JPS6339230B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for purifying long-chain acyl coenzyme A synthetase (abbreviated herein as acyl-CoA synthetase, EC.6.2.1.3). Acyl-CoA synthetase is an important enzyme located in the first step of fatty acid oxidation in vivo, and is involved in the following reactions. RCOOH + CoA + ATP → RCOCoA + AMP + pyrophosphate (R means an alkyl group or an alkenyl group) Its location is known in rat liver, Escherichia coli, Bacillus bacteria, yeast, etc., but it is a membrane-bound enzyme. Since it is unstable, various attempts have been made to purify it, but it has not been possible to extract it as a pure specimen. For example, D. Samuel et al.
Biochemistry) Volume 12, pages 576-582 (1970)
In Escherichia coli (Escherichia coli)
coli is hereinafter referred to as E.Coli. ) has been purified, but it has not been completely purified into a single protein, and the specific activity per protein is low. Also, J.Bar.Tana etc.
Biochemical Journal
Journal), Vol. 122, pp. 353-362 (1971), the long-chain acyl-CoA synthetase was purified from rat liver microsomes, but here too, the homogeneity was an electrophoretic problem, and the specific activity is also low. The present inventors were previously researching microbial acyl-CoA synthetase, and discovered that there are two types of this enzyme, one of which, acyl-CoA synthetase, is involved in the system that directly incorporates fatty acids into lipids.
We discovered that acyl-CoA synthetase is an enzyme involved in the β-oxidation system of fatty acids. [Mishin et al. European Journal of Biochemistry (European Journal of Biochemistry)
Biochemistry) Vol. 82, pp. 347-354 (1978)] Among these, the present invention relates to a method for purifying acyl-CoA synthetase. Below, Asil
CoA synthetase is called the enzyme. The present inventors have discovered that long-chain acyl-CoA of microbial origin
As a result of intensive research into methods for purifying synthetase, we discovered a method for obtaining the enzyme with extremely high specific activity and electrophoretically uniform properties, thereby completing the present invention. That is, the gist of the present invention is that long-chain acyl-CoA
Synthetase-containing Candeida lipolyteica
After disrupting the NRRL Y-6795 strain, it includes a step of solubilizing the membrane-bound enzyme with polyoxyethylene alkylphenol ethers and a step of separating the solubilized enzyme from contaminant proteins by affinity chromatography. The present invention provides a method for purifying long-chain acyl-CoA synthetase. This bacterium was entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on November 17, 1985, under the microbial accession number 9711 (FERMP-9711). One feature of the invention is the solubilization of the enzyme from the membrane with polyoxyethylene alkylphenol ethers and the separation of the enzyme from contaminating proteins by affinity chromatography.
Until now, there has been no report on the use of affinity chromatography in the purification of acyl-CoA synthetase, but when the present inventors used it for this enzyme, it was found that the present method is an extremely excellent method. One feature of the present invention is that the enzyme was obtained from Candeida lipolyteica NRRL Y-6795 strain. Candida Ripolitica NRRL Y-
The 6795 strain is an extremely excellent source for obtaining enzymes easily and inexpensively in large quantities, and in this sense, the present invention provides an industrial method for producing the enzymes. Another feature of the present invention is that the obtained enzyme is very pure and has a specific activity that is lower than that of conventional enzymes.
This is extremely high, about 50 to 100 times higher.
In other words, the E. coli enzyme mentioned above is not electrophoretically single even at the most purified stage;
Further, the specific activity was 106 mU/mg protein, and the specific activity of the enzyme in rat liver was 250 mU/mg protein. However, Candida purified by the method of the present invention
The enzyme of L. lipolytica is completely single electrophoretically and has an extremely high specific activity of 50 to 100 times. As shown in the reaction formula above, this enzyme produces an equivalent amount of AMP from free fatty acids, and if this AMP is measured using the reaction system described below, free fatty acids in blood can be measured by an enzymatic method. . (Refer to Clinical Chemistry Vol. 4, No. 2, p. 179 (1975), Japan Clinical Research Society) Therefore, the enzyme purified by the method of the present invention is extremely excellent as a diagnostic enzyme for use in measuring free fatty acids in blood. Furthermore, labeled acyl-CoA, which is important as a biochemical reagent, can be produced very advantageously by using the enzyme from labeled fatty acids. Next, details of the present invention will be explained. Acyl-CoA synthetase exists in a wide range of microorganisms and can be extracted and purified from them, but in the present invention, since the bacteria grow quickly, the enzyme has high activity, and it is easy to extract, we used Candeida lipolyteica NRRL Y-6795 strain. is used.
Candeida Ripolitica NRRL Y-6795
