones O) is used. , A,
T. , Zou, J .; -Y. , Cowan, S .; W. & K
Jeldgaard, M .; (1991) Acta Cr
ystallog. sec. A. 47, 110-11
A model of the molecule is constructed on three-dimensional graphics by using 9) and the like.

Further, it is necessary to refine the constructed molecular model using various programs. For example, X-PLOR (Brunger,
A. T. , Kuriyan, J. et al. & Karplus, M
(1987) Science 35, 458 to 460) and a manual correction using the program O can be used to make the structure closer to the correct solution. The goal is to reduce the value of the R factor, which is an index of refinement, to 20% or less. For example, in the case of SON, the refinement was completed when the R factor dropped to 18.6%.

In the three-dimensional structure of the thus obtained SON, several residues at the N-terminal and C-terminal of the protein molecule are deleted. In addition, some of the side chains of residues such as lysine protruding from the molecular surface have an ambiguous structure. However, these ambiguous parts are not parts that play a significant role in the function of the enzyme. In addition, about 34 bound waters were specified per asymmetric unit.

[0053]

The present invention will be described in more detail with reference to the following examples, but the technical scope of the present invention is not limited to these examples. [Example 1] Crystallization of sarcosine oxidase N As a material for crystallization, commercially available Bacillus sp.
Cillus sp. ) NS-129 <6
No. 71 (FERM BP-671)> derived "Sarcosine oxidase SOD-TE" (manufactured by Kikkoman Corporation) was used.

This SON was prepared in a 20 mM Tris-HCl buffer (pH 8.0) so as to have a protein concentration of about 30 mg / ml, and then was treated with a 20 mM Tris-HCl using a Mono Q column (Pharmacia). Buffer (pH 8.
0) in a linear concentration gradient of NaCl (50-5
(00 mM). The purified SON fraction was buffer exchanged with 10 mM Tris-HCl buffer (pH 8.0) using Centriprep-30 (manufactured by Amicon), and finally the protein concentration was about 60 mg /
It was concentrated to a volume of ml.

On the other hand, the precipitant solution was prepared in five concentrations so that the concentration of ammonium sulfate was in the range of 2.02 to 2.06 M in 0.01M increments, and the buffer was 1 solution.
A 00 mM Tris-HCl buffer (pH 8.5) was used.

The crystallization was carried out by a known hanging drop method as follows. That is, the ammonium sulfate solution containing the above Tris-HCl buffer was added to a tissue culture Limbro plate (Linbro plate, 24 wells).
1 ml was put into each well of the above to prepare a reservoir solution. Next, 5 μl of the SON solution was placed on the center of a silicon-coated cover glass having a diameter of 22 mm, and immediately the same amount (5 μl)
Was added and stirred quickly. The cover glass was gently inverted and the lid was closed so as to close the hole containing the reservoir. At that time, silicone grease was applied to the bonding portion to ensure the sealing. Since it is calculated that the concentration of ammonium sulfate in the reservoir solution is twice that of the protein solution, the water in the protein solution gradually evaporates and diffuses into the reservoir solution. Also, the concentration of ammonium sulfate in the protein solution gradually approached that of the reservoir solution, and the insoluble protein gradually crystallized. The crystals grow by standing at 20 ° C. for 2 days or more, and from each of the above-mentioned ammonium sulfate concentrations, columnar crystals satisfying the object of the present invention can be obtained, and as shown in FIG. About 0.2 ×
SON columnar crystals reaching 0.2 × 1.5 mm were obtained.
Since this enzyme has a covalently bound FAD, the columnar crystals have a yellow color.

Example 2 Collection of X-Ray Diffraction Intensity Data From the SON columnar crystals obtained in Example 1, a large and highly unitary crystal was selected and subjected to an X-ray diffraction experiment.
The columnar crystal used for the measurement had a thickness of 0.1 to 0.2 mm square and a length of about 1 mm. The SON
To collect diffraction intensity data by irradiating the columnar crystal with X-rays, an X-ray diffraction experiment was performed with a Weissenberg camera at the synchrotron radiation facility of the Tsukuba High Energy Physics Laboratory. The crystals were sealed in a 1.5 mm diameter glass capillary and fixed on a goniometer with viscosity. The wavelength of the X-ray was 1 °, the camera length was 429.7 mm, and the temperature was measured at 10 to 12 ° C. 20 × 40c as detector
An m ² imaging plate (manufactured by Fuji Film Co., Ltd.) was used, and data from the detector was read using a Fuji Image Reader BA100. The data was processed by using a program-wise (WEIS). FIG. 2 shows the S detected on the imaging plate.
3 shows a part of an X-ray diffraction image of ON.

The crystallographic parameters specified from the pattern of the obtained X-ray diffraction intensity data are shown below. The crystal system to which the columnar crystal of the SON according to the present invention belongs is a tetragonal system, the space group is P 4 ₁ 2 ₁ 2 and the lattice constant is a = b =
177.0 °, c = 72.6 °, α = β = γ = 90 °. The calculated solvent content of the crystal was 62.8%, and SON was 2 in the asymmetric unit.
It was presumed to contain molecules. The resolution of the data was about 2.7 °, which was at a level at which the three-dimensional structure including the direction of the side chain could be determined.

