Acyl-coa oxidase

Abstract

To provide acyl-CoA oxidase having sufficient Acyl-CoA oxidase activity and more suitable for use in clinical diagnosis and industrial use.SOLUTION: Disclosed herein is an acyl-CoA oxidase produced by strain ACO-1 (NITE P-03955).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a novel acyl-CoA oxidase. [Background technology]

[0002] The in vitro applications of acyl-CoA oxidase (EC.1.3.3.6) have been investigated in various technical fields, and some have been put to practical use.

[0003] An example of an in vitro diagnostic reagent is a reagent for measuring free fatty acids using acyl-CoA synthetase and acyl-CoA oxidase. A method is known in which acyl-CoA synthetase acts on free fatty acids (NEFA: non-esterified fatty acids) in a sample in the presence of ATP and CoA, acyl-CoA oxidase acts on the resulting acyl-CoA, and the amount of hydrogen peroxide produced is measured to quantify the free fatty acids (see Patent Document 1 and Non-Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 62-62160 [Non-patent literature]

[0005] [Non-Patent Document 1] Masaru Shimizu, "Synthesis of coenzymes using microbial enzymes and their applications", Journal of the Agricultural Chemical Society of Japan, 1985, Vol. 59, No. 12, p. 1273-1281 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide an acyl-CoA oxidase which has sufficient acyl-CoA oxidase activity and is more suitable for use in clinical diagnosis and industrial applications. [Means for solving the problem]

[0007] Means of the Invention As a result of intensive efforts to solve the above-mentioned problems, the present inventors discovered that the problems could be solved by acyl-CoA oxidase produced by the ACO-1 strain (NITE P-03955), and thus completed the present invention.

[0008] In order to achieve the above object, the present invention provides the following acyl-CoA oxidase. [1] Acyl-CoA oxidase produced by strain ACO-1 (NITE P-03955). Effect of the Invention

[0009] According to the present invention, it is possible to provide an acyl-CoA oxidase which has sufficient acyl-CoA oxidase activity and is more suitable for use in clinical diagnosis and industrial applications. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Hereinafter, an embodiment of the present invention (hereinafter, referred to as the present embodiment) will be described in detail. Note that the present invention is not limited to the following embodiment, and can be modified in various ways within the scope of the present invention.

[0011] The acyl-CoA oxidase according to this embodiment is an enzyme produced by the ACO-1 strain (NITE P-03955).

[0012] This strain was named ACO-1 strain to distinguish it from known strains, and was deposited at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Accession Number: NITE P-03955).

[0013] In producing the acyl-CoA oxidase according to the present embodiment, a microorganism is cultured in a nutrient medium to produce acyl-CoA oxidase in the cells or in the culture solution. After the culture is completed, if the product is in the cells, the cells are collected from the culture by filtration, centrifugation, or other means, and then the cells are disrupted mechanically or enzymatically using lysozyme or other means to obtain a cell extract. If the product is released outside the cells and is in the culture solution, the cells and insoluble components in the culture solution may be removed by centrifugation or filtration. If necessary, EDTA, PMSF, a protease inhibitor, and / or a suitable surfactant may be added to concentrate the aqueous solution of acyl-CoA oxidase, or the aqueous solution may be treated with ammonium sulfate fractionation, gel filtration, adsorption chromatography such as affinity chromatography, or ion exchange chromatography without concentration to obtain acyl-CoA oxidase with high purity. When determining the sequence of the produced acyl-CoA oxidase, known methods such as the Edman degradation method and mass spectrometry may be used.

[0014] The culture conditions for the microorganisms may be selected taking into consideration their nutritional and physiological properties, and in most cases, liquid culture is used, but from an industrial perspective, submerged aeration and stirring culture is advantageous. As nutrient sources for the medium, a wide range of nutrients commonly used for microbial culture can be used.

[0015] The carbon source may be any assimilable carbon compound, such as glucose, saccharose, lactose, maltose, fructose, glycerol, molasses, etc. The nitrogen source may be any usable nitrogen compound, such as peptone, meat extract, yeast extract, soybean protein, casein hydrolysate, etc.

[0016] The amount (concentration) of each component during culture can be appropriately selected within the range in which acyl-CoA oxidase is produced, following the method described in a non-patent document (Journal of Industrial Microbiology & Biotechnology (1999) 23, 456-475).

[0017] In addition, salts of phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, copper, etc., specific amino acids, specific vitamins, etc. may be used as needed.

[0018] The culture temperature can be appropriately changed within a range in which the microorganism grows and produces acyl-CoA oxidase.

