Novel Pyranose Oxidase Processes for the Production and Use Thereof and Test Compositions for Use Therein

Europaisches Patentamt (19) J European Patent Office @ Publication number: 0119332 A1 Office europeen des brevets EUROPEAN PATENT APPLICATION Application number: 83301376.6 @ intcL».-C12N 9/04, C 12 Q 1/26 Date of filing: 11.03.83 Date of publication of application: 26.09.84 @ Applicant: KYOWA HAKKO KOGYO CO., LTD., Bulletin 84/39 Ohtemachi Bldg., 6-1 Ohtemachi Itchome, Chiyoda-ku Tokyo 100 (JP) Inventor: Nakanishi, Toru, 145-42 Tomuro, Atsugi-shi Kanagawa-ken (JP) Inventor: Machida, Yozo, 500-50, Arai Hodogaya-ku, Yokohama-shi Kanagawa-ken (JP) Representative: Thomas, Roger Tamlyn et al, D. Young & @ Designated Contracting States : DE FR GB IT Co. 10 Staple Inn, London WC1V7RD (GB) @ Novel pyranose oxidase processes for the production and use thereof and test compositions for use therein. A novel pyranose oxidase exhibiting strong specific activity substantially only on glucose is disclosed, as well as a method for its preparation by fermentation of a microorganism of the genus Coriolus, Daedaleopsis, Gloeophyllum or Pleurotus. The enzyme is useful for the determination of glucose, and methods for doing this, as well as components for use therein are described. The present invention relates to the preparation and use of pyranose oxidases and, in particular, to the production of a novel pyranose oxidase exhibiting a stong specific activity on glucose alone. In the prior art a pyranose oxidase (EC 1.1.3.10) is known which catalyzes the oxidation of glucose, xylose and sorbose into glucosone, xylosone and 5-ketofructose, respectively (Methods in Enzymol, vol. XLI, p.170, (1975)). In oxidizing these substances, oxygen is consumed and hydrogen peroxide is formed and the enzyme is, therefore, useful for the total quantitative determintation of pyranoses such as glucose, xylose and sorbose but without distinction between them. This known enzyme is produced by culturing a microorganism belonging to the genus Polyporus (Methods in Enzymol, Vol. XLI p.170 (1975)). In contrast, the present invention provides a novel pyranose oxidase which shows strong specific activity which is substantially restric- ted to activity on glucose alone. According to the present invention, this novel pyranose oxidase is obtained by culturing a microorganism belonging to the genus Coriolus, Daedaleopsis, Gloeophyllum or Pleurotus and which is capable of producing the novel pyranose oxidase, in a nutrient medium until enzymatic activity is detected in the culture liquor and thereafter recovering the pyranose oxidase therefrom. The novel enzyme of this invention is useful for the quantitative determination of pyranose oxidase substrates, especially D-glucose, by oxidizing the substrate in the presence of oxygen and measuring the amount of a product of the reaction, usually the hydrogen peroxide, or the amount of oxygen consumed by said reaction. In the following description of the properties of the novel enzyme of this invention, the following method is used for the determination of the enzymatic activity. Assay Method A 3.0 ml sample is first prepared comprising 1.0 ml of 0.1 M tris- hydrochloride buffer (pH 7.0), 0.2 ml of a colouring reagent prepared in advance as hereinafter described, 0.1 ml of 1 M glucose solution, 0.1 ml of a solution of the pyranose oxidase at a known concentration, 0.1 ml of 0.1 mM flavinadenine dinucleotide and 1.5 ml of distilled water. Said colouring reagent is prepared by adding 0.1 M tris-hydrochloride buffer (pH 7.0) containing 10 m mol l4-aminoantipyrine (hereinafter referred to as 4AA) and 10 m mol/Zphenol, to an amount of a peroxidase which is sufficient to provide a peroxidase concentration in said buffer of 2000 U per 100 mI. The pyranose oxidase containing sample is incubated at 37°C and the increasing rate of the absorbancy at 500 nm is automatically measured on a spectrophotometer. If the molecular extinction coefficient of a quinoneimine pigment formed under these conditions is defined as 5.3 x 103, and one unit of enzymatic activity is defined as the amount of the enzyme that produces one µ mole of hydrogen peroxide per minute at a temperature of 37°C and at a pH of 7.0, then the enzymatic activity (A) of the pyranose oxidase in the sample can be defined by the following formula:- where A500 is the rate of increase of the absorbancy of the sample per minute at 500nm. The properties of the novel enzyme of this invention are also described hereinafter with reference to the accompanying drawings ,in which:- Figure 1 shows the relationship between the relative activity of the enzyme and the stable pH range. Figure 2 shows the relationship between the relative activity of the enzyme and the optimum pH. Figure 3 shows the relationship beween the relative