Example 3 Preparation of Electron Density Diagram In order to derive an electron density diagram of the molecules constituting the crystal based on the X-ray diffraction intensity data obtained in Example 2, the phase was determined by the heavy atom isomorphous substitution method. Decided. At this time, three types of heavy atom reagents (10 mM
HgCl ₂ , 10 mM AgNO ₃ , 2 mM K ₂ PtC
Using l ₄ ), X-ray diffraction intensity data was collected after back soaking for about 2 to 3 hours for SON crystals soaked with those heavy atoms for 2 to 3 days. AgNO ₃ was subjected to a chlorine ion (Cl ⁻ ) removal operation. In the process up to the phase determination, various programs of the program collection CCP4 were appropriately used.

Next, an electron density diagram of the SON was drawn using the initial phase obtained by the heavy atom isomorphous substitution method. This was further converted to a clearer electron density diagram by performing a DM process. As options for the DM processing, solvent fluttering, histogram matching, and the like were used.

Example 4 Modeling and Refinement Based on the electron density diagram obtained in Example 3, modeling was performed using a program O to construct a three-dimensional structure of the SON molecule. The model of the constructed SON molecule was refined using the program X-PLOR. And X-PLO
Using the refinement by R and the manual repair work using the program O, the structure repair was repeated. The value of the R factor, an indicator of refinement, fell to 18.6%.

In the three-dimensional structure of the SON thus obtained, three residues at the N-terminal and six residues at the C-terminal were deleted. In addition, the structure of some side chains of residues on the molecular surface was ambiguous. Also, about 34 bound waters could be identified per asymmetric unit.

FIG. 3 shows a model of the three-dimensional structure of the SON obtained through such a process. From this three-dimensional structure,
The active center of sarcosine oxidase can be inferred. Therefore, according to the present invention, it is possible to improve the enzyme by site-directed mutagenesis, targeting the residues near the active center based on the three-dimensional structure. Sarcosine oxidase can be obtained.

[0064]

The columnar crystal of sarcosine oxidase of the present invention has such a quality that X-ray diffraction intensity data with a resolution of about 2.7 ° can be obtained by performing an X-ray diffraction experiment. Can be elucidated to the level of the side chain.

Sarcosine oxidase is a useful enzyme used in the field of clinical medicine as a diagnostic enzyme for renal diseases and the like. By elucidating its three-dimensional structure, sarcosine oxidase remains near the active center of the enzyme. Since the enzyme can be improved by site-specific mutation targeting a group, the present invention is extremely useful industrially.

[Brief description of the drawings]

FIG. 1 is a photograph showing a columnar crystal of sarcosine oxidase N.

FIG. 2 is a view showing a part of an X-ray diffraction image of sarcosine oxidase N detected by an imaging plate.

FIG. 3 is a photograph showing the three-dimensional structure of sarcosine oxidase N.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Makoto Nishiyama 4-1-22-303 Shimobotani, Hoya-shi, Tokyo (72) Inventor Yasuji Koyama 1606-19 Yamazaki, Noda-shi, Chiba Prefecture (72) Inventor Tano Yu Kura1001 Shimotaga, Atami

Claims (8)

[Claims] 1. A columnar crystal of sarcosine oxidase having a grade capable of elucidating its three-dimensional structure down to the level of a side chain by X-ray crystal structure analysis. 2. The method according to claim 1, wherein the sarcosine oxidase is Bacillus.
2. The sarcosine oxidase columnar crystal according to claim 1, which is derived from Bacillus sp. NS-129. 3. The space group of the crystal is P4 ₁ 2 ₁ 2;
The sarcosine oxidase columnar crystal according to claim 1, wherein the lattice constants are a = b = 177.0 °, c = 72.6 °, and α = β = γ = 90 °. 4. The size of the crystal is about 0.2 × 0.2.
The sarcosine oxidase columnar crystal according to claim 1, which is up to 1.5 mm. 5. The method for producing columnar crystals of sarcosine oxidase according to claim 1, wherein ammonium sulphate is used as a precipitant in the purified sarcosine oxidase solution and crystallization is performed by a hanging drop method. 6. The concentration of ammonium sulfate is 1.9 to 2.
The production method according to claim 5, wherein the production method is 2M. 7. The method according to claim 7, wherein the sarcosine oxidase is Bacillus.
Sarcosine oxidase derived from SP (Bacillus sp.) NS-129, and the protein concentration of sarcosine oxidase is 10 to 50 mg / m2.
l, the concentration of ammonium sulfate is 1.9 to 2.2M.
The method according to claim 5, wherein 8. A three-dimensional structure of sarcosine oxidase obtained by performing an X-ray crystal structure analysis using the columnar crystal of sarcosine oxidase according to any one of claims 1 to 4.