[0019] The acyl-CoA oxidase in the culture can be collected and used as it is, but generally, when the acyl-CoA oxidase is present in the culture, it is used after separating the acyl-CoA oxidase-containing solution from the microbial cells by filtration, centrifugation, or the like. When filtration is difficult, the use of a filter aid such as perlite is considered for removing the microbial cells and insoluble components, and when centrifugation is difficult, the use of a flocculant such as a soluble polymer having an ion exchange group is considered, and it is a common practice to add an appropriate economical step. When the acyl-CoA oxidase is present in the microbial cells, the obtained culture is collected by means of filtration or centrifugation, and the microbial cells are then destroyed by mechanical methods or enzymatic methods such as lysozyme and zymolyase, and, if necessary, a chelating agent such as EDTA, a protease inhibitor such as PMSF, and / or a surfactant is added to solubilize the acyl-CoA oxidase and separate it as an aqueous solution.

[0020] The acyl-CoA oxidase-containing solution thus obtained may be precipitated by a fractional precipitation method such as vacuum concentration, membrane concentration, and further salting out with ammonium sulfate, sodium sulfate, or the like.

[0021] The precipitate is then dissolved in water and dialyzed using a semipermeable membrane to remove impurities with lower molecular weights. Alternatively, the precipitate can be purified by gel filtration using an adsorbent or gel filtration agent, adsorption chromatography such as affinity chromatography, ion exchange chromatography, etc., and the purified acyl-CoA oxidase can be obtained from the acyl-CoA oxidase-containing solution obtained by these methods by treatments such as vacuum concentration and freeze-drying.

[0022] It has been confirmed that the acyl-CoA oxidase according to the present embodiment has sufficient acyl-CoA oxidase activity and is practical. Therefore, the acyl-CoA oxidase according to the present embodiment is useful industrially, particularly as an enzyme for in vitro diagnosis.

[0023] The present invention will now be described with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0024] <Acyl-CoA oxidase (ACO) activity measurement method> [Reaction Reagent Mixture] 0.2M Tris-HCl buffer (pH8.0) 0.2ml 15mM 4-aminoantipyrine solution 0.1ml 0.2% (W / V) phenol solution 0.1 ml 0.1ml of 50U / ml peroxidase (POD) solution 1% (W / V) Triton X-100 solution 0.1 ml 5mM Palmitoyl-CoA solution 0.1ml Purified water 0.3ml [Enzyme dissolving dilution solution] 10 mM KH containing 3 mM ATP and 10 μM FAD 2 PO 4 -K 2 HPO 4 Buffer (pH7.0)

[0025] Dispense 1.0 ml of the above reaction reagent mixture into a reaction cell (1 ml black cell), preheat at 37°C for 5 minutes, then add 20 μl of the enzyme sample solution diluted B times with the above enzyme dissolution dilution solution, mix, and start the enzyme reaction at 37°C. After the reaction starts, measure the absorbance at 500 nm to determine the absorbance change per minute during the linear reaction (As / min). As a blank test, perform the same procedure using the above enzyme dissolution dilution solution instead of the enzyme sample solution to determine the absorbance change (Ab / min). The absorbance difference (As / min-Ab / min) between the absorbance change using this enzyme sample solution (As / min) and the absorbance change using the blank test (Ab / min) is defined as ΔA / min, and the enzyme activity is calculated using the following formula. The enzyme dilution ratio B is set so that ΔA / min≦0.060 Abs / min.

[0026] Enzyme activity (U / mL)={(ΔA / min) / (12.0×1 / 2)}×(1.02 / 0.02)×B 12.0: Millimolar extinction coefficient (cm) of quinoneimine dye at 500 nm 2 / μmol) 1 / 2:H 2 O 2 Coefficient for the production of 1 mole of quinoneimine dye from 2 moles 1.02: Total reaction volume (ml) 0.02: Amount of enzyme sample solution used in the reaction (ml)

[0027] <Production and activity measurement of acyl-CoA oxidase (ACO)> ACO-1 strain (NITE P-03955) was precultured in LB medium, the preculture solution was suspended in a medium containing glycerol, yeast extract, peptone, etc., and cultured at 34°C for 32 hours using a 2L culture device. The culture solution after the culture was centrifuged to collect the cells. The cells were then mechanically disrupted to solubilize the acyl-CoA oxidase, and the activity of the centrifuged supernatant was measured using the ACO activity measurement method described above. The enzyme activity was calculated to be 45.1 U / mL.

Claims

[Claim 1] Acyl-CoA oxidase produced by strain ACO-1 (NITE P-03955).