The culture method of the strain is not specified, and the culture medium may be one normally used for culturing microorganisms, containing a carbon source (e.g., glucose), a nitrogen source, phosphoric acid, potassium, magnesium, etc., and culture temperature and culture time. etc., normal conditions are also used. Furthermore, in the case of a microorganism that produces the enzyme in an inducible manner, an inducer (for example, a fatty acid) may be added to the medium. When cultured bacterial cells are collected by centrifugation, filtration, etc., the cells are disrupted by mechanical means such as a homogenizer, ultrasound, or trituration with glass beads, or by chemical means such as cell wall lytic enzymes, and then centrifuged. The bacterial fragments are removed by separation. Next, the supernatant containing the cell granules is treated with a surfactant to release the enzyme from the granules. Prior to this operation, the supernatant may be subjected to ultracentrifugation to precipitate the granule fraction and suspended in an appropriate buffer, and then the above treatment may be performed. Polyoxyethylene alkylphenol ethers are nonionic surfactants, and a preferred compound is Triton , hereinafter referred to as activator T. The average molecular weight is assumed to be 628.)
It is. The amount added is usually 0.1 to 1.0 W/V%. The solubilization treatment of the enzyme with such an activator is
It usually lasts 30 minutes to 2 hours. The temperature is low, for example 0
-10°C, preferably 0-4°C. At this time, in order to prevent inactivation due to solubilization, a phosphate buffer, an enzyme protectant such as mercaptoethanol, EDTA, etc. may be added to the treatment solution. The enzyme solubilized under these conditions is separated as a supernatant from the granule fraction by ultracentrifugation. Next, this liquid is purified by affinity chromatography, but a purification operation may be performed prior to this. Ion exchange chromatography using phosphocellulose is used as one such operation. This operation separates and removes anionic proteins and the like. In phosphocellulose column chromatography, a phosphate buffer is usually used to elute the enzyme, but it is preferable to desalt it prior to the next affinity chromatography. Next, this enzyme-containing solution is purified by affinity chromatography. The adsorbent used in this case is selected to be one that specifically adsorbs enzymes that have affinity for ADP, ATP, CoA, etc. That is, as a ligand, part of the structure
It has a portion that has structural similarity to ADP and/or CoA. Structural similarity here refers to the property of having an affinity for and specifically adsorbing enzymes that participate in reactions involving ADP, ATP, and/or CoA. Substances with structural similarity to ADP, ATP and/or CoA have the formula () 1-amino-4-(4'-aminophenylamino)-anthraquinone-2,3'-disulfonic acid sodium represented by, and Cibacron blue F3G-A having the following general formula () (trade name, manufactured by Ciba Geigy (hereinafter referred to as Ligand C)). (In the formula, R ₁ = H or -SO ₃ Na R ₂ = - SO ₃ Na or H) For this purpose, these are bonded to a support in a manner that does not impair structural similarity. preferred as an adsorbent. Although the spacer is not particularly necessary in this case, it may be inserted. Next, although the support for these ligands is not particularly limited, the pore size
Polysaccharides and porous glass are preferable because of their large size, mild binding conditions with ligands, high mechanical strength, chemical stability, and low nonspecific adsorption of proteins. . As the polysaccharide, Textran and its crosslinked products, agarose and its crosslinked products, etc. are particularly preferred, and bead-shaped ones are easy to handle. The most preferable adsorbent for affinity chromatography has the general formula () (In the formula, one of R ¹ and R ² represents a hydrogen atom, the remaining one represents a -SO ₃ Na group, and X represents a polysaccharide residue.) As a commercially available adsorbent, Bluecepharose CL-6B (trade name: Pharmacia Fain.
Manufactured by Chemicals (hereinafter abbreviated as BS)). This uses the aforementioned Ligand C as a ligand, and the support is Sepharose CL-6B (trade name: manufactured by Pharmacia Fine Chemicals Co., Ltd., main component: crosslinked agarose). After adsorbing the enzyme-containing solution on the affinity adsorbent column, ATP, ADP, AMP,
When a buffer solution containing CoA etc. is passed through, the protein is eluted, but this protein fraction has no activity. Next, when this column is treated with a solution prepared by adding the above ATP-containing buffer solution to a gradient condition of 0 to 1.5M sodium chloride, the protein is separated and eluted. At this time, the enzyme has a sodium chloride concentration of 0.05
It elutes as a single peak in the ~0.5M fraction. From the above results, it appears that the chromatographic separation of the enzyme is due to chromatographic separation that combines not only affinity but also ion exchange. Although the enzyme solution thus obtained has a considerably high specific activity, it may be further purified if necessary. In this case, Sephadex is usually used.
G-100 (trade name: Pharmacia Huain Chemicals Co., Ltd. (hereinafter referred to as G-100)) treatment, and further hydroxyapatite
Chromatography etc. are suitable. The acyl-CoA synthetase obtained by this method was extremely pure and gave a single band even in SDS electrophoresis. In the purification of acyl-CoA synthetase to date, deoxycholic acid, which has surface activity, has been used to solubilize the enzyme. However, the purity and specific activity of the enzyme were insufficient. However, it has now been found that it is possible to isolate an extremely pure enzyme very easily by combining the above-mentioned solubilization treatment with a surfactant and a purification method using affinity chromatography. Affinity chromatography has recently begun to be widely applied to the purification of enzymes, and is gradually being used for lipid metabolism-related enzymes, but it has never been used for such enzymes. However, when the present inventors applied affinity chromatography to this enzyme for the first time, they were able to identify proteins with similar physical properties, which had been difficult to separate from the enzyme using previous methods. For example ATPase