activity of the enzyme and the optimum temperature. Figure 4 shows the relationship between the relative activity of the enzyme and the heat stability. Figure 5 shows the relationship between the absorbancy and the glucose concentration. In further detail, the properties of the novel enzyme of this invention are:- (1) Stable pH range Samples of the enzyme were dissolved in three different buffers, to make up enzyme solutions having an activity of 100 mU/0.7 ml. viz a citrate buffer, a Tris buffer and a borate buffer. In this test buffer solutions were used having the pH values indicated in Figure 1. Each enzyme solution was incubated at 50°C for 30 minutes, cooled and neutralized prior to measurement of the residual enzymatic activity by the above described assay method. The present enzyme is stable at a pH of 5.0-7.4 as shown in Figure 1. (2) Optimum pH Samples of the enzyme were dissolved in Tris buffer solutions have the pH indicated in Figure 2 to make up enzyme solutions having an activity of 250 mU/3.7 ml. Each of the enzyme solutions was put into a BIOXYGRAPH cell (trade name for a cell for measuring oxygen concentration made by Kyusui Kagaku Laboratory, Japan), and 50 µl of 1 M glucose were added. Measurements were made to determine the amount of absorbed oxygen. The optimum pH for this enzyme was shown to be around 6.2. (3) Optimum temperature The same enzymatic reactions as described in the assay method were repeated for 5 minutes at the temperatures indicated in Figure 3, except that pyranose oxidase concentrations of 100 mU were used. The optimum temperature for the present enzyme was determined to be 30°C. (4) Heat stability The enzyme was dissolved in 0.6 ml of 0.2 M Tris buffer solution (pH 6.0) to make up an enzyme solution having an activity of 100 mU/0.7 ml. The enzyme solution was incubated for 30 minutes at the temperatures indicated in Figure 4 and measurements made to measure the residual enzymatic activity. As shown, the enzyme keeps 90% of its activity with treatment at 60°C for 30 minutes. (5) Substrate specificity The same reactions as described in the assay method for enzymatic activity were repeated except that an enzyme concentration of 2.5 U/ml was used, and, as the substrate, 0.1 ml samples of various carbohydrate liquors indicated in Table I were used, each such liquor having a concentration of 100 mM. The results are expressed as percentages of the glucose reaction and as will be seen the specificity of this enzyme with glucose is very high and substantially exclusive thereto with only minor levels of activity on mannote, galactose, sorbose and xylose. The specificity with respect to the latter two, in particular being a great deal lower than that of the known pyranose oxidase derived from microorganisms of the genus Polyporus (Table 1 of B.B.A. 167, 501-510 (1968)). (6) Electron acceptor In this phase, 1.4 ml of 50 mM tris-hydrochloride buffer (pH 7.0), 0.1. ml of 250 mU enzyme solution and 0.5 ml of electron acceptor solutions having various concentrations as indicated in Table 2 were put into a cell with Thunberg tube. After saturating with dissolved oxygen, the reaction solution was mixed with 0.5 ml of a 0.1 M glucose solution. The change in absorbancy per minute from 30 minutes to 90 minutes after mixing was determined and the activities of the electron acceptors are expressed as relative activity. As will be seen the optimum electron acceptor for this enzyme is oxygen. (7) Enzyme inhibition Various inhibitors were studied in the same manner as that adopted in determination of the optimum pH. A reaction solution comprising 3.4 ml of 50 mM tris-hydrochloride buffer (pH 7.0), 0.1 ml of enzyme solutin containing 280 mU of pyranose oxidase and 0.2 ml of 18.5 mM inhibitor solution were put into a cell for measuring oxygen uptake and 50µlof 1M glucose were added thereto and the sample incubated at 37°C. The results are given in Table 3. As shown the enzymatic action is substantially inhibited by Cu2+, Ag+, Ni2+ and p-chloromercuribenzoate, and to a lesser extent by Co2+,8-hydroxyquinoline and hydrogen peroxide, and not at all by Mg . (8) Isoelectric point The isoelectric point of the enzyme is 4.2 as determined by the isoelectric focussing method using carrier ampholyte at a pH of 3.5 - 10.0 (9) Molecular weight By the gel-filtration method on Sephadex G-200 (Registered Trade Mark) and using, as the standard protein, egg albumin, bovine blood serum, alcohol dehydrogenase, Y-globulin and xanthin oxidase derived from milk, the molecular weight of the enzyme is calculated to be about 220,000. (10) Km value According to the Lineweaver-Burk plot method using 2.5 U pyranose oxidase the Km value for glucose is determined to be 0.83 mM, which is about one-twelfth to one-twenty fourth of that of glucose oxidase (10 - 20mM).

Load more