It has been found that these compounds can be separated very easily. EXAMPLES Next, the present invention will be explained with reference to Examples, but it goes without saying that the present invention is not limited to these Examples. [Method of measuring activity] Activity was measured using Banis and Taub.
Tove method (Biochimica et Biophysica Acta)
Acta), Vol. 348, pp. 210-220 (1974). In other words, the reaction solution is Tris-HCl buffer (PH
7.5) 20 μmol, ATP 3 μmol, MgCl _{2 2} μmol, dithiothreitol 1.0 μmol, [U- ¹⁴ C] potassium palmitate (0.2 m Ci/mmol) 0.2 μmol, activator
T0.32μmol, CoA0.2μmol, total volume of enzyme solution is 0.2ml
And so. After preheating for 5 min, start the reaction by adding CoA or enzyme solution (25 °C), and incubate for 10 min.
Minutes later isopropanol-nheptane-1MH ₂ SO ₄
The reaction was stopped by adding 2.5 ml of 40:10:1.
Unreacted free fatty acids were extracted and removed with heptane. The aqueous layer was washed twice with 2 ml of heptane containing 8 mg of palmitic acid, and the radioactivity of the aqueous layer was measured. 1 μmol of palmitoyl in 1 minute under the above conditions
The amount of enzyme that produces CoA was defined as 1U. [Protein measurement method] The Lowry method was used. Example 1 Candida lipolytica NRRL Y-6795
The strain was treated with 2% glucose, 0.5% NH ₄ , H ₂ PO ₄ , 0.25
% _KH2PO4 , 0.1 _{% MgSO4}・_7H2O , 0.002%
24 flasks containing 500 ml of medium with the following composition: FeCl ₃ 6H ₂ O, 0.1% Bacto Yeast Extract (Difco USA) pH 5.2 were heated at 25°C at 72°C.
The cells were cultured with shaking under conditions of reciprocation/minute, and the cells in the mid-logarithmic growth phase were collected by centrifugation to obtain 250 g of wet cells. Next, after washing the bacterial cells with 0.1M phosphate buffer (PH6.5), 5mM mercaptoethanol and 1mM
The suspension was suspended in the same phosphate buffer containing EDTA and disrupted under liquid CO ₂ cooling using a Braun cell homogenizer (Merschungen, West Germany). (All operations below were performed at 0 to 4°C.) The homogenate thus prepared was heated at 8000G for 15 to
Centrifuge for 20 minutes, remove the supernatant, and add it further.
After ultracentrifugation at 230,000G for 1 hour, the precipitate was collected and suspended in a solution containing buffer and 2-mercaptoethanol EDTA at the above-mentioned concentration. (Total amount 72
ml, protein concentration 46 mg/ml, this fraction is called the granule fraction. ) Next, this suspension was mixed with a solution containing activator T, PH7.4 phosphate buffer, 2-mercaptoethanol, and EDTA, each with a final concentration of 5mM.
The concentrations were set to 50mM, 5mM, and 0.5mM. (Protein concentration: 11 mg/ml) The mixture was allowed to stand for 1 hour, then ultracentrifuged at 230,000 G for 1 hour, and 265 ml of the supernatant was collected. Next, this supernatant was diluted with 1.5 times the volume of 2mM activator T and 5mM 2-mercaptoethanol aqueous solution, and then a 20mM phosphate buffer containing 2mM activator T and 5mM 2-mercaptoethanol (this was referred to as buffer A) was added. ) was adsorbed onto a column (52 mm x 75 mm) of phosphocellulose p-11 (trade name: manufactured by Fatman). After washing the column with twice the volume of Buffer A, add 2.5 times the column volume of Buffer A and the same volume of 0.5M phosphate buffer (PH7.4) (containing 2mM activator T and 5mM mercaptoethanol).
Elution was performed with a linear gradient eluate between The flow rate at this time was approximately 90 ml/hr, and the fraction was 15 ml. The enzyme has a phosphate ion concentration
90ml of a fraction that appeared as a single peak between 0.05 and 0.15M and showed a specific activity of 0.28U/mg or more.
Collected. Next, in order to desalt this fraction, a column (38 mm
Combine all protein fractions eluted through a column of BS (16 mm × 350 mm) equilibrated with the same buffer and then
×100mm). The column was washed with buffer A equal to the column volume, and then with twice the column volume of buffer A containing 10 mM ATP. then
The flow rate was 30 ml/hr under linear gradient conditions using 3.75 times the volume of buffer containing 10 mM ATP and the same volume of this composition plus 1.5 M NaCl.
The enzyme was eluted in 10 ml fractions. The enzyme eluted as a single peak at NaCl concentrations between 0.1 and 0.3M. Fraction with specific activity 2.6U/mg or more
Combined 50ml. Next, the fraction of this eluate was ultraconcentrated to 10 ml using Diaflow Membrane Filter PM-30 (manufactured by Amicon Far East Co., Ltd., Tokyo), and then equilibrated with buffer A. G-
Buffer A was passed through a 100 column (27 mm x 870 mm) at a flow rate of 30 ml/hr, and the eluate was fractionated into 10 ml portions.
The elution pattern was as shown in Figure 1, and the enzyme activity coincided with the second peak. 40 ml of fractions showing a specific activity of 9 U/mg or more were collected. This fraction showed a completely single pand on SDS-polyacrylamide gel electrophoresis and was found to be pure acyl-CoA synthetase. Table 1 shows data at each purification step in the above method.

[Sephadex G-200 (trade name, manufactured by Pharmacia Fine Chemicals Co., Ltd.)]

2 Cofactor requirements ATP Mg ⁺⁺ CoA 3 Substrate specificity Table 2 4 Optimum pH 7.1-9.6

[Table] Reference Example 1 The amount of fatty acids was measured using the acyl-CoA synthetase purified by the method of Example 1. Reaction solution 1
Predetermined amount of potassium oleate, 100 μmol per ml
Tris HCl buffer (PH7.4), 5 μmol dithiothreitol, 1.6 μmol activator T, 7.5 μmol ATP,
10 μmol MgCl ₂ , 0.2 μmol phosphoenol potassium pyruvate 0.15 μmol NADH, 20 μg adenylate kinase, 30 μg pyruvate kinase, 30 μmol
g-lactate dehydrogenase, purified acyl
A solution containing 4 μg of CoA synthetase was placed in the cell of a spectrophotometer (Model 1101M manufactured by Eppendorf)25
After preheating at ℃ for 1 minute, 0.5 μmol of CoA solution was added (total volume: 0.5 ml) to start the reaction, and the change in OD334 nm due to the decrease in NADH was read from the recorder. The relationship between potassium oleate in the reaction solution and ΔOD334nm is as shown in Figure 2, and good linearity was obtained between 0 and 25 nmoles of oleic acid. Reference Example 2 Using the same method as in Reference Example 1, the relationship between 10 nmol of various long chain fatty acids and ΔOD334 nm was determined. The results are the theoretical value of △OD334nm as shown in Table 3.
0.246 (HU Bergmeyer)
Bergmeyer), Zeitschrift fu¨r Klinische Chemie.
und Klinische Biochemie), Vol. 13, pp. 507-508 (1975).

【table】 [Brief explanation of the drawing]

FIG. 1 is a graph showing the elution of acyl-CoA synthetase in Example 1. The horizontal axis is the volume of effluent (ml), and the vertical axis is the activity of acyl-CoA synthetase (U/ml) (on the left) and the protein (mg/ml). ml) (right side)
, and the symbol ● indicates the activity of acyl-CoA synthetase,
The circle indicates the amount of protein. FIG. 2 is a graph showing the relationship between the fatty acid amount and OD on reference side 1, where the horizontal axis is the fatty acid amount (nmole) and the vertical axis is ΔOD334nm.

Claims (1)

[Claims] 1. A step of pulverizing long-chain acyl-CoA synthetase-containing Candeida lipolyteica NRRL Y-6795 strain and solubilizing the membrane-bound enzyme with polyoxyethylene alkylphenol ethers, and the solubilized enzyme. 1. A method for purifying long-chain acyl-CoA synthetase, the method comprising the step of separating acyl-CoA synthetase from extraneous proteins by affinity chromatography. 2 Long-chain acyl described in claim 1
A method for purifying CoA synthetase, characterized in that the affinity chromatography is an affinity chromatography using a structural analog of ADP, ATP and/or CoA as a ligand. 3 Long chain acyl described in claim 2
In the method for purifying CoA synthetase, the ligand has the general formula () (In the formula, one of R ¹ and R ² represents a hydrogen atom, and the remaining one represents a -SO ₃ Na group.) 4 Long chain acyl described in claim 2
In the CoA synthetase purification method, the affinity chromatography adsorbent has the general formula () (In the formula, one of R ¹ and R ² is a hydrogen atom, the remaining one is -SO ₃ Na group, and X represents a polysaccharide residue.) How to do it.